DOI

Received: October 2022 – Accepted: November 2022

¹ Budapest University of Technology and Economics

Keywords

food industrial developments, cereal industry, cereal chemistry, Elek ‘Sigmond, Géza Binder Kotrba, Sándor Zoltán, János Holló, Lajos Fodor, Béla Sevella, László Telegdy Kováts, Radomír Lásztity, András Salgó, Beáta Vértessy

The end of XIXth century and the beginning XXth century was the golden age of Hungarian agriculture and agro-industrial sector and processes.

High number of factories with large capacities were founded in sugar-, starch-, and spirit industries and world-famous innovations were introduced in the Hungarian cereal and milling industry, breweries and in further other branches of food processing.

The developments of industrial microbiology and it’s conscious applications had also strong effects on the investments and innovations in yeast, beer and alcohol production and in other branches of bio-industries.

This age can be characterized by the innovations and methods development introduced in the world-wide recognised cereal- and milling industry:

• András Mechwart (1837-1907) innovation of striate rolling mill with chill-casting and manufacturing by Ábrahám Ganz,
• Károly Haggenmacher (1835-1921) 15 patents in milling industry included flat sieves and purifiers,
• Tamás Kosutány (1848 - 1915) test methods for detection of dough quality and rupture,
• Imre Pekár (1838 - 1923) „pekáring”, determination the colour of flour,
• Leo Liebermann (1852 - 1926) test method development for gluten strength determination,
• Jenő Hankóczy (1879 - 1939) innovation of Farinograph for determination of water absorption and flour quality,
• Ferenc Grúzl (1897 - 1972) innovation of Laborográf for measuring extensibility of dough,
• Mihály Vuk (1876 - 1952) methods develpment in cereal and flour chemistry.

Same age was the period the initiation and definition of biotechnology as a novel field of science. Károly Ereky (1878-1952) mechanical engineer defined the concept of biotechnology as can be followed below:

• “Biotechnology - (1918) all the activities, which produce products (convert raw materials) with the help of microorganisms.”
• “Living machine, biotechnological machine”

In this period of time claims the demand in knowledge of food industry, processing and biotechnology also in higher education:

In the Institutum Geometricum-Hydrotechnicumban (predecessor in title of BME) Lajos Mitterpacher gives lectures in „General nature study and economy” covering hemp, flax, wine, vinegar, alcohol and silk production and technology. Vince Wartha gives lectures of “Fermentation chemistry’’ in 1871. Later he gives (1873) lectures in “Agricultural chemistry” and with Gyula Klein introduce “Technical microscopy”, “Wine chemistry” and “Wine microscopy” subjects in curriculum.

In 1908 Vince Wartha and Lajos Ilosvay invited professor Elek ‘Sigmond (1873-1939) to establish a Department of Agricultural Chemistry at BME. Elek ‘Sigmond was that time the professor of soil science and agricultural chemistry in Hungarian Crop Production Institute at Magyaróvár. He organized a lecturer staff covering in those days most relevant industrial syllabus and subjects:

Gyula Szilágyi “Fermentation” and “Spirit-, Beer- and Vinegard Technology’’, Lajos Vázsony “Fermenting organisms”, Ede László “Chemistry of Enology”,Tuzson “Technical micology”, Mihály Vuk “Control of adulterated food and gusto products”, Telegdy Kováts „Protozoology”.

Elek ‘Sigmond recognized the latent of Mihály Vuk in the analysis of methods development and made a motion (1919) to establish a Department of Chemical Analysis at BME with the following justification:

„The Hungarian engineering need an extended potential in agricultural chemistry and in food chemistry in higher education”.

Based on this proposal the Department of Food Chemistry was founded in 22nd of September 1921.

The founder of Department Prof. Mihály Vuk (1876-1952) graduated as chemical engineer in Zurich (1898), after receiving his Dr. title he was assistant lecturer at BME and later worked with Tamás Kosutány and Elek ’Sigmond in Magyaróvár. Between 1903-1904 he was director of Budapest Casein Factory and since 1905 worked in Hungarian Institute of Chemistry. His life-work was acknowledged with the Kossuth Grand Prix in 1911 by the Hungarian Society of Engineers and Architects.

The characteristic research activities of Vuk era (1921-1948) were the followings:

Chemistry and technology of enology, Flour chemistry and flour improvers, Novel methods is food analysis (chemical preservers, thermal decomposition of lactose, rancidity of lipids, detection of adulteration). Mihály Vuk was the Dean of Faculty between 1928-29 and the TMB (Hungarian Academy of Sciences) awarded him with the DSc (doctor of scienses) title in 1952.

The second head of Department was László Telegdy Kováts (1902-1987) between 1950-1971, who listened the lectures of Mihály Vuk since 1923 and got his doctor tile in 1927 and employed in Hungarian Institute of Chemistry. Since 1942 he was appointed as director of Hungarian Sugar Ltd. Center. After his nomination as professor and head of Department (1950) two times were Dean of Faculty of Chemical Engineering (1952-55) and in 1957.

In hard circumstances after world war the restart of research were cumbersome. The main orientations of projects were the followings: Method developments for quality control of raw materials (Department panel), investigation of Maillard reactions (L.Telegdy Kováts, F. Örsi), analysis of vitamins and antioxidant effects (É. Berndorferné Kraszner), cereal reseaches (R. Lásztity, J. Varga), investigation of packaging material (M. Szilasné Kelemen, F. Örsi), methods development in rheology (J. Major, J. Varga), wine chemistry and tests (D. Törley, J. Nedelkovits), composition and biochemistry of mushrooms (D. Törley), food toxicology (J. Kovács), instrument and methods developments (Dep. panel).

The next head of Department Prof. Radomír Lásztity (1929-2018) took over this leading position from L. Telegdy Kovács in his age 42 in 1971. The talented new leader had quick scientific career (1951 assisted lecturer, 1961 candidate, 1968 DSc, 1969 full professor) and parallel took faculty and university positions (1966-70 Vice Dean of Faculty, 1970-76 Vice Rector of BME education affairs).

Prof. Lásztity had plenty of innovations in education, e.g. actuation of education in organizer chemical engineering, initiation of english language education, but the most important and relevant was the foundation and actuation of biochemical engineering education.

This education form and curriculum was figured out with Professor László Nyeste under dean’s leadership Prof. János Holló, recognized the increased importance of biological and biochemical skills beside the chemical and engineering skills and the importance of integrated knowledges.

According to this recognitions the name of Department was also changed to Dept. of Biochemistry and Food Technology.

The research profile of Departmant kept the strong orientation in cereal chemistry; mechanism of flour improvers (L. Telegdy Kováts L. – R. Lásztity), non-covalent interactions in gluten complex (R. Lásztity), isolation and determination of amino acid sequence of avenotionin (F. Békés – R. Lásztity), heterogenity and control functions of wheat proteases (A. Salgó), and novel research trends and projects arose:

• Protein–carbohydrate, protein–lipid, protein-protein interactions: Mechanism and role of formation brown colour products and methyl-glioxal (F. Örsi)., structure and role of lipoproteins in cereals (F. Békés, I. Smied), characterization of interactions (R. Lásztity– J. Nagy).
• Novel protein sources, enhance the biological value of proteins: Isolation, purificationamino acid composition, deficienciesdigestibility (bioavailability)biological value (in vitro)techno-functional characteristicsoptimization of recipies (Dept. panel)
• Development of analytical methods: Analysis of amino acid composition and biogenic amines. (A. Zsigmond, L. Sarkadi, E. Ungár), Methods development for detection of mycotoxins (Á. Bata), measurement the functional properties of proteins (S. Tömösközi – J. Nagy), development of air segmented and flow injection analytical methods (J. Varga, S. Tömösközi), innovation of electrophoretic method (E. Györey , M. Kárpáti).

The leadership of Department was received from Prof. Lásztity by András Salgó (1951) in 1993. The new head went through all the academic stair-steps and got his professor’s nomination in 1995 and parallel served the Faculty as Vice Dean (economic affairs) between 1993 and 1999.

The main tasks of this period are to deal with the negative processes affecting higher education after the regime change “Bokros package”, to develop the departmental infrastructure, to rebuild and renovate. Preparations for the Bologna process and the complete overhaul of the education system will begin. Projects aimed at strengthening the links between industry and academia are launched, and the Department is gradually involved in international, mainly European Union, research framework programmes and projects. (Lajos Bokros was a left-wing Hungarian minister of finance, during whose tenure the actors of Hungarian public life and science were forced to endure numerous financial restrictions. The Ed.).

Beside the traditional research orientations, novel innovation themas and projects were below:

• Investigation of functional properties of proteins: Methods development of emulsifying and foam forming activity and stability using conductometry. (S. Tömösközi et.al.), Micro-scale test methods and instrument developments in detection of cereal quality (J. Varga,S. Tömösközi, A. Salgó, F. Békés).
• Plant physiological (seed development, germination, stresses) research: Non-destructive spectroscopic methods development to follow the seed development and germination processes in wheat (A. Salgó – Sz. Gergely), Evaluation of post–harvest processes in fruits (P. Merész, T. Lovász, A. Salgó), Biochemical and chromosomal investigation of the effects of drought, cold and salt stresses in cereals, methods development for detection of biogenic amines (L. Sarkadi).
• Development of variety identification methods based on proteins: Gel electrophoretic method for variety identification in wheat, development of national wheat catalogue (Á. Kemény, M. Kárpáti, F. Örsi , F. Békés), Variety identification using HPLC method (O. Baticz, F. Örsi F), Variety identification with capillary electrophoresis (É. Scholz).
• Near-infrared based spectroscopic and imaging (NIR/MIR/FT-IR) methods developments: Qualitative and quantitative method for detection of proteins, composition, physiological status, methods networking. Industrial process control, detection of adulterations. (A. Salgó – Sz. Gergely).
• Molecular biological methods development and application in quality control of proteins: Genetic background the formation of gluten proteins, detection of gluten „contamination”, analysis of GM raw materials, developments using aptamers. (J. Gaugecz –T. Révay – A. Szarka- T. Mészáros).
• Technological innovation in development of health promoting cereal products. (S. Tömösközi et.al.)
• Vitamin C: metabolism research (A. Szarka et.al.).

Due to internal integration of Faculty (2007) basic changes were resulted in the life and operation of Department. The main aims of integration were to develop bigger, more integrated and efficient units, so instead of former 10 departments five were structured.

Department of Applied Biotechnology and Food Science was formed by the integration of Dept. Biochemistry and Food Technology (earlier Food Chemistry) and by Dept. Agricultural Chemistry. The integrated unit with 25 academic people, 20 technical staff and approx. 20-25 PhD student took extreme big education tasks and produced notable incomes from national and international projects and industrial resources.

The research profile of integrated Department was changed continuously according to alteration of scientific and industrial conditions and demands. At the end of integration process nine main thematic area were defined which were listened below:

1. Biochemical and molecular background of draught, salt és oxidative stresses in plant materials (cereals, tuberoses);
2. Research in cereal chemistry and technology, product developments, innovation of functional products.
3. Methods and instruments development of rapid-tests and dedicated analysis methods.
4. Non-destructive methods developments and applications in monitoring of biosystems and bioprocesses.
5. Non-food exploitation of agricultural and other wastes.
6. Theoretical projects in biology and industrial microbiology,
7. Development of up-stream and down-stream processes and operations in fermentation.
8. Development and application of targeted biodegradation processes and operations.
9. Development of engineering toolkit in environmental management

On account of integration procedure both former units maintained their research traditions, the inside synergic affects were firmed in the novel innovations and the partcipation of students in R+D+I projects were increased

The leadership of integrated department was taking over by Vértessy Beáta in 2015. She was the Vice Head of Institute of Enzymology Hungarian Academy of Sciences (later ELKH-TTK) and Head of Research Group of Genom Metabolism. Her scientific carrier was acknowledged by the Division Biology in Hungarian Academy of Science as candidate (1991) and DSc (2001).

The histories of Dept. Food Chemistry and Dept. Agricultural Chemistry were interlocked very organic way, so both organizations made significant services for the Hungarian food science during the last total 113 years.

Finally, the names of the heads of the departments providing training in food chemistry and technology should be listed here (the numbers indicate the dates of the department’s name changes):

• Agricultural Chemical Technology (1908)
  • Elek ‘Sigmond
  • Géza Binder Kotrba
  • Sándor Zoltán
  • János Holló
  • Lajos Fodor
  • Béla Sevella
• Food Chemistry (1921) later Biochemistry and Food Technology
  • László Telegdy Kováts
  • Radomír Lásztity
  • András Salgó
• Department of Applied Biotechnology and Food Science (2007-)
  • András Salgó
  • Beáta Vértessy

DOI

Received: September 2022 – Accepted: November 2022

¹ External Member of the Hungarian Academy of Science, FBFD PTY LTD NSW Australia

Keywords

model of wheat glutenin, tionions, arabidoxane, activan

2. Introduction

The rather unusual title of this paper requires some explanation: The author started to work as a demonstrator during his last semester of his chemical engineering studies in 1972 at the Department of Biochemistry led at that time by professor Telegdi-Kovats. He was employed as a sponsored postgraduate in July 1972 under Professor Lásztity, two weeks after the change of leadership of the department. He carried out research and was involved in undergraduate teaching, moving ahead on the academic ladder, achieving university doctorate and PhD till 1987 when he was invited to join the Wheat Research Unit of CSIRO (Commonwealth and Industrial Research Organization) in Sydney, Australia. Being involved cereal research there he had a permanent and a continuous relationship with the staff of the Department in Budapest, he was not only a witness but also a participant of the ongoing research there, meanwhile he had the opportunity to directly perceive the recognition of the Departments’ research activity on the international platform.

3. Early history

Before covering the story of cereal research carried out at the department, it is worth to mention an episode happened 50 years earlier than the establishment of the department but had and even has nowadays strong effect on its spirit and strategy.

The last quarter of the 19^th century is the era of success of the Hungarian milling industry, when the chilled-iron roller mills equipped with planar sieves, manufactured by the factory of Abraham Ganz spread around the world. A functioning model-mill, to demonstrate the revolutionary new technological solutions of the Ganz mills, with all their novel features, have appeared in Australia. The mill was capable to mill some kilogram grain for evaluating its quality. This equipment, representing one of the superior products of the Hungarian manufacturing industry of the XIX century is also a valuable symbol of the Australian grain industry developing in same time. However, the mill has an important role in the birth of cereal chemistry as scientific discipline, too [1].

In the 1890ies the Australian wheat breeder William Farrer and Fredrick Guthrie, the chemist working with Farrer introduced the concept that the quality of wheat also should be considered together with harvest yield as a criterion used during wheat breeding. Guthrie’s concept was to evaluate quality by applying identical methodologies and scaled down equipment and in the laboratory as the milling and baking industry practice in large scale [2]. So, it was inevitable that Guthrie should turn to this form of milling when he was asked to evaluate the milling qualities of a series of cross-bred lines of Farrer, in 1894. Guthrie took the model mill to his laboratory and started to evaluate Farrer’s wheat line on it: The Ganz mill became the very first test equipment to evaluate the milling quality wheat – the discipline of grain science born [3].

Guthrie’s model mill – after several decades of vicissitude – was taken back to Hungary in 2011 and now it shown as a part of the permanent exhibition of the history of the Ganz mills in Budapest at Foundry Museum of the Hungarian Technical and Transport Museum.

4. The beginnings

Cereal science is one of the most important research directions at the Department since its establishment under in the 20ies under the leadership of Miháyl Vuk. In the first quoter of the the XX. century, the Hungarian grain science was internationally well-recognised László Karácsonyi achieved internationally known results in investigations of bread staling, published in the then established American journal, Cereal Chemistry [4, 5], the Pekár flour colour measuring method, became widely known and applied in several countries [6], internationally one of the very first detailed information about the chemical composition of the Hungarian wheat and wheat flour was published [7]. Hankóczy’s dough rheology equipment had been developed in this period, revolutionising the wheat chemistry: he discovered the farinometer in 1905, the very first equipment which was capable to determine the extensibility of dough and gluten. After further development he established the Farinograph, for the simultaneous determination of the water absorption of the flour, the quality of the gluten and the dough development time [8,9]. His method and his invention has been spread around the world. He proposed the establishment of Hungarian wheat cadastral leading to quality-based wheat selection in breeding [10,11].

Under the leadership of Vuk Mihály, the department played important role in the development of methodologies for Hungarian food analytics and quality control. He published the first Hungarian food chemistry textbook with his co-workers, Zoltán Sándor and Károly Vas [12], he is the author of the first Hungarian wheat chemistry book [13].

5. Radomir Lásztity, the “Hungarian Pomeranz”

A new, internationally well recognised era started in 1951 when Radomir Lásztity joined to the department and especially from 1972 when he took over the leadership at the department. The number of staff doubled; the average age dropped by 30 years. In addition to his research activity, Lásztity had essential role in shaping the educational structure of the Chemical Engineering Faculty by establishing the bioengineering division, he had a mentor role of for the teaching and research stuff of Department. His teaching passion is best illustrated by the fact that throughout his carrier he always has found time to write numerous much-needed textbooks in English and Hungarian language as well as books for undergraduate and postgraduate students in co-authorship with colleagues from The Department or from other institutions. Since its publication in 1984 by CRC Press under the title “The Chemistry of cereal proteins”, it has become one of the most frequently mentioned handbooks in the literature worldwide. Radomír Lásztity’s most important works on this subject are presented in Figure 1 without claiming to be exhaustive.

The openness for new ideas of Lásztity is best demonstrated by the fact the Ganti’s Chemoton-theory on the principles of life has been part of the curriculum of bioengineering students at the faculty first time around the world as a part of a facultative subject of “Selected chapters of Biochemistry” under the name of Lásztity. Several awarded student works, thesis have been written on the related subject, and a self-study small group called “Kinetic Club”, sponsored by the MTESZ, has actively worked on topics related to pre-biology evolution, not only with students from the BUTE, but from SOTE and ELTE.

From the very beginning, Professor Lásztity’s personal research focused on the investigation od structure and rheological properties of dough and on the development of new apparatus and methodology for this task [14, 15, 16, 17, 18]. The most important groundbreaking result of his work was the development of a linear structural model of wheat glutenin [19] (Figure 2), which made his name one of the greatest of the era (Bloksma, Evans Ewart, Pomeranz).

Lásztity’s place and role in the world grain science pantheon was most succinctly articulated by Colin Wrigley, a world-renowned Australian representative of the profession, when he introduced Lásztity, who was an invited speaker at the annual Australian Grain Chemistry Conference in the 1990s, as the “Hungarian Pomeranz*” before his presentation (Pomeranz is the greatest figure of our profession of all the time, who, in addition to his own research, has enriched the field of cereal science with over 500 publications, teaching, educating generations of students at several universities in the USA as a scientific organizer. F. Bekes).

6. Priority research directions

Lásztity’s definition of wheat gluten is the most accurate description to date of this complex food constituent with an extremely intricate structure: Wheat gluten is a protein-lipid-carbohydrate complex formed during dough making when, as the results of mechanical input, several specific covalent and non-covalent interactions are formed among the hydrated components of flour. This definition illustrates Lásztity’s lifelong interest in the complex structure of foodstuffs, which has ultimately defined one of the most frequent areas of cereal research in the Department to date, the study of the composition and structure of protein-lipid and protein-carbohydrate interactions and their relationships to functional properties of dough.

6.1. Research on complex proteins in wheat

The Department’s numerous publications on this topic [20, 21, 22, 23, 24], and not least the methods for estimating baking properties based on these complex chemical interactions [25, 26] have generated considerable international interest. The Department has been recognised as one of the most important centres of research in this field. This area also includes the research on thionins, a lipoprotein of cereals with a particular composition and small molecular mess [27, 28], which resulted in two publications cited more than a hundred times in the international literature: one on the detailed structural model of the lipid complex of wheat purothionin [29], and the other on the amino acid sequence of the oat thionin, avenothionin [30]. The study on complex wheat proteins is still an active area of research in the Department trough international collaborations, in particular in on arabinoxylane and its interactions with wheat storage proteins [31, 32, 33, 34, 35, 36].

6.2. Cereal laboratory instrument development, collaboration with LaborMIM

Another very important research area of the Department, initiated by Lásztity, is the development of testing equipment and methodologies for the cereal industry. This activity has been carried out in the framework of a decades-long collaboration between the Department and the engineers of the Laboratory Instrumentation Works (LaborMIM) [16, 17, 18]. To quote the 1971-72 Yearbook of the Faculty of Chemical Engineering of the BUTE: “The department regularly carries out the determination of parameters for the design of new laboratory grain and flour testing instruments, as well as the measurement and expert evaluation of equipment and instruments for complete laboratories. In 1970, the Department carried out a study on the design of a micro-baking and moisture measuring equipment, an elastograph, a bread-crumb investigating apparatus, a hydrolysis equipment for rapid protein content determination. Contractor: the company named LaborMIM.”

Contributions to the creation of numerous grain testing instruments, in addition to a large number if scientific publications and patents, have significantly enhanced the reputation of the Department both in Hungary and abroad. For decades, Valorigraph was the basis for grain classification in Hungary, so the expert level of knowledge of the instrument provided a direct link woth the industry namely with the Department of Quality Control of the Hungarian Grain Trust, headed by Pál Kézdy, and the two relevant industry research institutes (Grain Research Institute, Baking Research Institute). Through these contacts, the Department has been able to obtain first-hand information on the development ideas of the cereal industry and to contribute to the solution of problems that arise. Two examples of these latter are worth mentioning here, because both topics, which were originally industrial problems, developed into basic research projects at the Department, which have been internationally acclaimed for several years. The severe mycotoxin infection appearing in the Hungarian cereal chain lad to start to deal with analytical problems of mycotoxin-contaminated grain, which activity later extended in the direction of mycotoxin detoxification [37, 38, 39, 40]. The Department’s research on in vitro biological value evolved from its expert advice on feed optimalisation activities for the Grain Trust, resulting in the development of a non-linear optimalisation methodology using novel chemical indices [41, 42], as an objective methodology for the formulation of both human food (e.g., baby food) and feedstuff [43, 44, 45].

LaborMIM’s grain analysers were widely used not only in domestic applications but also in contries behind the Iron Curtain: their purchase, unlike comparable analysers in Western countries, did not require “hard currency” in these countries. This was one of the reasons for close cooperation between the Department and researchers from neighhouring countries, especially the former Yugoslavia and Czechoslovakia.

In terms of content, the LaborMIM relationship has far exceeded the decades of cooperation based on grain industry instruments, with the Department actively involved in the development of other products of the company, such as liquid chromatographs and electrophoretic equipmint. One of the success stories of these activities was the BNW’s grand price-winning complex seed testing laboratory, which produced a world-class gel-electrophoresis apparatus and a computer program package for variety identification tasks based on a pattern recognition algorithm written under DOS environment [46]. An improved version of the program, converted to Windows, is still in use today by many research and breeding institutions worldwide under the name “PatMatch” [47].

7. Gluten workshops

In 1980, researchers from the Department were invited to participate in a exclusive International Wheat Gluten Workshop organised by INRA (Nantes, France), which was attended by the world’s leading wheat research institutions. The “International Workshop on Gluten Proteins” has become the highest level professional forum in the field, traditionally held every 3 years since 1980. The Department has been represented at all 13 Gluten Workshops held so far, and we organized the 3rd Workshop in Budapest in 1987. The 3rd Gluten Workshop in Budapest has been rated by the profession as one of the highest quality and best organised meetings to date [48].

8. The 80s, wheat research at the crossroads

The 2^nd Gluten Workshop held in Wageningen in 1984 was an important milestone in the history of the field: it was here that a Lásztity-Shewry polemics on the use of the word prolamin took place, raising much more general questions. Until then, cereal chemistry had classified cereal proteins according to the classical nomenclature laid down bt Osborne, based on their solubility.

It was the early 80s that plant molecular biology and genetics came to interpret the genetics of this complex croup of compounds, leading the British research group led by Miflin to formulate a genetically based grouping and new nomenclature for wheat proteins [49], (Figure 3). This provides a more precise designation/definition of the different protein groups in all respects, but it contains a serious problem -not at all important from the view of content – but with consequences: the prolamin designation of the alcohol -soluble proteins of the Osborne nomenclature is by Miflin et al with a completely new content to designate a different concept, causing a great deal of confusion in the literature published over the last 50 years.

Following Shewry’s presentation, Lásztity, recognizing the problem, made a comment that led to a long and substantive discussion, pointing out the great dilemma of that era of cereal chemistry: until then, cereal chemistry had focused on wheat and wheat flour as the raw material for bread-making, but with the advance of molecular biology/genetics, wheat as a biological object came to the fore. This meant that while the former approach focused on the properties of flour and in particular dough, based on complex interactions, latter focused entirely on the components, i.e. the individual genes representing the genetic make-up of the wheat plant, completely ignoring the fact that the techno-functional properties of wheat flour is manifested through the interactions between the products of these genes.

We reported on the Workshop in an article entitled “At a crossroad in wheat protein chemistry?” in the magazine, Food Industry [50]. Unfortunately, the evolution of the field over the next 10 years has proven our fears to be well founded, and even showed signs of splitting. Although both camps have achieved epoch-making results – an era when biochemical and then molecular markers have been introduced in plant breeding, NIR technique become routine method for the quantitative analysis important grain and flour components, when MacRitchie and his co-workers developed the reconstitution technique, the production of unique wheat proteins become available bacterial expression, wheat protein genes have been genetically altered – but at the same time the gap between basic research I cereal chemistry and applied research that can directly serve practical applications has widened considerably. The results of biotechnology, in particular following the successful solution of wheat transformation, have led to articles by leading experts predicting that in the near future genetic engineering will take over the function of plant breeding and that new genetically modified varieties will be created to meet the various needs of cereal technology. Of course, their prediction did nit come true at all.

It is no accident it was Peter Shewry, Olin Anderson and Rudi Appels, the most prominent pioneers in the application of molecular biology in crop science, who were first realize that the unprecedented potential of molecular approaches and tools could only be fully exploited, if we can not only manipulate the gene and detect its product in wheat, but also - using the tools and experience of traditional cereal science – we can follow and interpret their effect in wheat-based products. This realisation has opened a new chapter in cereal science, in which the Department has played a key role.

9. Development and application of small- and micro-scale dough-testing equipment and methodology

At the turning of 1980s and 1990s, British and American researchers solved the problem of isolating genes coding various gliadin as well as glutenin proteins and producing the encoded proteins at mg scale by bacterial expression. Around the same time, the 2g Mixograph, the first micro-scale dough tester, originally developed for early selection in wheat breeding, was found to be an excellent basic research tool: by adding a few mg of protein to the flour during dough mixing, its effect on mixing characteristics could be monitored and recorded in a sensitive and reproducible way [51]. By means of a consecutive reduction/oxidation process carried out directly in the mixing bowl, supplemented glutenin subunits could be incorporated into the polymeric structure of glutenin polymer and their effect on the rheological properties of the dough could be studied [52]. These novel test methods opened the way to investigate the relationship between the dough properties and the structure of added/incorporated natural and/or genetically modified proteins [53, 54].

The characterisation of wheat transformed with modified wheat genes [55] and the above-mentioned in vitro methodologies had two fundamental shortcomings: we did not have the means to mill gram quantities of transgenic seed into flour and there was no micro-scale equipment for the determination of one of the fundamental properties of wheat flour: water absorption, the parameter what is actually measured on macro scale using the Hankóczy Farinograph.

It was the time when members of the Department and the staff of two small Hungarian companies, engineers from the then defunct Labor MIM, joined forces with Australian CSIRO researchers [56] to create the world’s smallest laboratory micro-mill, capable of grinding even a single grain of wheat to flour [3, 57] and micro-version of Hankóczy’s Farinograph (micro-Z-arm mixer), capable mixing 4 gram of flour and determining mixing properties, including water absorption [58, 59, 60].

These developments are an integral continuation of the Department’s activity in this field, which has been carried out in the spirit of Guthrie and are the culmination of decades of cooperation with LaborMIM. The micro-mixer is a joined intellectual property of BUPA and CSIRO, the manufacturing rights were purchased by Newport and its successor, Perten. And which is now being further developed, manufactured and marketed under the name of Micro DoughLAb. More than 200 units are in operation in 41 countries worldwide. Nowadays, wheat transformation for research purposes, research on structure/function relationships of wheat proteins, QTL analysis on large sample population, molecular marker development, all basic and applied research areas where sample size is limited, are unthinkable without the use of Z-arm micro mixer.

The Department has been/is involved in a large number of international collaborations in various areas of cereal science using its micro-scale test equipment [61, 62, 63, 64, 65, 66]. Instrument development, development of new micro-scale equipment and methodologies is still an important area of research in the Department. Recent developments include a micro Zeleny sedimentation apparatus [26, 65, 66], a micro scale apparatus for gluten-washing with starch isolation options [67] and a complete micro scale test baking facility and methodology [68, 69, 70].

The significance of the Hungarian-Australian cooperation on micro-scale testing was perhaps most succinctly expressed by Harry Sapirstein, a Canadian researcher, who introduced the authors of a paper on micro scale studies (Tömosközi, Gras, Varga, Rath, Nánási, Salgó, Békés) at a conference in the USA with these words: “ They were the traitors to traditional cereal science who made contact with the gene-jokies – opening a new chapter in cereal science by their joined achievements.

10. Other international contacts

This overview of the Department’s international relations would not be complete without mentioning the activities of the Department’s staff in various international associations. Professor Telegdy-Kováts became a member of the Executive Committee of the ICC (Interntionl Ceral Chemistry Association, Vienna) as early as the 1960s, and was replaced by Professor Lásztity in 1978, even serving as President of the ICC for one election cycle. The Department, through Dezső Törley and András Salgó, was actively involved in the work of the FAO/WHO Codex Alimentarius. Budapest has been the venue for several highly successful ICC events, on one of which in 2002 András Salgó received the ICC’s prestigious Harold Perten Award as a recognition of his work in the field of near infrared (NIR) technique in cereal science [71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 84].

As a member of several largescale international collaborations organised by the ICC and the EU, including the MoniQA [81, 82, 83] and HealthGrain [84, 85, 86] projects the staff of the Department have worked and published results with researchers from numerous countries in europe. Among these links, a special place is occupied by the cooperation with the sister department of BOKU in Vienna, which has been established over several decades and includes not only research but also the training of chemical engineering students [33, 34, 87, 88, 89, 90, 91, 92].

11. Conclusion

The broadening concept of cereal quality, such as the need to study nutritional and health related properties, continues to reinforce the interdisciplinary nature of food science. New disciplines and new methodologies are being integrated int traditional research/development processes.

As illustrated above, the research palette of the Department has been enriched accordingly with new colours. Large-scale instrumentation techniques, indirect methods for online control have been added to the research topics, as well as the study of allegenic/toxic components of food materials, including cereals.

Building on the Department’s long tradition, these are carried out on an international scale, often in the framework of international cooperation.

12. References

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[45] Békés, F., Hidvégi, M., Lásztity, R., and Tóth, A. (1985): Moglichkeiten der Optimierung von Futterungskosten unter Berucksichtigung der biologischen Beurfnisse. Muhle + Mischfutter. 122. pp. 628-630.

[46] Békés, F., Kemény, A., Kemény, S., Merész, P., Demeter, L. and Varga, J. (1988): Gelelektroforezis-kiserletek szamitogepes mennyisegi kiertekelese I. Relativ mobilitas skalak osszehasonlito vizsgalata. Elelmezesi Ipar 42. pp. 121-129.

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[48] Lásztity R., Békés F. (1980): Proceeding of the 3rd International Workshop of Gluten Proteins. World Publ. Comp., Singapore

[49] Shewry P.R., Tatham A.S., Forde J., Miflin B.J., Kasarda D.D. (1980): The primary structures, conformations and aggregation properties of wheat gluten proteins. In: Proceedings of 2nd Workshop on gluten proteins, Eds: Graveland A, Moonen JHE, pp. 51-58, TNO, Wageningen, The Netherlands

[50] Lásztity R., Békés F., Kemény, A. (1986): Valaszút elött a búzafehérje kémia? Élelmezési Ipar 140: pp. 121-122.

[51] Békés F. and Gras P.W. (1992): Demonstration of the 2-gram Mixograph as a research tool. Cereal Chem. 69. pp. 229-230.

[52] Békés, F., Gras, P.W., and Gupta, R.B. (1994): Mixing properties as a measure of reversible reduction/ oxidation of doughs. Cereal Chem. 71. pp. 44-50.

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[57] Békés F., Southan M.S., Tömösközi S., Nánási J., Gras P.W., Varga J., McCorquodale J., Osborne B.G. (2000): Comparative studies on a new micro scale laboratory mill. Proc. 49th RACI Conference, Eds Panozzo J.F., Ratcliffe M., Wootton M., Wrigley C.W. pp. 483-487., RACI, Melbourne.

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[60] Haraszi, R., Békés, F., Bason, M.L., Tömösközi, S, Varga J, Salgó A, Dang., J.M.C., Blakeney, J.L.: Dough mixing studies on the micro Z-arm mixer. pp. 219-222. In: ‘The gluten proteins’, Proc. 8th Gluten Workshop, Eds.: Lafiandra, D., Masci, S. and D’Ovidio, R., RS-C. Chambridge, UK.

[61] Uthayakumaran, S.,Tömösközi, S., Savage, A. W. J., Tatham, A., Gianibelli, M. C., Stoddard, F.L., and F. Békés. (2001): Effects of gliadin fractions on the functional properties of wheat dough depend on molecular size and hydrophobicity. Cereal Chem. 78. pp. 138-141

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[63] Morgounov, A.I., Belan, I., Zelensky, Y., Roseeva, L., Tömösközi, S., Békés, F., Abugalieva, A., Cakmak, I., Vargas, M., Crossa, J. 2012. Historical changes in grain yield and quality of spring wheat varieties cultivated in Siberia from 1900 to 2010 Can. J. Plant Sci. 93. pp. 425- 433.

[64] Oszvald, M., Balázs, G., Pólya, S. Tömösközi, S., Appels, R., Békés, F., Tamás., L. 2013. Wheat Storage Proteins in Transgenic Rice Endosperm. J. Agric. Food Chem. 61, pp. 7606−7614.

[65] Cavanagh, C.R., Taylor, J., Larroque, O., Coombes, N., Verbyla, A.P., Nath, Z., Kutty, I., Ramplin, L., Butow, B., Ral, J.P., Tömösközi, S., Balázs, G., Békés, F.,Mann, G.,. Quail, K., Southan, M., . Morell, M. K. Newberry, M. (2010): Sponge and Dough Bread Making: Genetic and Phenotypic Relationships with Wheat Quality Traits. Theor. Appl. Gen. 121: pp. 815-828

[66] Roy N., Shahidul I., Ma J., Lu M., Török K., Tömösközi S., Békés F., Lafiandra D., Appels R., Ma W. (2018): Expressed Ay HMW glutenin subunit in Australian wheat cultivars indicates a positive effect on wheat quality. Journal of Cereal Science 79: pp. 494-500

[67] Tömösközi S., Szendi Sz., Bagdi A., Harasztos A., Balázs G., Appels R., Békés F. (2013): New possibilities in micro-scale wheat quality characterisation: micro-gluten determination and starch isolation Proc. 11th Internat. Gluten Workshop, Beijing, Eds.:He, Z., and Wangm D., pp. 123-126. CIMMYT, Mexico City

[68] Németh R., Bánfalvi A., Csendes A., Kemény S., Tömösközi S. (2018): Investigation of scale reduction in a laboratory bread-making procedure: Comparative analysis and method development. Journal of Cereal Science 79: pp. 267-275

[69] Németh R., Farkas A., Tömösközi S. (2019): Investigation of the possibility of combined macro and micro test baking instrumentation methodology in wheat research. Journal of Cereal Science 87: pp. 239-247

[70] Langó B., Jaiswal S., Bóna L, Tömösközi S., Ács E., Chibbar R.N. (2018): Grain constituents and starch characteristics influencing in vitro enzymatic starch hydrolysis in Hungarian triticale genotypes developed for food consumption. Cereal Chemistry 95: pp. 861-871

[71] Gergely S., Salgó A., (2003): Changes in Moisture Content during Wheat Maturation—What is Measured by near Infrared Spectroscopy? J. Near Infrared Spectrosc. 11: pp. 17-26

[72] Gergely S., Salgó A. (2005): Changes in carbohydrate content during wheat-maturation-what is measured by near infrared spectroscopy? J. Near Infrared Spec. 13: pp. 9–17.

[73] Juhász R., Gergely S., Gelencsér T., Salgó A. (2005): Relationship Between NIR Spectra and RVA Parameters During Wheat Germination. Cereal Chem. 82: pp. 488-493

[74] Gergely S., Salgó A. (2007): Changes in protein content during wheat maturation—what is measured by near infrared spectroscopy? Journal of Near Infrared Spectroscopy 15: pp. 49-58

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[77] Juhász R., Salgó A. (2008): Pasting behavior of amylose, amylopectin and their mixtures as determined by RVA curves and first derivatives. Starch-Stärke 60 (2), pp. 70-78

[78] Schmidt J., Gergely S., Schönlechner R., Grausgruber H., Tömösközi S., Salgó A., Berghofer E. (2009): Comparison of Different Types of NIR Instruments in Ability to Measure β-Glucan Content in Naked Barley. Cereal chemistry 86 (4), pp. 398-404

[79] Hódsági M., Gergely S., Gelencsér T., Salgó A. (2012): Investigations of native and resistant starches and their mixtures using near infrared spectroscopy. Food Bioprocess Technol. 5: pp. 401–407.

[80] Salgó A., Gergely S. (2012): Analysis of wheat grain development using NIR spectroscopy. Journal of Cereal Science 56: pp. 31-38

[81] Bugyi Zs., Nagy J., Török K., Hajas L., Tömösközi S. (2010): Towards development of incurred materials for quality assurance purposes in the analysis of food allergens. Anal. Chim. Acta, 672: pp. 25–29.8

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[89] Bagdi A., Balázs G., Schmidt J., Szatmári M., Schoenlechne R., Berghofer E., Tömösközi S. (2011): Protein characterization and nutrient composition of Hungarian proso millet varieties and the effect of decortication Acta Alimentaria 40: pp. 128-141

[90] Schoenlechner R., Szatmari M., Bagdi A., Tömösközi S. (2013): Optimisation of bread quality produced from wheat and proso millet (Panicum miliaceum L.): by adding emulsifiers, transglutaminase and xylanase. LWT-Food Science and Technology 51: pp. 361-366

[91] Bender D, Fraberger V, Szepesvári P., D’Amico S., Tömösközi S., Cavazzi G., Jäger G., Domig K.J.,·Schoenlechner R. (2017): Effects of selected lactobacilli on the functional properties and stability of gluten-free sourdough bread European Food Research and Technology 244: pp. 1037-1046

[92] Békés F., Schoenlechner R., Tömösközi S. (2016): Ancient wheats and pseudocereals for possible use in cereal-grain dietary intolerances. In: Wrigley, C.W., Batey I., and Miskelly D. (Eds) Cereal Grains Assessing and Managing Quality (2nd Edition) pp. 353-393, Elsevier, Amsterdam

DOI

Received: October 2022 – Accepted: November 2022

¹ Budapest University of Technology and Economics, Faculty of Chemical Technology and Biotechnology, Department of Applied Biotechnology and Food Science, Cereal Science and Food Quality Research Group

Keywords

cereals, protein, micromolecule, wheat starch, gluten, grain quality, FODMAP

2. Development of grain qualification instruments and methods suitable for testing small amounts of samples

Practically until the beginning of the 20^th century, the concept and content of grain quality were primarily limited to the development of cultivation indicators, possibly cleanliness, and the sensory assessment of the quality of foods made from the crop. As the milling industry, and later the baking industry became medium, then large-scale, quality, especially wheat quality became a critical issue [2]. At the end of the 19th century and at the beginning of the 20^th century, research aimed at promoting technological developments, then aimed at influencing technological quality, as well as examining the relationships between composition and quality began. A key issue in the development of the field has become the development of methods and instruments that provide objective results suitable for determining technological quality (milling behavior, rheological properties of doughs made from ground flour, etc.), including the determination of gluten content, gluten strength and kneading properties, in which Hungarian researchers have also played a pioneering role on a global scale from the beginning [3].

By the last quarter of the 20^th century, it became clear that to investigate the relationship between composition and quality, and to understand unique rheological factors or those influencing the quality of the complex product, it is not enough to apply the „analytical approach”, that is, knowledge of the macro- (e.g., protein or gluten content) and micro-composition (e.g., amino acid or protein subunit composition and size distribution). Determining the properties of the individual protein molecules was the next step in both understanding quality and improving it. Individual protein molecules and groups can also be produced using more traditional separation methods, but the biotechnological development of the last decades of the last century made it possible to use molecular biological methods and to express individual gluten proteins as well [4, 5].

The possibility of examining the unique role of proteins, and macromolecules in general, has also created a new demand for the development of methods and measuring devices that are suitable for studying the complex rheological properties and final product quality of small amounts of samples. Not long after, the need for micro methods also appeared in the fields of breeding using traditional or biotechnological methods (e.g., the possibility of qualification in the earlier phases of breeding), then in research and development for different purposes (e.g., examination of the role of macromolecules, the effect of additives or treatments) and partly in routine analytics.

First, the 2 g version of the Mixograph, standardized mainly in the American and Australian regions, with a mixing bowl containing needle mixing elements, was completed [6], which was successfully used as a revolutionary new research tool to identify the role of expressed proteins [5, 7], in incorporation experiments [8], and in the testing of breeding lines [9], among other things. As a result of the successful application of micro methods, and in parallel with these, measuring devices and methods suitable for testing other parameters (e.g., one-dimensional extensibility [8,10], multi-dimensional extensibility [11], intensive kneading [12]), in addition to normal kneading properties have also appeared. This process can be viewed in a way that test techniques with small sample requirements proved their right to exist in this period and gained wider acceptance [13,14].

The employees of our department managed to connect to this fantastically exciting field almost from the beginning. Despite the success of using the 2 g Mixograph, it became necessary for both Australian developers and international researchers using the method to move on. It is an undoubted professional fact that the physico-chemical effects of needle and Z-arm (farinograph and variograph) kneading are partially different [15, 16]. On the other hand, it is indisputable that the use of qualification methods based on standard Z-arm mixers, including the sample preparation for mono- and biaxial extensibility tests, is significantly more common and widespread than mixograph methods [17, 18].

Therefore, the possibility arose to continue instrument and method development at the Australian CSIRO (Commonwealth Scientific and Industrial Research Organisation) institute and with its partners, which at that time also covered micro extensograph [8,19] and micro baking processes [20], together with the Hungarian specialists experienced in valorigraph measurement technology. A part of the funds necessary for the joint work was provided by the OMFB (Hungarian National Technical Development Committee) grant awarded in Hungary, which was considered to be a significant amount at the time. In addition to the staff of CSIRO and BME, Metefém Szövetkezet and Lab-Intern Kft. from the Hungarian side, and Newport Scientific Pty Ltd., the developer and manufacturer of rapid viscosity analyzers (RVA) from the Australian side, took part in the collaboration. The most significant part of the consortium work was aimed at the development of the hardware and software of the micro Z-arm mixer, its working name being the “micro-valorigraph and suitable for the determination of the kneading properties of 4 g of flour, and the related measurement methods. At the same time, we can only take advantage of the possibility of testing small amounts of material, especially in the field of breeding, if we have a suitable sample preparation procedure (grinding and separation). Therefore, on the Hungarian side, the tender also included the design of a micro mill and sieving machine, which is suitable even for grain-by-grain grinding.

Scientific articles usually do not include the details and steps of instrument development. This may partly be due to commercial or intellectual property protection reasons, and researchers and systems evaluating research work rarely give such developments their proper due. In cereal science research, as everywhere else, the results obtained using the measuring devices have a real scientific value, and today there is also talk about the reliability, validation, and performance characteristics of the application of the methods. Naturally, we will not go into the technical details here either. However, we would like to emphasize that it is a very long road and hard work from the birth of an idea to its realization. A lot of brainstorming, consultation, and laboratory experiments are necessary to find the right proportions, lengthy design and mechanical work are required to produce the small parts, miniaturize the drive mechanism, to solve thermostatic or automatic water feeding, which was considered to be a novelty at the time, to solve the processing of the analytical signal, to create the control and evaluation software, then from the creation of the prototype to establishing the conditions for serial production. This path was followed for all instrument development, but due to the learning process, the path leading to the development of the micro-Z arm kneading unit was, by definition, the most difficult. The more than 5 years, during which the idea gave birth to the prototypes of the first working micro-devices, partially with domestic cooperation, were hard but, especially in retrospect, incredibly exciting and useful in terms of gaining knowledge and experience. In addition to the colleagues named in the articles, software, and hardware development colleagues, and in the experimental work and testing, many of our students took part, and they also deserve a huge thank you from us. Maybe not from a professional point of view, but definitely a symbolic milestone in the development of the micro valorigraph and the micro mill was the Hannover Industrial Fair organized between April 21-28, 2001, where it was possible to present the functional equipment to the international public for the first time (Figure 1).

Back to the grain science orientation, the initial focus was on validating the justification for instrument and method development, and on comparisons with the results of standard methods using large amounts of samples. The first scientific papers were published, first in the form of conference articles and presentations, and later in the form of scientific manuscripts in Hungarian and English [21, 22, 23, 24, 25, 26, 27, 28, 29, 30]. From the micro valorigraph prototype, Newport Scientific Pty Ltd. Later launched an improved version of the prototype suitable for routine testing purposes under the name micro-doughLab, which was marketed by the Perten Company until 2020. In addition, the micro mill manufactured by METEFÉM Cooperative has also reached many countries and research sites. Much information on the development and application results of the microvalorigraph and the mill can be found in the current and previous [31] issues of the Journal of Food Investigation (Élelmiszervizsgálati Közlemények), in the writings of Ferenc Békés. Therefore, in the following, we will give a brief overview of the later developments of instruments and methods and their role in research so far, mentioning some examples.

Subsequent instrument developments are mainly linked to Hungarian activities, primarily from the cooperation of the BME-ABÉT Cereal Science and Food Quality Research Group and Lab-Intern Kft., partly also with the involvement of grant funds. The second member of the instrument family was the instrument suitable for the automated and small sample quantity (0.4 g of flour) version of the standard Zeleny sedimentation test (SediCom® System). With the help of the sedimentation test, we follow the swelling process of the polysaccharides and proteins of the flours, which can be linked to certain quality properties of the wheat. The method is widely used around the world, the sedimentation value is considered to be a general acceptance and quality classification parameter [32, 33]. As a result of the instrument development, a modular, automatic, combined measuring technique, containing digital signal processing and capable of handling both macro and micro test tubes and the related processing software were developed, a first at the international level, followed by the development and validation of the measurements. The prototype of the instrument also received an innovation award at the ICC conference held in Vienna in 2005 [34, 35, 36].

The determination of gluten content is one of the oldest and still most frequently used wheat qualification methods [37]. In this area as well, there was a demand, mainly from breeding professionals, for the development of a method with a significantly smaller sample requirement than the standard one, which enables the screening of lines in significantly earlier breeding phases. With a cereal chemistry approach, it is also possible to further study the isolated gluten. Not to mention that another main actor in the hydration and dough formation processes is starch, and the investigation of its properties and role has gained new momentum when varieties with, for example, special carbohydrate composition (low amylose or waxy) coming to the fore. Previously, there was no gluten-washing equipment available, which was suitable for both the examination of smaller than standard sample quantities and the separation of the washed-out starch. With this in mind, we also launched the development of our combined macro and micro gluten-washing instrument, later named the GluStar® System, together with Lab-Intern Kft., which debuted at the 13^th Gluten Workshop in China in 2011 [38].

The rheological behavior of the dough structure formed after the hydration of ground wheat (water absorption, product-dependent optimal consistency, dough formation process, time, stability, etc.) is an extremely complex process in itself, we know a lot and we don’t know or understand even more. This is also a reason why only a total of several rheological tests (kneading tests, mono- and biaxial extensibility, viscosity, falling number, etc.) can provide more detailed information about dough properties [39]. However, it is still the final products that are consumed directly, the quality of which is the result of even more complicated, not unrelated factors. The quality and composition of the raw material, the biochemical and physico-chemical processes that take place during leavening (fermentation) and baking (heat treatment), then cooling and storage (aging) all influence the quality of the final product in ways that are partly known and partly still being researched. The question becomes particularly exciting if it is not white flours, but, e.g., whole grinds, also containing shell parts, whose properties are investigated. Here, in addition to protein and starch, the (bio)chemically much less reactive fibers, hydrophobic lipids, and even the different ionic strength, pH, etc., also enter the picture as structure-forming factors [40]. In order to be able to carry out comparative tests between the quality of the raw material and the quality of the final product, and to enable the examination of the influencing factors, reproducible laboratory bakery tests are needed. Such standard tests have been used in domestic and international practice for a long time [41, 42, 43]. However, the biggest problem is exactly the precision (repeatability, reproducibility) of the implementation of the methods. Currently used standard methods are based on manual execution, with all its “beauty” and reliability problems. Improving the reproducibility of the methods can be achieved by reducing the occurrence of random errors, primarily by at least partially eliminating the human factor. An obvious solution seems to be the at least partial automation of the test loaf production operations (kneading, shaping, leavening, baking, cooling) and evaluation methods (volume, height, texture, etc.).

From the point of view of our topic, it is important that the required amount of samples for traditional baking industry tests is also large, which can be problematic from the point of view of both research and development and breeding. Of course, improvements were also made earlier for instrumentation [44] and size reduction [10,13,45, 46, 47]. However, the possibility of a complex, partly automated, traditional standard and reduced sample quantity laboratory testing was missing from the toolbox of cereal research. Based on these considerations, as well as with the courage gained from the results of previous instrument developments, the development of methods and instruments suitable for performing automated macro and micro baking tests was launched, again with the help of Lab-Intern Kft.’s employees. Following the determination of the possible extent of size reduction [46] and after rejecting several technical ideas, the instrument version with computer control and data collection was completed in 2017, which is suitable for carrying out leavening and baking under controlled conditions after kneading [48]. The measurement method developed by us includes the standardized farinograph dough preparation, kneading, and leavening, then the evaluation of the dimensions of the finished test loaves based on laser scanning, the evaluation of the pore distribution of the crumb based on digital image processing, as well as the characterization of its texture using an instrumental texture testing method [49, 50].

By supplementing our own instrument, built with the cooperation BME, with equipment from other manufacturers, we managed to create a wheat and grain testing capacity for the characterization of the rheological and final product properties of small quantities of samples that is rare even on an international scale (Figure 2). In the following, some of the research results obtained using micro instruments and methods are presented.

In parallel with the development of instruments and methods with small sample requirements, naturally, their application in various research areas also began. In the beginning, in accordance with the original objectives of the instrument developments, the role of wheat gluten proteins was investigated with variety of comparison experiments, the dosing of expressed proteins, or by studying genetically modified samples. Some of these experiments were performed using a wheat matrix [16, 51, 52, 53, 54]. Later, the unique role of wheat proteins was studied by following the changes in the rheological properties of gluten-free rice dough matrix as a result of dosing or experimental genetic modifications [55, 56, 57].

In addition to grain proteins, the properties of other plant proteins were also studied with the help of micro-instruments. The goal of these researches, for example in the case of wheat germ or pseudocereal proteins, was to determine to what extent and in which direction high-protein isolates added to wheat flour in order to increase its nutritional value and modify the technological behavior of it. Behind the experimental ideas, however, there was also a basic research idea. Among other things, we tried to understand how the gluten modifying effect of the isolated less hydrophobic germ, legume and pseudocereal (e.g., amaranth) proteins with better surfactant activities and containing primarily albumins and globulins can be explained [25, 58, 59].

In many respects (e.g., biodiversity, increasing nutritional value, expanding product range, handling food safety problems, etc.), we consider the development of the technical classification of grains (e.g., spelt, triticale, rye, oats, millet) and pseudocereals (amaranth, buckwheat) currently produced in small quantities in more detail than before to be an important and exciting line of research, as well as getting to know the genetic and environmental variability of these crops and understanding the macromolecular relationships behind these properties. The use of equipment requiring small sample amounts is also of great help in this respect, while also improving cost-effectiveness in the process. Without claiming to be exhaustive, we mention here as examples the results of research aimed at the complex technological qualification of triticale varieties [60] and our experiments aimed at improving the kneading properties of dough matrices made from gluten-free, buckwheat and millet flours [61, 62] (Figure 3).

Apart from proteins, the quality of bakery and other products intended for direct consumption is also fundamentally influenced by the other two groups of macromolecules, starch and non-starch carbohydrates (dietary fibers). The issue becomes particularly acute in the case of the slow but welcome spread of health-conscious eating, where the consumption of whole-grain, fiber-rich cereals is constantly increasing. However, relatively little is known about the structure-forming properties of the fiber components. Our equipment with small sample requirements is also of great help in our research aimed at getting to know them. Some of the recent and current research topics were directed at the investigation of the role of arabinoxylanes, the defining fiber components of wheat flours, in wheat flours and gluten-free matrices [63, 64, 65, 66]. Recently, a basic research project was launched, which aims to extend the reduction and reoxidation incorporation technique previously developed by Békés et al. To fiber components [67] (Figure 4). It may also be interesting to investigate the changes in the dough-forming properties of fiber-rich flours due to heat treatment [68]. Heat treatment is a well-known process for changing the technological properties of wheat flours or, for example, to increase the shelf life of oatmeals, however, little is known about the molecular processes that take place in the case of fiber-rich samples, and their effects on technological behavior.

Naturally, it is impossible to highlight and mention every application example from the decades-long professional history in such an overview. We would like to apologize to those colleagues and cooperating partners whose joint work and results could not be included here for reasons of scope. But perhaps the above can provide some overview of the possibilities of using grain testing micro methods, the possible directions of development and the results achieved so far.

3. Possibilities for improving cereal quality and classification

In the past decades, together with domestic and foreign research institutes, universities, economic enterprises and social organizations, we have participated in and initiated many large-scale R&D programs, the objectives of which included the modernization and harmonization of cereal, especially wheat, classification system, as well as the exploration of the application possibilities of new qualification methods and instruments. Perhaps one of the most influential of these was the launch of the Pannonbúza programs (Pannon Wheat Programme). In the framework of these, in the 1990s and 2000s, we determined the detailed composition and technological properties of the wheat varieties that played a decisive role in domestic public cultivation and breeding programs and compared them with the quality requirements of domestic standards, on the one hand, and those appearing in international trade, intervention and on stock exchanges, on the other hand. It has been clearly proven that although the majority of domestic wheat varieties performs exceptionally well in the traditional Hungarian and partly in Central European classification systems, it is more difficult to comply with the alveographic or extensographic parameters, for example, included in export requirements. A related problem is that wheat acceptance and classification and, consequently, the breeding practice in Hungary was determined by the completely coherent, professionally sound domestic wheat standard centered on gluten quality and farinographic value. This did not include the new requirements for wheat for export. At the same time, a significant part of domestic wheat production (30-50%) is sold internationally. Further problem was the obligation to harmonize methods is a legal consequence of joining the EU. However, for example, the farinograph or valorigraph values that have been used to this day and have become ingrained in domestic practice, in a completely justifiable way from a professional point of view, were not included in the international standards, and the evaluation of the curves also differed somewhat.

As an attempt to resolve all this, first, the system of quality requirements for the Pannon Wheat Trademark was developed in two categories (Figure 5) [69,70]. Although this was not accepted by the economic reality of the time, the results and the approach were successfully transferred to the process of renewing the standard for domestic bread wheat. As a result of long discussions and negotiations, the new wheat standard that is still in force and contains one of the most detailed requirements at the international level [71]. Its most important innovation is considered to be the possibility of classification according to alternative approaches (farinographic, extensographic, alveographic), as well as the updating and harmonization of the related test standards with international regulations, on the one hand, and with the relevant chapter of the Hungarian Food Codex, on the other hand [72]. We would like to believe that, with this solution, we have succeeded in creating a system that is able to exploit the quality advantages of the Hungarian range of varieties, and at the same time encouraging the cultivation of quality wheat which better aligns with sales requirements [73, 74].

The assessment of the nutritional role of wheat and other cereals has changed significantly in recent decades. In the past, the production of white flour containing mainly the kernel and the production of baking, confectionery, and pasta industry products, and other foods from them was decisive. Therefore, the majority of methods suitable for characterizing compositional and technological quality also served this (gluten) protein and starch „centered approach” (see above). A more complete understanding of the role of dietary fibers and bioactive ingredients and the spread of conscious nutrition has resulted in the rise of whole grain flours and flours richer in fibers and non-starch carbohydrates, and also of cereal-based foods. However, the definition of dietary fiber did not develop until the beginning of the 2000s [75], and the methods suitable for their determination then became standard, routine procedures. However, it is also clear that the various fiber components (cellulose, hemicelluloses, pentosans, pectins, lignin, in other groupings, soluble and insoluble fibers) have different nutritional and physiological properties, and their functions influencing technological behavior also differ, and the clarification of these issues required numerous methodological developments and the adaptation of new analytical techniques. We first used in our joint programs with companies and research institutes aimed at the development of health-promoting grain-based products, for example, the mixolab measurement technique suitable for determining the primarily protein-dependent kneading properties and the mainly starch/non-starch carbohydrate-dependent viscous properties to characterize the rheological behavior of milling industry wheat fractions of a new type, rich in aleurone layer, and to examine the function of fibers, among other things (Figure 6), [76, 77]. Also, these researches made it necessary to adapt methods mainly based on separation techniques and suitable for the quantitative and later qualitative determination and comparison (e.g., molecular size, solubility) of individual fiber components, first arabinoxylans (AX), then β-glucans and, currently, arabinogalactan (peptides), then their further development [78-80].

The centuries-old good reputation of Hungarian wheat cultivation and quality was founded first on conscious selection, land varieties adapted to growing conditions, and later, with the development of breeding methods, on the creation of genetically stable wheat varieties with excellent baking quality. The breeding of the “Bánkúti” wheat varieties [81], which are known to have excellent baking quality, can be considered a milestone in this process, and their quality characterization and the exploration of the molecular background behind the good quality have been partially achieved [82, 83, 84]. In recent decades, these varieties have lost their importance in public cultivation, mainly due to their agrotechnological properties. At the same time, due to their excellent technological quality, they still play an important role in breeding programs, or at least they can. However, exploring the composition and technological potential of old species, lines and ancient wheat varieties (e.g., spelt, emmer) using a modern approach and testing methods is an even less well-developed field. Based on such considerations, together with breeding houses and economic enterprises, we launched our R&D program aimed at characterizing old wheat genotypes from a new perspective and improving their use, as well as our complementary basic research. In these, too, we used and further developed our new methods for determining rheological properties and carbohydrate (fiber) content. It was found, for example, that while the kneading characteristics of the Bánkúti varieties and lines are similar with a few exceptions, there are significant differences in their viscous behavior (Figure 7a) [54]. Also, was found to be new information that the white flours of the Bánkúti lines have significantly different soluble arabinoxylan contents, and in some cases, their AX content exceeds that of the international comparative varieties (Figure 7b), [85,86].

4. Improving the utilization possibilities of small grains and pseudocereals – two examples

Large grains, including primarily wheat, have a system of qualification criteria based on a significant tradition and knowledge base, and a widely accepted, standardized method and tool background that serves it. However, grains (such as rye, oats, sorghum, millet, triticale) or pseudocereals (amaranth, quinoa, buckwheat), which are currently produced in significantly smaller quantities but are of growing importance due to special nutrition needs, selection expansion, or even fashion, do not have a detailed qualification procedure. Little is known about the differences between species and varieties, compositional and technological quality differences, effects of growing areas, and quality stability. In many cases, even the definition of the quality requirements corresponding to the specific purpose of use is incomplete. For this reason, together with domestic and foreign research centers and colleagues, several basic and applied research and development programs have been launched, to develop the classification methods of small grains, on the one hand, and in the direction of the product, developments promoting the broadening of their utilization, as well as to learn about the molecular background in order to be able to improve their technological behavior, on the other hand. Due to space limitations, the research and development potential and possibilities of these areas will be presented with the help of two examples. Within the framework of our recently completed project, the classification methods of domestically grown and partly domestically bred oat and rye varieties were developed further and, by applying them, the variability of the varieties’ compositional and technological properties was examined. In milling industry experiments, by examining 54 fractions, we „drew up” the fraction map of milling and identified some new rye flours that had more favorable properties than traditional flours in terms of nutritional value and health-promoting composition. The rheological and baking industry final product quality of the doughs were characterized in detail, and with the help of the results, it was possible to manufacture new types of products that are favorable from both a compositional and sensory point of view (Figure 8) [87].

Our other example shows the possibility of improving the nutritional and technological properties of gluten-free dough matrices. The dough-forming properties of gluten-free raw materials (e.g., millet or buckwheat) are relatively weak, in the absence of structure-building macromolecules (gluten proteins), the viscous structure of dough matrices necessary for product preparation is provided by carbohydrates and, in many cases, added hydrocolloids. The technological behavior of this “fragile” molecular system is adversely affected by the presence of fibers. Among other things, this is the reason why the nutritional value of gluten-free baking and pasta industry products is in many cases less favorable compared to the gluten-containing version. At the same time, it is known, for example, that fiber-forming arabinoxylans are capable of forming a macromolecular network partially similar to the gluten structure by connecting their side chains in an oxidative environment. In our experiments, it was possible to prove that hydrogen peroxide produced in enzymatic reactions, in a suitable concentration, can induce the polymerization of arabinoxylans added to gluten-free dough matrices, and thus improve the kneading properties of the dough and the complex quality of the final bakery products, while the nutritional value also increases with the addition of fibers (Figure 9) [62, 66].

5. Excerpts from research activities related to food safety

The complex assessment of cereal quality is further complicated by the emergence of special expectations and food safety requirements (e.g., being gluten-free, low content of fermentable oligo-, di- and monosaccharide and polyol (FODMAP) content, etc.) arising from digestive system disorders (celiac disease, allergies, irritable bowel syndrome) in some consumer groups.

The quality of life of persons with celiac disease, also known as people allergic to gluten, can be ensured by maintaining a gluten-free diet. According to current legislation, the gluten content of gluten-free products cannot exceed the value of 20 mg/kg. Maintenance of the production process, product qualification, and food safety are not possible without the use of analytical methods. ELISA tests operating on the immunoanalytical principal can be considered the most common routine method, but it is also possible to use measuring techniques operating on the basis of molecular biology (based on DNA determination), or using a mass spectrometric detector either alone (MALDI-TOF) or coupled with a separation technique (HPLC-MS). Different ELISA kits and methods operating on other principles usually provide significantly different results. The harmonization and validation of the methods are hindered by the fact that neither a reference method nor a reference material is available. Recognizing this, our research group started a research work aimed at comparing the methods, exploring the reasons for the differences between the results, investigating the effects occurring during product manufacture, and then producing native wheat flour and model products labeled with isolated protein-based reference material. The research work that began more than ten years ago has been carried out in an international cooperation, first as a sub-program of a European framework program, and then as an independently maintained joint work. The most important result is that it was possible to produce a matrix based on wheat flour for the first time, which is likely to meet the requirements of reference materials [88, 89, 90, 91]. Work is still underway with the inclusion of other gluten-containing cereals (rye, barley, oats).

From the point of view of FODMAP composition, the majority of cereals have an unfavorable perception. It is a particular problem that the FODMAP content of flours, which are favorable in terms of nutritional value and rich in fiber, is usually higher. At the same time, there is almost no information on the variability between cereal species and varieties, on the technological properties of variants with lower FODMAP content, on the joint development of changes in the FODMAP content and fiber composition of flour fractions, and on the effect of the operations used during the technological process (fermentation, heat treatment, pH change, etc.). A comprehensive research project was also recently launched by us in collaboration with others to jointly examine the non-starch carbohydrate (fiber) composition and the content and composition of short-chain carbohydrates also containing FODMAPs. Here again, it became necessary to execute a multi-stage methodological development (Figure 10), by using which it became possible to compare rye and oat varieties, flour fractions, heat-treated products and bakery end products, among other things [92, 93].

6. Conclusion

It is difficult to write a short summary at the end of such a large-scale paper, reviewing the research work of several decades and shamelessly exceeding the limits of its scope. We would like to believe that the results presented as examples add something to the development of grain testing methodology, with their use we can get a little closer to understanding the behavior of complex dough and product matrices, and we can contribute to broadening the use of cereals and pseudocereals, to the development of their value-added utilization. In addition, we would like to repeatedly emphasize the importance of cooperation at the individual and institutional level. It is impossible to implement this intensive creative process without our university, research institute, and industrial partners. We would like to thank all our partners for the opportunity of cooperation so far and, hopefully, in the future.

7. Acknowledgement

Special and particular thanks to our colleague Erika Szűcsné Makay for decades of professional and personal cooperation. We are grateful to our predecessors for their guidance and support, to the former members of the research group and the department for the opportunity and results of the fantastic joint creation, to our professional partners for the experience and fruits of joint work, and to our supporters for the resources, hopefully spent on a good cause.

8. References

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[65] Bagdi A., Tomoskozi S., Nystrorm L. (2017): Structural and functional characterization of oxidized feruloylated arabinoxylan from wheat. FOOD HYDROCOLLOIDS 63 pp. 219-225. DOI

[66] Rakszegi M., Balazs G., Bekes F., Harasztos A., Kovacs A., Lang L. Bedo Z., Tomoskozi S. (2014): Modelling Water Absorption of Wheat Flour by Taking into Consideration of the Soluble Protein and Arabinoxylan Components, CEREAL RESEARCH COMMUNICATIONS 42 : 4 pp. 629-639. DOI

[67] Bender D., Németh R., Cavazzi G., Túróczi F., Schall E., D’Amico S., Török K., Lucisano M., Tömösközi S., Schönlechner R. (2018): Characterization of rheological properties of rye arabinoxylans in buckwheat model systems. Food Hydrocolloids, Volume 80, pp. 33-41. DOI

[68] Németh R., Bender D., Jaksics E., Calicchio M., Langó B., D’Amico S., Török K., Schoenlechner R., Tömösközi S. (2019): Investigation of the effect of pentosan addition and enzyme treatment on the rheological properties of millet flour based model dough systems. FOOD HYDROCOLLOIDS 94 pp. 381-390. DOI

[69] Németh R., Fekete-Papp R., Orosz K., Jaksics E., Szentmiklóssy M., Török K., Tömösközi S. (2022): Investigation of the Role of Arabinoxylan on Dough Mixing Properties in Native and Model Wheat Dough Systems. Periodica Polytechnica Chemical Engineering, 66(3), pp. 437–447. DOI

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[73] MSZ 6383: Búza és durumbúza élelmezési célra

[74] MAGYAR ÉLELMISZERKÖNYV (Codex Alimentarius Hungaricus) (2020): 2-201 számú irányelv, Malomipari termékek.

[75] Tömösközi S. (2012): Az új búzaszabványról. Martonvásár 24, pp 24-25.

[76] Tömösközi S. (2013): Az új magyar búzaszabvány szerepe a minőségszemlélet formálásában. AGROFÓRUM EXTRA 24:50. pp. 46-47.

[77] Codex Alimentarius 2010. Guidelines on nutrition labelling CAC/GL 2-1985 as last amended 2010. Joint FAO/WHO Food Standards Programme, Secretariat of the Codex Alimentarius Commission, FAORome.

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[80] Rakszegi M., Balazs G., Bekes F., Harasztos A., Kovacs A., Lang L. Bedo Z., Tomoskozi S. (2014): Modelling Water Absorption of Wheat Flour by Taking into Consideration of the Soluble Protein and Arabinoxylan Components, CEREAL RESEARCH COMMUNICATIONS 42 : 4 pp. 629-639. DOI

[81] Török K., Szentmiklóssy M., Tremmel-Bede K., Rakszegi M., Tömösközi S. (2019): Possibilities and barriers in fibre-targeted breeding: Characterisation of arabinoxylans in wheat varieties and their breeding lines. JOURNAL OF CEREAL SCIENCE 86, pp. 117-123. DOI

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[85] Juhász A., Larroque OR., Tamás L., Hsam SLK., Zeller FJ., Békés F. et al. (2003): Bánkúti 1201 - an old Hungarian wheat variety with special storage protein composition. Theor Appl Genet107. pp. 697–704. DOI

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[95] Pauk, J. Lantos Cs., Ács K., Gell Gy., Tömösközi S., Búza Hajdú K, Békés F. (2019). Spelt (Triticum spelta L.) In Vitro Androgenesis Breeding for Special Food Quality Parameters. In: Al-Khayri, J., Jain, S., Johnson, D. (eds) Advances in Plant Breeding Strategies: Cereals, pp. 525–557 Springer, Cham. DOI

9. List of basic and applied projects

2020-2024 National project‚ Exploring of the genetic, compositional and processing potentials of spelt’ (Project ID: OTKA K 135343)

2017 – 2021 National project (GalgaGabona), “Developments to improve the conditions of human utilization of oats and rye in terms of food safety, agrotechniques, processing technology and nutritional value“ (Project ID: 2017-1.3.1-VKE-2017-00004)

2015 – 2019 “Consortional assoc. New aspects in wheat breeding: improvement of the bioactive component composition and its effects“ (Project ID: OTKA K112179)

2015 – 2018 Austrian - Hungarian bilateral project, “Improving gluten-free dough by a novel hemicellulose network“ (Project ID: OTKA ANN 114554)

2016 – 2018 Austrian - Hungarian bilateral project, “Fundamental study on the structure, rheological and functional properties of model gluten-free dough and products based on modified carbohydrate systems“ (Project ID: TÉT_15-1-2016-0066)

2013 – 2016 National project, “Quality characterization and applicability study in market-oriented breeding of old wheat genotypes“ (Project ID: AGR_PIAC-13- 2013-0074)

2012 – 2014 Austrian - Hungarian bilateral project, “Improvement and optimisation of the nutritional value and technological properties of gluten-free products – study on the effect of newly developed food additives and alternative crops“ (Project ID: TÉT_10-1-2011-0731)

2009 – 2013 National Project - “Development of breeding, agricultural production and food industrial processing system of Pannon wheat varieties” (Tech_09-A3-2009-0221)

2010 – 2012 National Project - “Development of quality-oriented and harmonized R+D+I strategy and functional model at BME” (Project ID: TÁMOP-4.2.1/B-09/1/KMR-2010-0002)

2009-2012 National project - “Development of R+D environment and tools for improvement the technology- and knowledge transfer activity at Budapest University of Technology and Economics” (Project ID: TÁMOP-4.2.1-08/1/KMR-2008-000)

2009 - 2012 Hungarian Scientific Research Fund - „The relationships of breadmaking quality properties of wheat with the composition of gluten and pentosan” (Project ID: OTKA CK 80334)

2009 – 2012 National Project - “Health Promotion and Tradition: Development of raw materials, functional foods and technologies in cereal-based food chain” (Project ID: TECH_08_A3/2-2008-0425)

2010 – 2011 Austrian – Hungarian Action Fund - Use of pentosans for the production of bread, bakery goods and gluten-free bread of enhanced nutritional value (Project ID: 77öu12)

2009-2011 EU-supported national program “Development of curriculum for MSc education on the area of Food safety and quality” (Project ID: TÁMOP-4.1.2-08/2/A/KMR-2009-0011)

2009 – 2011 Hungarian – Turkish bilateral project – “Improvement of quality and safety of cereals and cereal based food products” (Project ID: TR-16/2008)

2007 – 2011 EU FP6 - MoniQA Network of Excellence (Monitoring and Quality Assurance in the Food Supply Chain (Project ID: FOOD-CT-2006-036337)

2008 – 2009 Austrian – Hungarian Bilateral project - “Functional foods from underutilized cereals and pseudocereal; optimisation of processing parameters and evaluation of its health promoting properties” (Project ID: AT-12/2007)

2003 – 2008 Hungarian Scientific Research Fund - “The effect of low molecular weight polypeptides on the polymerization degree distribution of polymeric glutenin” (Project ID: OTKA K 42703)

2004-2007 National R+D Program - „Scientific program for development of Pannon wheat quality - (Project ID: GAK-ALAP-00126/2004)

2004 – 2005 Scientific-technical cooperation, Austria – Hungary - „Comparison of different amaranth species (regarding chemical composition, functional and sensory properties) for the production of amaranth beverages and amaranth bread” (TéT Program, Project ID: A-20/03)

2001-2005 National R+D Program - „Improvement the utilisation of basic materials in cereal industry” (Project ID: NKFP (4/035/2001)

2001 – 2005 Hungarian Scientific Research Fund – “Development of micro-scale methods for determination of cereal quality” (Project ID: OTKA K 34486)

2003 – 2004 Scientific-technical cooperation, Poland – Hungary “Study of functional, rheological and surface properties of mixed protein systems containing wheat and other plant protein fractions” (TéT Program, Project ID: Pl 05/99)

2001 – 2004 Hungarian Economic Competitiveness Program – “Pannon Wheat Program” (Project ID: ALAP1-00126/2004)

2000 – 2003 Hungarian Scientific Research Fund - “Investigation of nutritional and functional properties of pseudo cereals” (Project ID: OTKA T-032650)

1996-2000 National Committee for Technical Development (OMFB) Program – “Development of micro-scale Z-arm mixer and laboratory mill” (Project ID: 96-97-68-1354)

1994 – 1998 PHARE-PMU Program – “An educational, retraining and continuing education project in the field of food and pharmaceutical industries and environmental protection” (Project ID: HU-94.05 0101-L015/20)

DOI

Received: October 2022 – Accepted: December 2022

¹ Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science, Research Group of Cereal Science and Food Quality, Hungary
² Karlsruhe Institute of Technology, Institute of Applied Biosciences, Department of Bioactive and Functional Food Chemistry, Germany
³ Biotask AG, Germany
⁴ University of Natural Resources and Life Sciences, Austria
⁵ AGES, Austria
⁶ MoniQA Association

Keywords

gluten, celiac disease, immunoanalytics, validation, reference material

2. Introduction

Wheat and other cereals, such as rye, barley and oats, have long been staple foods in the human diet due to their significant contribution to our daily energy, protein and fiber intake and their high contents of certain vitamins and bioactive phytochemicals [1]. Gluten is a collective term referring to a protein fraction of wheat, rye, barley (and their crossbred varieties, such as triticale) that plays a very important role in the baking quality of these cereals [2]. However, these very same proteins (in certain cases alongside other protein types) are able to trigger different hypersensitivity reactions in susceptible people. Of these, the most important one is celiac disease (CD), with a global prevalence of 1%. CD is a chronic autoimmune disorder that appears in genetically predisposed individuals upon dietary gluten exposure. Due to the inflammation of the small intestine and the atrophy of the intestinal villi, CD can manifest in a range of symptoms, like malabsorption-induced nutritional deficiencies, anemia, gastrointestinal and other atypical phenomena (e.g. neurological disorders, infertility, etc.). Currently CD is incurable; its only treatment is a lifelong gluten-free (GF) diet [3, 4].

To support CD patients in complying with their diet, it is mandatory for food producers to indicate the presence of gluten in their products, or the absence thereof if the product was specifically manufactured to meet the special needs of the celiac customer. According to current EU regulations, in line with the recommendation of Codex Alimentarius, products with a gluten content below 20 mg/kg can be labelled GF [5, 6, 7]. The law defines gluten as “a protein fraction from wheat, rye, barley, oats or their crossbred varieties and derivatives thereof, to which some persons are intolerant and which is insoluble in water and 0,5 M sodium chloride solution” [6]. Nevertheless, this definition does not do justice to the utter complexity of gluten. This complexity originates from the high number of protein subunits that gluten consists of and the genetic and environmental variability. The two major components of gluten are the alcohol-soluble prolamins (what gliadins, rye secalins, barley hordeins and oat avenins) and the alcohol-unsoluble glutelins (in wheat: glutenins). While gliadins are monomeric proteins, glutelins are large, aggregated biopolymers [2, 8, 9]. Celiac-toxic epitopes with various immunogenicity have been identified in both protein groups, with certain gliadin epitopes showing the highest levels of toxicity. In vitro and in silico methods indicate a high number of potentially toxic epitopes, but in patients, more than 90% of immune reactions are caused by a few so-called immunodominant epitopes. Besides the sheer number of epitopes, the estimation of toxicity is further hindered by the fact that the number of these epitopes may vary within and across cereal species [10, 11, 12].

Monitoring the compliance of food products sold as GF with the 20 mg/kg threshold requires such analytical methods that are able to quantify gluten accurately and reliably in this low concentration range. There are several suitable methodologies including PCR (polymerase chain reaction) or liquid chromatography coupled to mass spectrometry. However, the method-of-choice in routine analysis is the ELISA (enzyme-linked immunosorbent assay) which is based on the formation and detection of a specific antigen-antibody complex. The advantages of the method are its specificity, sensitivity, ease of use and relatively low cost. While gluten quantification has no reference methods, Codex Alimentarius recommends ELISA as the method to be used for this purpose [5, 13].

Because of the lack of reference methods, more and more ELISA assays appeared and the advent of the methodology brought along a number of studies drawing attention to the fact that different ELISA methods may provide different results when analyzing the same sample, which became an important reliability issue [14, 15, 16].

3. Specific problems of gluten analysis

The variability of gluten ELISA results is caused by several intertwined factors that affect method development and validation, as depicted in Figure 1.

The core of the problem is in part the pathomechanism of CD and the complexity of gluten proteins. As described in the Introduction, celiac disease can be triggered by a high number of epitopes located on a number of protein subunits. The amount of these epitopes expressed in different cereal species and cultivars strongly depends on genetic and environmental factors. This is one of the reasons of the lack of reference methods and reference materials, which makes method development and validation very difficult. These issues together lead to the variability of methods, which finally peaks in the observed variation of the analytical results. Major elements of this methodological variability are the applied antibodies, the sample preparation steps and the calibrating materials. Beside these, we must also keep in mind the effects of complex matrices and food processing procedures on protein structure and solubility, which may further increase measurement uncertainty through modifying the extractability and immunoaffinity of proteins. Overcoming these obstacles requires an urgent harmonization of gluten analytical methods of which a key element is the development of suitable reference materials (RMs) [17, 18, 19, 20, 21].

4. Reference material development efforts for the quantification of gluten

The problem of gluten reference materials has been in the focus of the researchers of the field for more than 20 years, but a universally accepted, certified reference material is still out of reach [17, 22]. An important milestone of the reference material development was the creation of the so-called PWG (Prolamin Working Group)-gliadin. PWG gliadin was isolated from a mixture of the 28 most common European wheat varieties. It is a high-purity, very well characterized material, and is commonly used as an ELISA calibrating material to this day. However, it could not obtain the status of a certified RM due to uncertainties about the reproducibility of its production and its long-term availability [17, 23].

Our research group joined the RM development efforts in 2008 as a member of the Allergen Working Group of the EU FP6 MoniQA Network of Excellence (FOOD-CT-2006-036337). In the first phase of our work, we developed such RM candidates that contained a known amount of gluten in a processed food matrix (Figure 2). As a gluten source, we used the above-mentioned PWG gliadin [24, 25]. This RM candidate was subsequently used in a number of experiments that aimed to identify the factors that act as sources of error in ELISA and thus contribute to analytical uncertainty [14, 19, 26, 27, 28].

As the questions of the disadvantages of PWG gliadin emerged, the Reference Material Working Group, coordinated by our research group under the auspices of the legal successor of the MoniQA project, the MoniQA Association (https://www.moniqa.org), contemplated that the development of a brand new reference material is necessary. The Working Group decided to start the work from scratch and to rethink this problem from the basics. For this, two questions had to be answered. The first one was which wheat variety or varieties are the most promising to represent a global sample population considering genetic and environmental variability. The other one was the question of the format of gluten in the new RM: should it be a flour, a gluten isolate of a gliadin isolate. To answer these questions the Working Group collected 23 commercial wheat varieties from a number of countries and continents. The samples were thoroughly characterized with a complex analytical methodology (including chemical composition, two gluten ELISAs and separation techniques). Based on the results a set of selection criteria including both qualitative and quantitative elements was created that we used to choose five such wheat cultivars that we deemed suitable to be used in a reference material [29, 30]. Then, flours, gluten and gliadin isolates made from the five varieties and their mixture were further analyzed in terms of protein composition with no significant differences found. As a conclusion of this work, we chose the flour of the mixture of the five varieties as a new RM. Its application is supported by its easy preparation that is also feasible in pilot plant conditions without changes in quality and that it contains not only gliadins but every other protein types as well [31, 32]. This is particularly advantageous because a common criticism towards gluten ELISAs is that they usually provide their results in gliadin units that are recalculated to gluten using a multiplying factor of two. This approach comes from the theory that the prolamin to glutelin ratio in gluten is 1:1. However, a growing body of evidence suggests that this ratio can be very different, which may also cause inaccuracy when calculating the results [33]. This explains that the latest method developments are moving to the direction of using several antibodies simultaneously to be able to detect not only prolamins but glutelins as well [34]. The flour reference material that is now available for analytical applications through the MoniQA Association fits this approach well.

Therefore, in case of wheat, a considerable progress occurred in the RM development. However, not only wheat but rye and barley also triggers celiac disease. While there is a lot less information about these cereals in this context, the available studies indicate that gluten antibodies show different affinity towards rye and barley prolamins. This may lead to under- or overestimation of the gluten content of samples with rye or barley contamination that makes it necessary to create new reference materials developed specifically for rye and barley [35, 36].

By recognizing this demand, our international research group is now working on the repetition of the experiments described for wheat, this time for rye and barley. So far, we have collected more than 120 barley and more than 50 rye samples and analyzed their chemical composition, gluten content as per ELISA and protein content and composition determined by separation techniques. We used the results for setting up new selection criteria. The selected seven rye and eight barley cultivars are currently being analyzed [publication underway]. The expected outcome of this work is the development of new rye and barley RMs that independently or in combination with each other and the wheat material could help to improve the conditions of gluten analysis.

5. A short but important detour: oats and the gluten-free diet

The previous sections covered the analytical aspects related to wheat, rye and barley. In the celiac context we must also involve oats and its controversial role in the gluten-free diet. An improper GF diet may be accompanied by nutritional problems such as the reduced intake of fibers, vitamins and minerals, an increase in saturated fatty acids and a higher glycemic load [37]. These disadvantages could be counterbalanced by the ingestion of oats due to their high fiber and antioxidant, and relatively high unsaturated fatty acid content [38].

Oats are generally considered safe for celiac patients because they contain significantly less prolamins than wheat, and contrary to the analogous proteins of wheat, oat avenins are less resistant to digestive enzymes [39]. The vast majority of clinical studies dealing with the capacity of oats to trigger CD also conclude that oat consumption in moderate amounts (20-25 g/day for children, 50-70 g/day for adults) is safe for celiac patients in remission [40, 41]. However, some other studies found that in certain cases, oats can pose a risk for celiac consumers and, while only a low amount, but some oat avenin epitopes were found to be able to induce CD. It is also important to note, that genetic and environmental variability is also present for oats, which can affect the presence of potentially toxic epitopes. Thus, it becomes necessary to screen the presence of toxic epitopes in oat varieties [42, 43, 44, 45].

This controversy also appears in international law. While Australia and New Zealand explicitly rejects the inclusion of oats in the GF diet [46], in the EU, the US and Canada it is permitted to introduce the so-called “pure oats” specifically produced for CD patients in the diet [6, 47, 48]. The issue of pure oats production is of paramount importance as different studies found that 13-88% of commercial oat products are contaminated with gluten to various extents. Contamination may occur at any step of the production chain [49, 50].

Pure oat production must be handled with exceptional care and is built on two pillars. In one hand, it must be ensured that seeds do not contain toxic epitopes, which requires pre-screening of oat varieties and the development of a suitable analytical methodology [51]. In the past couple of years, our research group got involved in these tasks [52, 53]. On the other hand, gluten contamination must be avoided in the entire production process. This requires serious efforts and compliance to special protocols that aim for the complete elimination of the risk of gluten contamination (e.g. confirming seed purity, safety lanes between land plots, the application of dedicated machinery and tools, segregated storage and processing, etc.) (Figure 3) [54]. The detection of the presence of unwanted gluten in oats is yet another analytical challenge. While ELISAs using the R5 antibody are suitable tools for this purpose, because they do not cross-react with oats but they do recognize wheat, rye and barley (with the limitations described earlier), in case of oats a specific difficulty is that only a few contaminating grains can pose a health risk. To mitigate this risk, sampling protocols developed specifically for oats have been established [55, 56].

In conclusion, the role of oats in the gluten-free diet keeps being a matter of debate to this day. While the nutritional benefits of oat consumption are beyond doubt, the safety of oats must be further assessed in clinical trials. Another important task is the improvement of pure oats production protocols and the related analytical methodologies.

6. Summary

Gluten proteins have long been an integral part of our diet through the consumption of cereals, they are very important in determining the quality of a range of staple foods, thus they deserve to be in the center of cereal science for a long time. In the last couple of decades, they also came to the forefront due to their connection to diseases such as celiac disease, non-celiac gluten sensitivity or wheat allergy. In this paper, we focused on disorders treated by a gluten-free diet, primarily celiac disease, to demonstrate the special food safety and analytical challenges represented by gluten.

While we have at our disposal a relatively large repertoire of analytical methods, it is very important to be aware of their possibilities and limitations, especially in case of the routinely used ELISA. Some of these limitations can be at least partially eliminated or improved with new findings of protein chemistry, immunology and clinical studies, which corroborates the need of the continuation of the research efforts presented in this article. However, others will always be present due to the innate characteristics of the methodology, which makes it necessary to create new analytical solutions, of which a good example is the quick evolution of proteomics [58].

Consequently, the handling of gluten as a food safety problem requires a multidisciplinary approach. It needs the close cooperation of clinical research, lawmakers, food producers, food analytics and a range of other areas. Our research group integrated in this system through the improvement of the conditions of gluten analysis with the goal of making gluten quantification more reliable and as a result of that, to contribute to the safety and better quality of life of people living with celiac disease.

7. Acknowledgements

While this paper is more of a review in nature, it mentions a number of research results that have been created by the Research Group of Cereal Science and Food Quality operating at the Department of Applied Biotechnology and Food Science of the Budapest University of Technology and Economics in cooperation with its partner institutions both inland and abroad during the past about 15 years. The authors wish to express their gratitude to every project, institution and colleague that contributed to these works in any way. We are especially thankful to those nearly 40 students of ours who took part in these research projects during the preparation of their BSc, MSc or PhD theses.

The rye and barley reference material development research presented in this paper was partly funded by the National Research, Development, and Innovation Fund of Hungary under Grant TKP2021-EGA-02.

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DOI

Received: October 2022 – Accepted: November 2022

¹ Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science, Research Group of Cereal Science and Food Quality

Keywords

carbohydrates, FODMAP, starch, HPLC, polymer, fibre content, characterisation, plant proteins

2. Introduction

The range of our plant-based foods is wide, and the consumption of them is necessary to cover our energy intake, they also partially provide our protein and fibre source, but they also have many ingredients that have a beneficial effect on our body. Unfortunately, they may also contain molecules that can be associated with disorders affecting the digestive system (e.g. allergies, celiac disease, irritable bowel syndrome).

In the field of food analytics, we have a number of methods available to characterize foods, but we are faced with a difficult task when we try to analyse and understand such complex material systems like cereal-based dough and bakery products. Because targeted sample preparation, maintaining the native structure during sample preparation, selective separation of different components, or detection of components can be a serious challenge.

In the BME ABÉT Research Group of Cereal Science and Food Quality, within the separation techniques, we mainly deal with the examination of proteins and carbohydrates, which covers the characterization of raw materials or, for example, the interpretation of phenomena experienced during rheological and technological behaviour. Our research goals and tasks can be divided into three large groups: the determination of nutritional value and the quantity of health-supporting ingredients, the characterization of carbohydrates and proteins that fundamentally determine technological behaviour, and the examination of certain ingredients that are critical from a food safety point of view.

3. Characterization of carbohydrates

Among plant carbohydrates, starch has fundamental importance, which is relevant from a nutritional point of view due to our energy needs, and it is a key component in technology, for example, it affects the quality of products made from grains, and in many cases, in its native or modified form, it is an important additive in dough improvement. In the case of non-starch carbohydrates, dietary fibres can play an important role both nutritionally and technologically, depending on their quality. In the case of grains, the most prominent representatives are arabinoxylan, β-glucan and arabinogalactan peptides. Small molecular size carbohydrates are also important for energy intake (e.g. sugars), and can also determine the sensory properties of products. However, some members of this group (FODMAP - fermentable oligo-, di-, monosaccharides and polyols) can pose a food safety problem for certain consumers. [1, 2]

Starch, its structure, amylose/amylopectin ratio and size can provide information about its technological properties. The size of the starch particles is related to the water absorption, which in dough systems determines the dough’s extensibility and resistance. Among the starch constituents, amylose results in a firmer gel. However, amylose is more involved in the aging process than amylopectin, so its high ratio reduces the shelf life of products. [1, 3]

The quantification of starch and its constituents and the characterization of their molecular properties are a serious challenge, as it is hardly possible to apply selective solution and to determine it and its constituents specifically. Enzyme kits are available to determine the total amount of starch and the amylose/amylopectin ratio (amylose content) [4, 5], but their reliability is questionable for the reasons mentioned above. For our research, we perform starch characterization with size exclusion high-performance liquid chromatography (HPLC), which allows us to obtain information on the size of the starch and the amylose/amylopectin ratio. We have used this in many research projects, for example to study wheat with high amylose and high amylopectin ratios produced by plant breeding. In addition, we also participated in a project focusing on the technological implementation of wheat-based starch production, in which case we mainly solved the examination of the variability of raw materials and the monitoring of starch production in this way. [6, 7] (Related projects: GINOP-2.1.1-15-2016-00855; OTKA K112179)

Fibres play a role in forming the technological properties of cereal based products, but their positive effects on our bodies are also crucial. For assessing the role of fibres in shaping nutritional and technological behaviour, it is important to characterize them from several points of view. We are currently characterizing arabinoxylans in detail using separation techniques, whose composition and size provide valuable information. The ratio of xyloses, which form the backbone of the molecule, and arabinoses, which make up the side chains, basically determines the properties of the polymer, e.g. solubility and size [8]. Information on the composition and ratio of the monomers that make up the polymer, as well as their absolute amount, is obtained using the gas chromatography method. In addition to the composition, it is also important to map the size of the fibre molecules in order to examine their effect on the technological properties. For this, the polymer structure is left unchanged, and the size distribution can be examined by size exclusion liquid chromatography. It is currently an unsolved problem that, it is only possible to determine the properties of soluble fibres in this way. It is still questionable how to characterize the insoluble fibres present in the vast majority of cereals. We encounter similar methodological problems for example in the case of examination of HMW glutenins (high molecular weight gluten proteins).

Numerous publications are available in the literature on the evolution of the total fibre content, but much less on the amount of individual fibre constituents. In our group, several projects are connected to the determination of arabinoxylans, during which we mainly mapped the effect of genetic and environmental factors, thus the variability of these molecules (Figure 1). [9, 10, 11]. (Related project: OTKA K112179)

In addition to the raw material characterization, we also examined the influence of the fibre-forming agents on the technological behaviour. One of the defining research tasks of our group in recent years was the forming of a dough structure similar to gluten-containing grains in the development of gluten-free products. Since the gluten-free raw materials lack the proteins that give the appropriate structure of the dough, this must be replaced somehow. During our research, we attempted to at least partially replace this with carbohydrates, namely by forming an arabinoxylan network (Figure 2). Our liquid chromatography method proved to be suitable for tracking this, proving the formation of the network and the changes that occur in the polymers as a result of enzyme treatment (Figure 3). [12, 13, 14] (Related projects: OTKA ANN-114554; TÉT_15-1-2016-0066)

Irritable bowel syndrome, which can be classified as a disorder affecting the digestive system, can be associated with the consumption of short-chain carbohydrates whose collective name is FODMAP [16]. In the past period, we have focused on mapping these in cereals, the qualitative and quantitative analysis of them can be solved using liquid chromatography methods.

There are many plant-based foods that can be considered high risk in terms of FODMAP content. Most of the cereals can be classified here, but we know almost nothing about the genetic and environmental variability of the FODMAP content of cereals. In recent years, during research work with our partners in the breeding and milling industry, we have begun to develop analytical methods suitable for the determination of these components in cereals. As a result, with adapted and improved methods, we are now able to provide information on the small molecular carbohydrate composition of different grain species and varieties, as well as different milling fractions. This enables mapping the quantitative and qualitative variability of these components. (Related project: 2017-1.3.1-VKE-2017-000)

4. Examination methods of plant proteins

The classification of plant proteins is traditionally based on the Osborne fractionation, which separates individual proteins according to their solubility. Although this does not always mean homogeneous groups in terms of function, size and composition, so, for example, in the case of cereal proteins, other classification including other aspects has also been developed. [17, 18]

In our group, we mainly deal with the understanding of the techno-functional properties of different proteins, and with the characterization and development of the analytical environment of proteins which are critical in food safety aspect. We mainly use liquid chromatography methods based on size exclusion and reversed-phase separation to examine the protein composition.

In case of wheat, the number of sources related to protein composition available in the literature is large, but interestingly the amount of knowledge about very similar and closely related grains (e.g. rye, barley, oat) is much smaller. Therefore, dealing with these kind of grains is a much more difficult task in our research, because we can rely less on the methods and data described in the literature. So the interpretation of the results and the identification of the obtained chromatographic peaks are often difficult due to the lack of knowledge. The development and application of liquid chromatography methods takes place in parallel with the mapping of the detailed compositional, rheological and technological behaviour of oats and rye. As a result, we can get a more detailed picture of the protein composition and its variability of these grains (Figure 4). [19] (Related project: 2017-1.3.1-VKE-2017-000)

However, we can use these methods not only for the characterization of raw materials. When developing products with appropriate technological characteristics, it is essential to understand the interactions between the components in the dough system. The researches at our department has contributed to understanding of the gluten complex formation [20]. But there are still many questions about the relationship between proteins and carbohydrates, the role of fibres and the integration of molecules added from an external source into the dough system. Understanding the exact mechanism of them is essential for product development aimed at improving technological properties, and chromatographic methods can be a good tool for it. They are well suited for monitoring the effect of various technological treatments in product development as well.

An example of this is the examination of the effects of the kneading, dough rising and baking steps, as well as the arabinoxylan addition in terms of protein composition in gluten-free product development. [21] (Related project: OTKA ANN-114554)

Last, but not least, components critical to food safety (e.g. proteins that cause celiac disease) can also be examined with the help of separation techniques. The lack of reference materials is a serious problem with regard to the reliability of analytical methods suitable for detecting/determining gluten contamination. For several years now, we have been working with companies producing immunoanalytical rapid tests and foreign partner research groups to solve this problem. An essential part of the development is the mapping of the genetic and environmental variability of the proteins that cause celiac disease, as well as the changes in the physico-chemical properties of the proteins during food production. The former was mainly investigated using reversed-phase HPLC, while the latter was studied using size-exclusion liquid chromatography. A more detailed analysis of the proteins was first carried out in case of wheat, however, rye and barley proteins are also involved in the disorder, which are currently being investigated in our research group. [22, 23, 24, 25] (Related project: FOODCT-2006-036337)

5. Acknowledgements

We would like to thank all former and current colleagues, PhD students and graduate students who contributed to the expansion of our research group’s knowledge of separation techniques. Special thanks to Gábor Balázs and Anna Harasztos, who were key members of the group for a long time, and to whom we owe the development of many methods that are still used today.

We can maintain the financing of the works with the help of numerous research projects and grants, of which the most significant ones of recent years are the following:

• “Exploring of the undefined genetic, compositional and processing potentials of spelt in different environments” OTKA 135211 project
• TKP2021 funding programme, BME-EGA-02 and BME TKP-BIO 2020 projects
• “GalgaGabona project: Developments to improve the conditions of human utilization of oats and rye in terms of food safety, agrotechniques, processing technology and nutritional value” project (2017-1.3.1-VKE-2017-00004)
• “Improving gluten-free dough by a novel hemicellulose network” (OTKA ANN 114554) (FWF I1842-N28)
• “Fundamental study on the structure, rheological and functional properties of model gluten-free dough and products based on modified carbohydrate systems” (TÉT_15-1-2016-006)
• “New aspects in wheat breeding: improvement of the bioactive component composition and its effects” (OTKA 11279)(FWF-I1842-N28)
• “Vállalatok K+F+I tevékenységének támogatása: Minőségorientált komplex ipari termelési rendszer és modell kifejlesztése, új módosított keményítő kialakítása, illetve új rostalapú feldolgozott termék hasznosításának kutatása” című projekt (GINOP-2.1.1-15)
• “Health Promotion and Tradition: Development of raw materials, functional foods and technologies in cereal-based food chain” (TECH_08_A/2-2008-0425)
• “Development of quality oriented, harmonized educational and R+D+I strategy and operational model at the Budapest University of Technology and Economics” (TÁMOP-4.2.1/B-09/1/KMR-2010-0002)
• MoniQA Network of Excellence (FOOD-CT-2006-036337)

6. References

[1] Goesaert H., Brijs K., Veraverbeke W.S., Courtin C.M., Gebruers K., Delcour J.A. (2005): Wheat flour constituents: how they impact bread quality, and how to impact their functionality. Trends in Food Science & Technology 16 (1-3) pp. 12-30. DOI

[2] Khan K., Shewry P.R. (2009): Wheat: Chemistry and Technology. 4th ed. AACC International, Inc.

[3] Gray J.A., Bemiller J.N. (2003): Bread Staling: Molecular Basis and Control. Comprehensive Reviews in Food Science and Food Safety 2 (1) pp. 1-21. DOI

[4] McCleary B.V., Charnock S.J., Rossiter P.C., O’Shea M.F., Power A.M., Lloyd R.M. (2006): Measurement of carbohydrates in grain, feed and food. Journal of the Science of Food and Agriculture 86 (11) pp. 1648-1661. DOI

[5] McCleary B.V., Charmier L.M.J., McKie V.A. (2018): Measurement of Starch: Critical Evaluation of Current Methodology. Starch – Stärke 71 (1-2) 1800146. DOI

[6] Jaksics E., Paszerbovics B., Egri B., Rakszegi M., Tremmel_Bede K., Vida Gy., Gergely Sz., Németh R., Tömösközi S. (2020): Complex rheological characterization of normal, waxy and high-amylose wheat lines. Journal of Cereal Science 93 102982 pp. 1-11. DOI

[7] Fekete D. (2021): A fajtahatás vizsgálata a búzakeményítő előállítás laboratóriumi modellezése során nyert termékekben. MSc Diplomamunka. Budapesti Műszaki és Gazdaságtudományi Egyetem, Budapest.

[8] Saulnier L., Sado P.-E., Branlard G., Charmet G., Guillon F. (2007). Wheat arabinoxylans: Exploiting variation in amount and composition to develop enhanced varieties. Journal of Cereal Science 46 (3) pp. 261-281. DOI

[9] Török K., Szentmiklóssy M., Tremmel-Bede K., Rakszegi M. Tömösközi, S. (2019): Possibilities and barriers in fibre-targeted breeding: Characterisation of arabinoxylans in wheat varieties and their breeding lines. Journal of Cereal Science 86 pp. 117–123. DOI

[10] Tremmel-Bede K., Szentmiklóssy M., Tömösközi S., Török K., Lovegrove A., Shewry P.R., Láng L., Bedő Z., Vida Gy., Rakszegi M. (2020): Stability analysis of wheat lines with increased level of arabinoxylan. PLoS ONE 15 (5) pp. 1–15. DOI

[11] Szentmiklóssy M., Török K., Pusztai É., Kemény S., Tremmel-Bede K., Rakszegi M., Tömösközi S. (2020): Variability and cluster analysis of arabinoxylan content and its molecular profile in crossed wheat lines. Journal of Cereal Science 95 103074 pp. 1-8. DOI

[12] Bender D., Nemeth R., Cavazzi G., Turoczi F., Schall E., D’Amico S., Török K., Lucisano M., Tömösközi S., Schoenlechner, R. (2018): Characterization of rheological properties of rye arabinoxylans in buckwheat model systems. Food Hydrocolloids 80 pp. 33-41. DOI

[13] Németh R., Bender D., Jaksics E., Calicchio M., Langó B., D’Amico S., Török K., Schoenlechner R., Tömösközi S. (2019): Investigation of the effect of pentosan addition and enzyme treatment on the rheological properties of millet flour based model dough systems. Food Hydrocolloids 94 pp. 381–390. DOI

[14] Farkas A., Szepesvári P., Németh R., Bender D., Schoenlechner R., Tömösközi, S. (2021): Comparative study on the rheological and baking behaviour of enzyme-treated and arabinoxylan-enriched gluten-free straight dough and sourdough small-scale systems. Journal of Cereal Science 101 103292. DOI

[15] Németh R., Bender D., Jaksics E., Calicchio M., Langó B., D’Amico S., Török K., Schoenlechner R., Tömösközi S. (2019): Investigation of the effect of pentosan addition and enzyme treatment on the rheological properties of millet flour based model dough systems. Food Hydrocolloids 94 pp. 381-390. DOI

[16] Ispiryan L., Zannini E., Arendt E.K. (2020): Characterization of the FODMAP-profile in cereal-product ingredients. Journal of Cereal Science 92 102916. DOI

[17] Osborne, T. B. (1907): The protein of the wheat kernel. Publication No. 84. Carnegie Institute: Washington, DC

[18] Shewry P.R., Halford N.G., Lafiandra D. (2003): Genetics of wheat gluten proteins, Advances in Genetics 49 pp. 111–184. DOI

[19] Járó K. (2021): Különböző zab minták fehérjeösszetételének jellemzése nagyhatékonyságú folyadékkromatográfiás módszerekkel. MSc Diplomamunka. Budapesti Műszaki és Gazdaságtudományi Egyetem, Budapest.

[20] Lásztity R., Békés F., Örsi F., Smied I., Ember-Kárpáti M. (1996). protein-lipid and protein-carbohydrate interactions in the gluten complex. Periodica Polytechnica Chemical Engineering 44 (1-2) pp. 29-40.

[21] Németh R. (2019): Sütőipari minőség meghatározására alkalmas műszer- és módszerfejlesztések és alkalmazásuk búzaalapú és gluténmentes modelltermékek vizsgálatára. Doktori értekezés. Budapesti Műszaki és Gazdaságtudományi Egyetem, Budapest.

[22] Török K., Hajas L., Bugyi Z., Balázs G., Tömösközi S. (2015). Investigation of the effects of food processing and matrix components on the analytical results of ELISA using an incurred gliadin reference material candidate. Acta Alimentaria 44 (3) pp. 390–399. DOI

[23] Hajas L., Scherf K.A., Török K., Bugyi Z., Schall E., Poms R.E., Koehler P., Tömösközi, S. (2018): Variation in protein composition among wheat (Triticum aestivum L.) cultivars to identify cultivars suitable as reference material for wheat gluten analysis. Food Chemistry 267 pp. 387–394. DOI

[24] Schall E., Scherf K.A., Bugyi Z., Hajas L., Török K., Koehler P., Poms R.E., D’Amico S., Schoenlechner R., Tömösközi, S. (2020): Characterisation and comparison of selected wheat (Triticum aestivum L.) cultivars and their blends to develop a gluten reference material. Food Chemistry 313 126049. DOI

[25] Schall E., Scherf K.A., Bugyi Z., Török K., Koehler P., Schoenlechner R., Tömösközi S. (2020): Further Steps Toward the Development of Gluten Reference Materials – Wheat Flours or Protein Isolates? Frontiers in Plant Science 11 906. DOI

DOI

Received: October 2022 – Accepted: November 2022

¹ NIR Spectroscopy Group, Department of Applied Biotechnology and Food Science, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics

Keywords

infrared spectroscopy, multivariate data analysis, imaging

2. Introduction

As a young adult, the Győr ”Festival of Photographers” [1] left me with many, many good memories. The medium consisted of lights and sounds, i.e., waves, it evoked a feeling and opened a window to new, unknown worlds, giving me a better understanding of it. In our small research group, we are striving to do the same: with the help of the infrared range of electromagnetic radiation we seek to understand the chemical (and often physical [6, 7, 8]) history of the substance under study through the interaction of waves with the matter investigated, as so many have done [4, 5] since Sir William Herschel [2, 3].

2.1. Characteristic NIR absorption bands

Almost all functional groups needed to study a biological system provide signals in the range closer to red, also called near infrared (NIR), of non-destructive molecular (or vibrational) spectroscopy, which requires little or no sample preparation (Figure 1). Life-giving water provides signals through its O–H groups; structural or reserve proteins through the C=O and N–H groups of the peptide bonds between amino acids and through their side chains; lipids through their saturated (C–C) or unsaturated (C=C) carbon-carbon bonds, and through the C–H vibrations of their large numbers of methyl (–CH₃) and methylene (–CH₂–) groups, absorbing the photons with wavelengths characteristic of them, and thus moving to higher energy levels. Carbohydrates, as polyhydroxy-oxo compounds, are a bit like mules: the vibrations of the groups listed above (O–H, C=O, C–H) appear together.

2.2. Multi variate data analysis in the NIR technology

And this brings us to the second so-called field of our interdisciplinary science after (N)IR spectroscopy, multivariate (data) analysis (MV(D)A), the magical world of chemometrics and statistics (i.e., mathematics as a common denominator), which we are constantly learning from Professors Sándor Kemény and Károly Héberger (professors at the Budapest University of Technology and Economy. The Ed.), still amazed by their knowledge, experience and pedagogical sense [9, 10]. In our area, the need for these sciences stems from two main sources.

Firstly, the macro-components listed above (together with the micro-components) make up the whole, the living. Given the profile of our department, it is mainly these complex (plant, animal, human) systems that are studied “as is” or in a processed form (crops, foods, tissues), always resulting in complex, envelope-like spectra. The analysis of these (e.g., identification or assignment of spectral peaks) is inconceivable without smoothing with moving averages, derivative-sensitized peak resolutions or baseline shifts eliminated by normalization. From another professional topic, an example is the case number diagram for the SARS-CoV-2 pandemic, where the change in the epidemic data provided a good indication of the current epidemic situation by plotting the moving averages (Figure 2).

Secondly, spectrum-based identification or the construction of qualitative or quantitative models also requires mathematical tools: sometimes only a single correlation calculation is needed, sometimes more serious vector algebra or operations with matrices.

It may be clear from the above that living in the age of Industry 4.0 (Figure 3) and circular economy [13], whether it is precision agriculture, the food industry using knowledge-based technologies, or the pharmaceutical industry with its PAT and QbD approach, non-destructive spectroscopic sensors (NDSS) are coming to the fore as the main tools, be it fiber-optic sensors in tubes (Figure 4) or cameras mounted on conveyor belts measuring in the UV/Vis/NIR range.

3. The application of infrared technics at the Department

The transition from foods to drugs (either this way or that) is a recurring theme in our work, especially if we look back at our career over the decades since the 1990s. For example, the experience gained in the measurement of wines, sparkling wines, liqueurs, spirits [19], beers and palinkas [20] has been useful in the development of techniques for real-time monitoring of the fermentation processes of monoclonal antibodies [21, 22]. If we had been working at product manufacture, i.e., upstream, we had to help in purification, i.e., downstream, if we were able, for example in the qualification of column packings used in preparative chromatography [23]. In all cases, the goal is, as in the eternal struggle of the world (living inside us), to recognize the difference between good and bad (based on experience = data (data-driven)), to find the cause of being bad (error analysis), to eliminate it and put it on the right track (golden batch). But perhaps the smartest thing to do, in the spirit of “better safe than sorry”, is to take preventive action (predictive maintenance) based on the warning signs before bad things happen. Even the latter can be very helpful, when the focus is not necessarily on the accuracy and precision of the specific metric, but on its variability, dynamics and trend.

It can be mistaken to think that on-site (in situ), immediate (just-in-time) measurements are only the prerogative of producers of high value-added biotech products. Agricultural sensors can form the same kind of network as IoT (internet of things) devices in a smart home. The added value is smaller: but the material flows can be orders of magnitude higher and, as we know, look after the cents and the euros will look after themselves. Whether it’s a feed mixer with a batch approach, working with dedicated recipes [24] or a continuous bioethanol plant with a constant quality goal, there are NIR instruments containing no moving optical elements (thus unaffected by vibrations and shocks). Both of these examples are based on corn and/or wheat, which is not surprising given the development of NIR spectroscopy. These are the basic food materials the investigation of which pioneered the diffuse reflectance NIR analysis of solid materials by Karl Norris of the USA and Phill Williams of Canada in the 1960s and 1970s [5]. Looking a little further, it is perhaps worth quoting Harari’s thoughts here, citing the foreword of Péter Hahner: “[...] it was not man who domesticated wheat, but rather wheat domesticated man, since homo sapiens has been living in houses since he switched to cereal production” [25].

Wheat plays an important role not only in the life of mankind, but also in the life of our group at the Department: the first NIR spectrometers were obtained mainly to measure wheat, thanks to János Varga and later András Salgó [26, 27], and the Cereal Group of Sándor Tömösközi provided us with constant ammunition in the field of cereal research [28, 29]. During the maturation dynamics of wheat, changes of the main components (moisture, proteins, carbohydrates) were followed over time by NIR spectroscopy [30, 31, 32], then by capillary electrophoresis with the help of our colleague Éva Scholz, then by liquid chromatography [33].

The life of a plant seed often continues in the mill, losing its compactness, its integrity, being broken into elemental pieces, and being reborn in different forms. Chaff is separated from wheat, the husk and germ from the kernel, meal from flour, these are the grinds fractions used for our daily bread, or for the formation of our reserves (Figure 5).

The need came from plant breeders to replace the grinding of 200 g of wheat grain for classical wet chemistry analyses with the processing of 10 g of material. In the initial stages, only a few spikes of grain are available, so analytical possibilities are severely limited. This led, thanks to domestic developments [35], to the birth of a micro-mill, the milling properties of which were compared by means of the NIR spectra of the milling fractions with that laboratory mill, which is able to grind 200 g of seed, [36]. After two decades, we are still interested in similar questions: how grinding and milling affects NIR spectra through light scattering due to mesh size, which can be used to profile different laboratory grinders, helping the decision of users when switching to a different model [8], or how to monitor a large-scale milling process, following pasta flour yields [37], or the heat treatment of the resulting fractions [38].

To the frequently arising question “What’s in the sample?”, NIR photons, still non-destructive but already with the energy needed for proper penetration, have helped many times, whether for investigations through packaging materials, or for looking into smaller or larger green samples (watermelon, algae). The former is necessary because, in many places, people work with substances that are hazardous to health and must be removed from the packaging material before use in order to identify and qualify them. For this reason, model systems have been developed where the extent of absorption of plastics can be detected, and their impact can be reduced by variable selection and/or mathematical treatments [39]. Of course, we also help our colleagues where we can, in our area of expertise. Pál Maák (BME TTK, Institute of Physics, Department of Atomic Physics) and his colleagues have built a prototype (Figure 6) that uses NIR lasers to measure the sugar content of watermelons – an order of magnitude leap in measurement sensitivity compared to the apple, kiwi and orange analysis methods of foreign researchers. Here, we provided support in measuring the sugar absorption peaks, the wavelength – indirectly the choice of lasers. Here, we have provided support in measuring the “sugar peaks” and selecting the wavelengths and thus, indirectly, the lasers [40]. To Áron Németh (BME VBK ABÉT, Laboratory of Fermentation Experiments) and his colleagues we were able to offer a solution in the mid-infrared (i.e., analytical) range for the selection of algal species and product yields (i.e., specific lipid amounts as sources of biodiesel) under small-scale cultivation conditions [41]. We strive to maintain integration not only at the level of research groups within the department and the university, but also with national and international universities and research institutes through our professional and grant activities, seeking new applications for the spectroscopic techniques we use [42].

Integration can be achieved not only between people but also between data sets. Just as we demonstrated in 2006 and 2006 how to combine optical NIR spectra and rheological viscosity curves [44, 45], we are working on new solutions for data fusion 15 years later with Pál Péter Hanzelik, representing large-scale industrial background (MOL Nyrt. – Hungarian gas and oil company. The Ed.) with coordination and active participation [46] and Zsombor Kristóf Nagy, who enables connection within the BME-FIEK framework and also involves our research group in the work of the Pharmatech Pharmaceutical Technology Laboratory. Similarly, a fruitful collaboration, now going back to several years, has been established with Márton Bredács, a pillar of the Polymer Competence Center Leoben (PCCL) which works closely with the University of Leoben, in the multisensory selection and classification of plastic wastes using several spectroscopic sensors (Vis, NIR, Raman) [47].

A significant milestone in the life of the group of our Department was the advent of imaging technology. From the early 2000s, presentations and exhibitors at international conferences and publications proliferating in the field showed that “commercialization” (i.e., mass production and wider marketing after prototypes and limited series) had begun. Sticking to what we had been doing, we investigated plant seeds [48], but in addition, a more classical human line was also initiated by the persistent work of Endre Kontsek and Adrián Pesti (our former students, now at the Institute of Pathology, Forensic and Insurance Medicine of Semmelweis University). After studying kidney and gallstones [49], as suggested by our doctors, we turned our attention to soft tissues, tumors and cancerous cell lines [50, 51], hoping to contribute to the development of digital pathology or, as a long-term goal, to the development of real-time tumor identification during surgery. We owe a lot to Alfréd Kállay-Menyhárd and his colleagues (in chronological order, Péter Müller, Dóra Tátraaljai, József Hári and Balázs Kirschweng) from a co-institute (BME VBK FKAT Plastics and Rubber Laboratory), the assignments from whom have contributed to our research on plastics microscopy, be it biodegradable polylactic acid/thermoplastic starch (PLA-TPS) mixtures (Figure 7) or the failure analysis or reverse engineering of various products (adhesives, foils, medical instruments, electric engine coatings).

And speaking of failure analysis, Péter Gordon and his enthusiastic team (BME VIK ETT EFI-labs) are a factor in this area who cannot be ignored, their sample preparations and documentations greatly assist not only our imaging tasks listed above, but also, for example, the cross-sectional analysis of laminated packaging materials (Figure 8).

Still on the subject of plastics, in terms of their environmental aspects, it is microplastics that we are most confronted with these days in everyday and scientific news media. Microplastics (and the chemicals they bind) that enter the food chain through contamination of the inanimate environment can also be harmful to health. In collaboration with Gábor Bordós and his colleagues (WESSLING Hungary Kft.), we have developed a methodology for the application of the infrared range to monitor different types of our waters [55, 56].

As in the previous example, we always come back to man. And once the disease is found, in Western medicine, the next step is the medicine, the pill, and like all good things, these are counterfeited as well. With József Horgos (WESSLING Hungary Kft.) we started to map out this area (literally) [57, 58], and later on we received support from Szilvia Lohner (OGYÉI) in the form of test substances. However, not only counterfeiting, but also (non)compliances of technological origin and the model systems built for formulation development can also be investigated in terms of the presence and distribution of ingredients. The aim is to have a machine vision that classifies not only in the UV and Vis, but also in the NIR range, estimating drug release based on models [59, 60].

One of the most beautiful challenges in terms forecasting is predicting earthquakes. If we recall the earthquake of June 28, 1763, which shook Komárom, Győr and Zsámbék, with a magnitude of 6.3 and claiming 63 lives, and which led to the order to circle the herma of St. László in the city of Győr to avoid further earthquakes (Figure 9), the context becomes clear [61]. I consider the collaboration with István János Kovács and his colleagues, who are members of the Research Institute of Earth and Space Physics at ELKH and the Lithospheric Fluid Research Laboratory at ELTE TTK, a godsend.

Solids and liquids in mixtures, solutions or even colloidal form, often organized into living systems, as complex biological systems are often measured, as could be seen in the examples so far, but gases (“mobile volatiles”) have been left out so far. We cannot take the credit for the measurements this time either. Our geologist colleagues have created that so-called Lithospheric Physics Unit (the first integrated geodynamic station in Central Europe) [62], enabling the very exciting time-series analysis of the measured IR spectra (and other data). However, we should not forget the question to be answered: what can be seen on the changes in soil CO₂ levels, which as a fluid also exhibits tidal movements, and may suddenly become more pronounced due to tectonic movements. Could this be a harbinger of earth movements that are being triggered by the collision of African and European plates? Parts of the big puzzle are the fluids trapped in rocks, which contain CO2, water vapor and other gases, in the study of which we can help with our background in microscopy [63].

The picture of the work in our Department would not be complete without a few words about the research on linear and non-linear relationships. In real life: our nervous system, equipped with our sensory organs (as peripheral organs), helps us to survive the challenges of everyday life by learning non-linear solutions. In machine language: an artificial neural network (ANN) with multisensory sensors and machine learning (ML) with nonlinear solutions operates decision trees.

What would László Telegdy-Kováts László (Galgóc, December 5, 1902 – † Budapest, May 11, 1987, chemical engineer, college professor) (Figure 10) say, who is remembered by CSEMADOK as follows: “He matriculated from the high school of Nyitra in 1919. Obtained his engineer’s diploma from the Faculty of Chemical Engineering of the Technical University of Budapest in 1925. Became an assistant professor to Elek Sigmond (1873–1939), a chemical engineer and professor of agrogeology at the Technical University of Budapest, where he initially worked on soil biology. In the late 1920s, for an extended period he worked in England, where he became familiar with the modern methods of mathematical statistics and experimental design. In 1935, his interest turned to food chemistry, and as a researcher and a ministerial official, his main focus became the management and development of food technology. In 1950, he was appointed professor in the Department of Food Chemistry at the Faculty of Chemical Engineering of the Technical University of Budapest. He educated generations of engineers. His papers on the theoretical and practical issues of food quality are still relevant today. He is also credited with the development of novel food analytical methods. His attention also extended to food packaging technology. He considered the dissemination of scientific knowledge to be an important task and for many years he was president of the Society for Dissemination of Scientific Knowledge (TIT)” [66]. We hope he nods in agreement [67]. In Figure 10, we want to illustrate the technical possibilities in the field of optics that can be exploited through the application of artificial intelligence [67].

4. Conclusion and acknowledgement

Our research group's exploratory bioengineering nature combines the attributes of our specializations (applied biotechnology, health protection, food quality and environmental protection specializations), of which we were able to flash a picture of each within the framework of this article. We hope that we have created a good feeling by opening some window at a time while the movie continues...

* * *

First and foremost, I would like to thank András Salgó, the father of the NIR Spectroscopy Group: without him we would not be here either. Nor would we be here without those who, for many years, have entered the doors of K.II.3. or, more recently, of Ch 165 as PhD students and/or colleagues, namely (perhaps in chronological order) Gábor Sárossy, Réka Juhász, Tímea Gelencsér, Mária Hódsági, Eszter Izsó, Mónika Berceli, László Párta, Éva Szabó, Bence Kozma, Gabriella Besenyő and János Slezsák. Over the past 25 years, I have received a lot of strength, encouragement and examples, in addition to the members of the group, from Sándor Tömösközi, it was is understanding patience and trust in me that made it possible to take stock and this snapshot. I hope that the colleagues and students who have been part of our group think back on us as fondly as we think back on them, and we would like to thank the time spent working together, as we have also learned a lot.

I would like to thanks Sándor Nógrádi, and Géza Tóth (†), (Servitec Kft.) with whom we were able to experience the networking possibilities of NIR spectrometers, in addition to their agricultural and food applications, and gained insight into applications in the pharmaceutical industry, and to Miklós Lipták and Sándor Varju (PER-FORM Hungária Kft.) for broadening our vision and possibilities through access to analytical IR and microscopy techniques, thus opening the way to numerous collaborations with academic, research and industrial partners.

Dedicated to the memory of my Godfather

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DOI

Received: October 2022 – Accepted: November 2022

¹ Budapest University of Technology and Economics, Laboratory of Biochemistry and Molecular Biology, Department of Applied Biotechnology and Food Science

Keywords

programmed cell death, harpin proteins, acrolein, ferroptosis-like cell death, heat stress

2. Introduction

Protecting plants from biotic stress caused by pathogens and abiotic stress caused by adverse environmental effects is an important and increasing challenge for agriculture. Therefore, it has become important to understand the background biochemistry better. Thus, the development of new, more effective ways of plant protection and the enhancement of their stress tolerance could also be possible, thereby increasing the security of food supply.

3. Harpins in plant protection

Chemical pesticides can cause long-term and severe environmental effects. Therefore, efforts are being made to replace them with environmentally friendly biopesticides. One promising avenue for biopesticide development could be the use of the elicitors named harpin proteins. Harpins are acidic, heat-stable, cysteine-poor but glycine- and leucine-rich proteins produced by plant-pathogenic bacteria with a type III secretion system, such as Pseudomonas syringae and Erwinia amylovora [1, 2, 3]. When purified harpins are applied directly to plants at low concentrations (~100 nM) or expressed in plant cells, they induce the expression of immune response genes that make the plant more resistant to pathogens. In addition, other beneficial effects have been described in harpin-treated plants, such as higher yields and more intensive growth [4].

The exact cause of the beneficial effects of harpins is currently elusive. However, earlier results from our research group suggest that harpin treatment can induce biotic stress in Arabidopsis thaliana suspension cell cultures. This results in a so-called oxidative burst. In the course of this phenomenon the amount of reactive oxygen species (ROS) suddenly and significantly increases, which induces the antioxidant system of the cells. Our data indicated that the expression of VTC2 and 5 enzymes, which catalyze the rate-determining step of ascorbic acid biosynthesis, were increased. The expression and activity of L-galactono-1,4-lactone dehydrogenase (GLDH), that catalyzes the final step of the biosynthesis, were also increased. Presumably, as a result of the above, the ascorbic acid content of the harpin-treated cells was higher than that of non-treated cells. The activity of the enzymes of the ascorbate-glutathione cycle, which is a central element of the plant antioxidant system, is also enhanced [5]. Based on these, harpins can induce biotic stress in plants without an actual pathogen infection, which, among other things, induces the plant antioxidant system, thereby enhancing resistance to real pathogens.

4. The ferroptosis-like cell death

Harpin treatments at higher concentrations (>250 nM) induce hypersensitive response (HR) in plants [3, 4, 5]. HR in nature occurs when a plant is resistant to a particular pathogen (incompatible interactions). In this case, the immune system of the plant recognizes the pathogen and destroys its own attacked cells in a quick, programmed manner to protect the plant as a whole. In 2019, Dangol et al. reported that the HR during the incompatible reaction between rice (Oryza sativa) and the fungus Magnaporthe oryzae is characterized by lipid peroxidation and accumulation of ROS and ferric ions. However, treatment of the cells with the iron chelator deferoxamine or the lipophilic antioxidant ferrostatin-1 prevented the iron-dependent ROS accumulation and lipid peroxidation, leading to the complete attenuation of the HR cell death. On the base of these observations, Dangol et al. hypothesize that the so-called ferroptosis-like cell death is involved in incompatible plant-pathogen interactions [6].

Ferroptosis is a form of iron-dependent, caspase-independent programmed cell death in mammalian cells that was first described in 2012 [7]. Ferroptosis has unique morphological and biochemical features that differ from other forms of cell death, and its specific inducers (e.g. erastin and RSL3) and inhibitors (e.g. ferrostatin-1) have also been identified. The cell death process can be triggered by the depletion of cellular glutathione by erastin, or by the inhibition of the enzyme glutathione peroxidase 4 (GPX4), which plays a key role in the elimination of lipid peroxides. Regardless of the mode of induction, the initiated process is characterized by increased ROS production, lipid peroxidation and elevated cellular labile iron pool. Due to these properties, ferroptosis can be inhibited by lipophilic antioxidants (e.g. ferrostatin-1, liproxstatin-1, α-tocopherol) and iron chelators (e.g. deferoxamine). [8].

In 2017, Distéfano et al. described a cell death process in plants that is very similar to ferroptosis [9]. They treated Arabidopsis thaliana root hairs at 55 °C for 10 minutes and found that heat stress induced a decrease in glutathione levels, the shrinkage of mitochondria – a unique morphological marker of ferroptosis in mammalian cells – and cell death. The authors also showed that the rate of heat stress-induced cell death was significantly reduced in root hairs pretreated with the ferroptosis inhibitors ferrostatin-1 or ciclopirox olamine (iron chelator). However, the same inhibitors could not prevent cell death caused by 77 °C, H2O2 treatment or salt stress. The inhibitors also did not affect reproductive or developmental cell death. Based on these findings, the authors conclude that cell death induced by moderate heat stress is a unique process in plants and have named it ferroptosis-like cell death based on its high similarity to ferroptosis.

However, it is important to point out that ferroptosis-like cell death, in contrast to the caspase-independent ferroptosis of mammalian cells, appears to be a caspase-like protease-dependent process (Figure 1) [9, 10, 11].

5. Outlook

As the average temperature of the Earth rises, heat stress and heat stress-induced ferroptosis-like cell death are expected to play an increasingly important role in the life and death of plants. By studying and better understanding this cell death process, we may be able to develop targeted defences against it and thus increase the heat stress tolerance of our crops.

One may wonder how thermotolerant desert plants protect themselves against ferroptosis-like cell death. For example, the leaves of the creosote bush (Larrea tridentata), which grows in the deserts of the USA and Mexico, contain high levels of nordihydroguaiaretic acid (NDGA), a pan-lipoxygenase inhibitor lignan. It has been described earlier that lipoxygenases play an important role in ferroptosis through the enzymatic catalysis of lipid peroxidation. Thus, lipoxygenase inhibitors are expected to have an inhibitory effect on the cell death process. The inhibitory effect of NDGA on ferroptosis has already been demonstrated in mammalian cells, but has not yet been tested in plants [13,14].

As mentioned above, ferroptosis-like cell death is also involved in the HR during some pathogen attacks. Thus, a better understanding of ferroptosis-like cell death may be highly important for developing more effective biopesticides, hereby our research may help in the defence against both abiotic and biotic stresses.

6. Acknowledgement

This research was funded by the National Research, Development, and Innovation Fund of Hungary under Grants K 123752, 2018-1.2.1-NKP-2018-00005, RRF-2.3.1-21-2022-00015, and TKP2021-EGA-02.

7. References

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[5] Czobor, Á.; Hajdinák, P.; Szarka, A. (2017) Rapid Ascorbate Response to Bacterial Elicitor Treatment in Arabidopsis Thaliana Cells. Acta Physiol. Plant. 39, (2), 62, DOI

[6] Dangol, S.; Chen, Y.; Hwang, B.K.; Jwa, N. (2019) Iron- and Reactive Oxygen Species-Dependent Ferroptotic Cell Death in Rice- Magnaporthe Oryzae Interactions. Plant Cell 31, (1), 189–209, DOI

[7] Dixon, S.J.; Lemberg, K.M.; Lamprecht, M.R.; Skouta, R.; Zaitsev, E.M.; Gleason, C.E.; Patel, D.N.; Bauer, A.J.; Cantley, A.M.; Yang, W.S.; et al. (2012) Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death. Cell 149, (5), 1060–1072, DOI

[8] Hirschhorn, T.; Stockwell, B.R. (2019) The Development of the Concept of Ferroptosis. Free Radic. Biol. Med. 133, 130–143, DOI

[9] Distéfano, A.M.; Martin, M.V.; Córdoba, J.P.; Bellido, A.M.; D’Ippólito, S.; Colman, S.L.; Soto, D.; Roldán, J.A.; Bartoli, C.G.; Zabaleta, E.J.; et al. (2017) Heat Stress Induces Ferroptosis-like Cell Death in Plants. J. Cell Biol. 216, (2), 463–476, DOI

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[14] Galluzzi, L.; Vitale, I.; Aaronson, S.A.; Abrams, J.M.; Adam, D.; Agostinis, P.; Alnemri, E.S.; Altucci, L.; Amelio, I.; Andrews, D.W.; et al. (2018) Molecular Mechanisms of Cell Death: Recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 25, (3), 486–541, DOI

DOI

Received: October 2022 – Accepted: November 2022

¹ Ministry of Agriculture, Department of Food Economics and Quality Policy

Keywords

food industry, Hungarian Food Guide, digital food strategy, regulation, origin, Hungarian product, FOPNL, imitations, GHP

2. Brief history of the food industry

2.1. A brief history of the food industry up to privatisation

Industrial food production is worth talking about from the period after the reconciliation (1867). In general, industry, including the food industry, underwent significant development in the second half of the 19th century. At the beginning of the 20th century (1913), the food industry employed 15 per cent of the total workforce, while its contribution to the value of production was close to 40 per cent. The sugar industry and the milling industry were given a boost, and, combined with the also prospering machinery industry, these sectors were characterised by world-class technology in Hungary. The changes up to the First World War were marked by the emergence of factories built with foreign capital and the development of a food industry linked to large estates. The further development of the sector was strongly influenced (some of whose effects are still felt today) by the development of infrastructure (Budapest is the centre of the sector), the presence of foreign capital, and the modernisation of domestic trade and credit.

After the First World War, a dual structural development started, with almost 80 per cent of production being carried out to European standards using large-scale industrial methods, and the rest in technically highly heterogeneous small-scale plants. The export orientation of large firms was already evident, while small firms concentrated primarily on local supply. The food industry at the time was characterised by a desire for an optimum combination of different production processes, for example, a distillery processing by-products were added to a canning plant, and the steam from sugar factories was used not only for concentrating sugar beet juice but also for producing tomato concentrate. By the end of the 1930s, in addition to the traditional Hungarian export product (wine), there were already well-known and respected brands such as Hertz, Pick, Zwack Unicum and Arany Fácán - also good exports, especially to Western European markets.

The agrarian reform of 1945 fundamentally broke down the large landed estate system, creating an agrarian structure that limited the realisation of modern production. However, unity of land ownership and land use was achieved. The attempted Soviet model forced collectivisation in the 1950s which was not successful, and a domestic version was developed. The 1960s saw the gradual emergence of the Hungarian agricultural model, with the modernisation of agriculture and expansion of production.

The food industry, which evolved between 1945 and 1968, was characterised by fundamental peculiarities:

• Recovered from the war damage, production exceeded the pre-war level already in 1949, and by 1960 it had doubled: in 1959 the food industry’s production value totalled HUF 19.6 billion, accounting for about 22 per cent of the total industrial production. In 1959, the canned fruit industry produced 53 578 t of canned fruit, the meat industry 179 834 t of raw meat and 35 246 t of fat, the poultry industry 21 452 t of poultry carcasses and 432 million eggs, the dairy industry 2 512 000 hl of drinking milk, 16 531 t of butter, 13 732 t of cheese and the sugar industry 320 890 t of sugar;
• Private ownership was abolished and state ownership dominated - large socialist factories were set up, producing partly semi-finished foodstuffs and partly food preparations;
• Autonomy of enterprise management was abolished and planned management was introduced;
• The volume of production increased;
• New technologies were created, giving rise to new specialised sectors;
• A scientific base for product and production development in the food industry was established in the form of a network of research institutes organised by sector.

The period 1969-1991 was characterised by the following developments in the Hungarian food industry:

• The production quality of Hungarian agriculture has increased significantly;
• The Soviet Union was facing a persistent food shortage, so it took the initiative to specialise production, i.e. through bilateral interstate institutions a permanent market was provided for Hungarian products;
• The economic objective was to improve living standards and increase domestic food consumption;
• A change in the system of economic governance, which increased the autonomy of enterprises, although the central budget also diverted and redistributed the results;
• Until the mid-1980s, the volume of food production increased steadily, with a surplus of 35-40 per cent produced for export (with state subsidies). The continuous growth of the food industry until 1980 was not economically sound, and the lack of financial basis for development was accompanied by a lack of quality development. The possibilities for the extensive development of the Hungarian food industry were exhausted by the end of the 1980s.

2.2. From the privatisation of the food industry

The economic and social changes of the late 1980s and early 1990s shook the food industry to its foundations. Production levels fell by 10-15 years. The main problem was that many companies had not been exposed to market developments and the need to adapt to them. The main problems arose from the illusion of inefficient, quantity-centred production and seemingly secure markets due to the requirements of planned management.

The privatisation process involved both professional and financial investors. Since the profitability of the Hungarian food industry was low, it was mainly professional investors who were interested. In those sectors where there was a secure domestic market, simple production technology or easy to automate the operations, or a secure supply of raw materials, it was easy to privatise, while there were few investors in, for example, the canning industry, which was based on the Soviet market, or the already highly competitive baking industry. Privatisation has completely transformed the ownership structure of the food industry, with state assets being completely reduced and replaced by foreign capital. The number of people employed began to fall significantly because:

• The newly created companies were no longer bound by the employment obligation;
• In some sectors, the volume of production has fallen substantially, and with it the demand for labour;
• There was also less demand for manual labour in automated sectors (confectionery, beer, sugar, oil, tobacco);
• Some activities (maintenance, catering, security, etc.) were “outsourced”.

Following the privatisation period, the food industry has steadily declined in importance, with output as a share of the national economy as a whole falling from 12.5 per cent in 1992 to less than 9 per cent in 2003. A similar trend can be observed in the size of the turnover of products sold by the food industry. The number of people employed in the food industry has also been steadily declining, from employing nearly 9 per cent of the national economy in the early 1990s to 5.8 per cent in 2003. The largest employing sectors were baking, meat processing, poultry processing and dairy products.

The period under review was characterised by a fragmentation of food businesses, with a steady decline in the number of farms and a parallel steady increase in the number of holdings. The reasons for this were the disappearance and restructuring of the ‘socialist large enterprises’, the adaptation of the newly created enterprises to market conditions and the pursuit of economies of scale.

The performance, mechanisation and export orientation of the food industry is determined by the ownership of the firms concerned. The dominant components are foreign capital, state ownership and the ownership of domestic joint ventures and their development. Depending on the year, these three factors accounted for 80-90 per cent of the registered capital. State ownership has declined from nearly 45 per cent to a few per cent and is now almost negligible. In contrast, the share of foreign capital and domestic partnerships has been steadily increasing. In 1992, foreign capital accounted for 31.7 per cent of registered capital, in 1995 it exceeded 50 per cent, and in 1997 it was as high as 60 per cent. Then the decline started, and was particularly steep after 2002 [1].

2.3. The current situation

The food industry is a highly populated and heterogeneous sector, with more than 4,000 enterprises producing in 33 different sectors. The sector is characterised by a dual structure dating back to the 1800s, with a mix of micro and small companies, which are involved in local supply and filling niche markets, and medium and large companies, which are able to produce homogeneous and large quantities of products, which can be sold in retail chains and exported.

The number of companies and employment in the sector has been steadily declining in recent years, while efficiency has been increasing, with total sector turnover exceeding HUF 4,000 billion and food industry profits increasing several-fold. The sector’s products play an important role, with domestic consumers spending 30 per cent of their income on food products and foreign markets are also important, with more than a third of the sector’s revenue coming from export sales. The positive shift in the sector’s performance and results is a promising trend, but it has mainly been able to grow relative to itself, lagging behind other manufacturing sectors and the international food industry.

For an industry to be successful, it needs to be able to compete in both domestic and international markets. This requires knowledge of the specificities of the area and targeted interventions and development. The food industry, despite being part of the manufacturing sector, has very different characteristics from other sectors. As an integral part of the agricultural sector, it is difficult to understand it in isolation and it is therefore worth analysing and studying the whole food chain.

It has a close symbiotic relationship with agriculture through its demand for raw materials and is also influenced by the performance of other industries (packaging, additives, etc.). The sector is confronted with both domestic and international expectations. For example, in Hungary, there is a clear demand from consumers for a wide range of quality products, preferably domestic, which is also supported by the need for operators to generate higher added value and improve operational efficiency - which also encourages exportability. At the international level, it is the EU expectations, currently mainly the evolving elements of the Farm to Fork Strategy-F2F, which will influence the competitiveness and international opportunities of the industry in the long term. In addition, the regulatory and funding environment, the results of science education and the presence of public authorities are also driving activity horizontally along the whole chain [2].

2.4. International context – F2F

On 20 May 2020, the Commission’s Communication on a Farm to Fork (F2F) strategy to guide the transition to a sustainable food system was published as part of the European Green Deal. The European Green Deal and the “Farm to Fork” strategy address many dimensions of the food system, from animal husbandry and crop production to food labelling and international trade.

Aim of the F2F strategy is to make food systems more sustainable by reducing negative impacts on the environment.

In order to enable consumers to make responsible choices for ‘healthy’ and sustainable food, the Commission has set the objective of harmonised mandatory introduction of Front of Pack Nutritional Labelling (FOPNL) and proposes to extend the origin labelling requirements, taking full account of the impact on the single market.

To improve the EU food environment and promote the shift to a “healthier” diet, the Commission will also continue to develop nutrient profiles that limit the promotion of foods high in fat, sugar and salt through nutrition or health claims.

In order to achieve the objectives set out in the strategy, appropriate instruments, support and regulation are needed to avoid competitive disadvantages [3].

2.5. Situation in Hungary

The ever-increasing efficiency and effectiveness of the food industry is an ongoing trend. Not only in nominal terms, but also in real terms.

Even with the difficulties and constraints experienced during the first waves of COVID, the sector managed to grow - due to export sales Table 1. Around one fifth of the companies are engaged in export activities, typically medium and large companies, which is why 80 per cent of employment and more than 90 per cent of total sector turnover is attributable to these firms. A secure export base is generated by those sectors where processing can be based on high-quality domestic raw materials. These are the ones that have also contributed significantly to the value of production: meat, dairy and fruit and vegetable processing are the most affected.

However, different sectors have different feedstock situations, income levels, ownership structures and performances:

• Food industry earnings have grown dynamically in recent years, but are still below the national average. Within the industry, there is considerable variation: there are some sectors where the average salary is around HUF 200 000 gross and others (mainly in highly mechanised areas) where it exceeds HUF 600 000 gross per month (Figure 1);

• The manufacturing sector includes nearly 25 thousand enterprises, of which nearly 4 thousand are in the food industry. As in previous years, micro and small enterprises dominate in terms of numbers, together accounting for more than 90 per cent of enterprises, while medium and large enterprises are the most important in terms of the sector’s operation. These two groups account for less than 10 per cent of total turnover (although their share is increasing as the number of micro-enterprises declines), but they still account for more than 80 per cent of total domestic turnover and more than 90 per cent of export turnover in 2020, and therefore the vast majority of food industry profits. They provide employment for more than 70 per cent of the sector’s workers and concentrate nearly 80 per cent of the sector’s wealth;
• The presence of foreign capital is important in the sector, although 91 per cent of enterprises are 100 per cent domestically owned. Foreign capital is prevalent in medium and large enterprises, so these capacities should be taken into account for exports and for the production of homogeneous and large quantities of commodities. Given the different corporate cultures, the direction and characteristics of sales, and the propensity to innovate, these should be taken into account when designing regulations and support schemes.

Tools

3.1. Support for the food industry

Support for the food industry is complex. For the period 2014-2020, which closed at the end of 2020, the sector benefited from both operational programmes and domestic funding. Businesses have different opportunities depending on their size (e.g. EU funding for large companies is minimal and limited) and the type of product they produce (annex, non-annex products).

3.1.1. Nationally funded grants 2014-2020:

• The Large Business Investment Support (LBIS) programme, coordinated by the Ministry of Finance, was created to support investments by large domestic companies with a capital shortage and by small and medium-sized enterprises of the size of large companies that are likely to be affected by the planned investment and which make a significant contribution to the growth and modernisation of the Hungarian economy, including the manufacturing and construction industries. The scheme was launched in 2015 and has been active in the food sector every year;
• The Investment Promotion Earmarking (IPE), operated by the Ministry of Foreign Affairs and Trade (MFAT), aims to support projects that improve the competitiveness of the Hungarian economy and attract working capital to create jobs;
• To offset the economic impact of the coronavirus epidemic:
  • As part of the Economic Protection Action Plan, the National Food Economy Crisis Management Programme (NFECMP), which was developed by the Ministry of Agriculture as part of the HUF 8 billion budget, 1457 food processing enterprises received nearly HUF 6.8 billion in aid;
  • The Competitiveness Enhancement Support Programme (CESP), announced by the MFAT and implemented by National Investment Agency Nonprofit Ltd. of the HIPA.

3.1.2. Operational programmes grants:

• Under the Economic Development and Innovation Operational Programme (EDIOP), food companies could apply for a number of grants, such as for technological innovation, employment promotion, efficiency gains, higher value-added products, capacity expansion or R&D;
• The Rural Development Programme (RDP) is open to micro and small enterprises other than farmers producing annex products and to farmers. Dedicated calls for proposals have helped operators in the sector to access resources;
• Almost three quarters of the aid comes from RDP and EDIOP sources, but the aid values for the large business sector (LBIS), which is only moderately eligible for EU funds, and the export promotion scheme (IPE), which typically targets medium-sized and large companies, are also relevant for the sector.

Double target of the 2021-2027 resource planning:

• Recapture domestic markets;
• Enhancing export capacity.

It also comes with a financial resource.

In the 2014-2020 funding period, the sector has been granted more than HUF 468 billion in national and EU funding. This was an outstanding amount compared to previous years and could also help the sector to put viable businesses on a growth path. The current (2021-2027) funding period plans to provide even more resources, with HUF 750 billion from the Rural Development Program alone earmarked for food sector development. After several rounds of consultation with industry players and stakeholders, the following intervention points have emerged for the planning of development directions:

• More efficient and profitable production structures can be created through the preference for robotisation and modernisation, and the domestic production of higher value-added products can change the structure of export-import favourably;
• Industrial development makes sense if there is consumer demand for the products produced. To achieve this, it is important that consumers are well informed and aware, but this requires awareness campaigns or brands to guide their choices;
• In addition, it is essential to increase resource efficiency, reduce the environmental burden, and include labour and skills in the preferences.

Unlike in previous support periods, for 2021-2027, support for the food industry will be concentrated in one place and coordinated. Due to a high share of national funding, two dedicated funds for the sector were opened in 2021, a small-scale capital equipment purchase and a HUF 320 billion fund for complex developments, with up to HUF 5 billion in non-repayable grants. The feed sector was excluded from previous calls, but as it is part of the food industry and is a sub-sector typically based on domestic raw materials with a significant export value, a separate call was made for it.

In addition to these, there are also calls for farmers and the food industry, which provide opportunities for development through a specific sub-area. This is the case, for example, for calls relating to quality schemes. Products produced under quality schemes are of a higher quality than other similar products that meet the minimum requirements of the legislation. The aim of the quality schemes is to help ensure that products produced under quality schemes recognised by the European Union and Hungary are known and recognised by consumers and can be marketed more effectively by producers. In addition to the products associated with the protected designations, there is currently one quality scheme in Hungary, the High Quality Food Trademark Scheme, which is nationally recognised and notified by the EU.

As with the two calls already launched in 2021, we plan to repeat these in 2023 and 2025, possibly adapting the calls in the light of experience gained. The aim is to produce higher value-added products, have more profitable and efficient food companies, increase exportability and reduce imports.

3.2. Recent issues

3.2.1. Codex Alimentarius Hungaricus

Our main aim in developing the industry is to ensure high-quality food, while at the same time reducing the market for poor, low-quality food. The Codex Alimentarius Hungaricus is a reliable source of information for consumer awareness and responsibility. One of its main aims is to provide guidance, both to producers and to consumers, but this is also an effective way of ensuring the smooth flow of international trade, fair competition in the market and the enforcement of national specificities. Therefore, our priority is to continuously review and amend the regulations and directives of the Codex Alimentarius Hungaricus.

On the basis of the previous practical experience of the Codex Alimentarius Hungaricus Committee and its Committees and the suggestions of the food industry, it was justified to change the composition of the Board in such a way that food businesses are better represented, taking into account the operational efficiency of the Board. As a result, the functioning and composition of the Codex Alimentarius Hungaricus Committee and its Committees have been changed, making the work more transparent and streamlined, and giving greater weight to the presence of industry. From a professional point of view, the new regulations have been developed with a focus on high quality.

The 2010s brought significant changes in the regulation of meat products and bakery products, this year brought the renewal of the dairy regulation, but further changes are underway for meat products and a comprehensive revision for quick-frozen fruit and vegetables.

3.2.2. Digital Strategy for Food

The food industry is the first largest sector in terms of production value in Europe and the third largest in Hungary. It also plays a major role in employment and it is a significant user of agricultural resources. Therefore, increasing productivity and efficiency in the food industry is of utmost importance, and Industry 4.0 and the application of digitalisation offer many new opportunities. This will lead to new products, new services, new solutions and approaches through more efficient use of resources, improved quality parameters, and new skills and competencies. All this could even improve the international competitiveness of domestically produced food.

In 2019, the Digital Food Strategy was launched, with inter-ministerial collaboration, academic and other scientific workshops, stakeholders and professional organisations. A general assessment of the current digital situation in the sector has been carried out and intervention points and lines of action have been identified. Their impact will be measured in the coming years, adopted in November 2022.

3.3. Other issues (research, horizontal regulations, sub-areas)

3.3.1. Regulation, origin, Hungarian product

Food labelling is a complex area that includes, in addition to the information on food packaging, the advertising, packaging, presentation and display of food products. The role of food labelling is to provide the consumer with objective, accurate and truthful information about the product.

The issue of origin labelling is becoming increasingly prominent, as shown by the increasing number of laws on mandatory and voluntary origin labelling in the EU harmonised food legislation and in the Member States’ regulations over the last 20 years.

For a number of foods (e.g. beef, veal, fish, fruit and vegetables, honey, olive oil, etc.) origin labelling is mandatory, while in other cases origin labelling is mandatory where failure to indicate it would mislead consumers or where the country of origin of the food is indicated but the origin of the primary ingredient is different from this information. The determination of origin for food labelling purposes is done through product-specific rules or, in the absence thereof, horizontal rules.

The rules on the origin labelling of foodstuffs are in some cases closely intertwined with those laid down in the EU Customs Code [4].

In Hungary, voluntary labelling legistlation is in force as defined in the Vm Decree No.74/2012 (25.VII.) of the Rural Development on the use of certain voluntary (hereinafter: MRD Decree), which defines the terms “Hungarian product”, “domestic product” and “product processed domestically” and the related requirements, but does not impose the obligation to use a trademark or logo.

These three categories provide the consumer with information on whether the product originates exclusively or partially in Hungary or whether it has been processed in Hungary. The MRD Decree also regulates claims referring to the above-average quality or specific quality characteristics of a product [5].

National and international research shows that origin information is one of the most sought-after labelling elements by consumers. According to a survey of the Hungarian population conducted by the National Food Chain Safety Office, 82.12% of respondents pay attention to the origin of Hungarian food products, 56.32% of whom take the origin of the product into account before making a decision, 25.80% of whom only buy certain products, and 17.88% of the adult population do not care about the origin of the product [6].

3.3.2. Front of Pack Nutritional Labelling - FOPNL

According to Regulation (EU) No 1169/2011 of the European Parlaiment and of the Council on the provision of food information to consumers (hereinafter: Labelling Regulation), nutrition labelling (energy, fat, saturates, carbohydrates, sugars, protein, salt) is generally mandatory for the vast majority of pre-packaged foods from 13.12.2016, in order to enable consumers to make an informed and health-conscious choice. The mandatory nutrition labelling is usually on the back of the food packaging. Under the Labelling Regulation, certain elements of the nutrition labelling may be repeated in the principal field of vision in one of two ways: a.) energy or b.) energy and fat, saturated fatty acid, sugar, and salt.

The Labelling Regulation allows the use of graphical forms and symbols in addition to the mandatory presentation. The main purpose of such schemes is to help consumers towards a healthier diet [7, 8].

The voluntary FOPNL on the front of packs is not harmonised at the EU level, so there are several such labelling schemes on the EU market [8], with different forms of presentation and different purposes, and therefore not comparable, as they are based on completely different principles.

Nowadays, FOPNL nutrition labelling has been the subject of continuous and constant interest at the national, EU and global level. As nutritional science evolves year by year, there is a need for legislation to adapt to these changes.

The FOPNL nutrition labelling is a complex and sensitive area, so it is important to see how the systems used in different Member States (e.g. in order to achieve these objectives, a study by the Food Economics and Quality Policy Department of the Ministry of Agriculture, using 800 products within 8 product categories, has examined the potential impact of the introduction of FOP schemes in EU Member States on the domestic food industry and on typical domestic products.

The survey showed that the NutrInform Battery can be considered balanced and objective for Hungarian products, as it provides consumers with a numerical indication of how much energy, fat, saturated fat, sugar and salt a serving of a product provides compared to the daily reference intake. Keyhole would not be strongly discriminatory, given that it only uses positive discrimination. There is a significant risk of introducing Nutri-Score, given the negative discrimination caused by the colours and categories. It classifies a number of traditional Hungarian products in categories E, D, C, including those with a single portion of less than 100 g as a basis for assessment.

3.3.3. Food Substitutions

The Food Economy and Quality Policy Department of the Ministry of Agriculture has carried out a representative consumer survey to examine whether consumers can clearly distinguish between the original and substitute products in the product range of milk, cream, yoghurt, butter, milk powder, cheese, stuffed meat products and meat patties and whether the current legislation regulates imitations adequately or whether amendments are needed. The research has shown that in some product categories there is an insufficient distinction between traditional and imitation products, and that the appearance and naming of products can often be misleading. Some products may have product names that are not in line with legislation and regulations and may use inappropriate graphic elements.

3.3.4. GHP- Good Hygiene Practice

Good practice guides, which food business operators use on a voluntary basis, play an important role in EU food safety legislation. Microbiological rules have changed frequently in recent years. The individual industry guides deal with microbiological requirements in broad general terms, but there is a need for a detailed summary of the subject, with a common set of requirements for microbial testing and subsequent procedures, and therefore a guide for food businesses has been developed.

4. References

[1] Síki J., Tóth-Zsiga I. (1997): A magyar élelmiszeripar története, MezőGazda Kiadó, Budapest

[2] Kapronczai I., Bojtárné Lukácsik M., Felkai B. O., Gáborné Boldog V., Székelyné Raál É., Tóth P., Vágó Sz. (2009): Az élelmiszerfeldolgozó kis- és középvállalkozások helyzete, nemzetgazdasági és regionális szerepe. AGRÁRGAZDASÁGI TANULMÁNYOK, 2009: (9) pp. 129

[3] Farm to Fork Strategy. Hozzáférés/Acquired: 18.07.2022

[4] Kuti B., Fehér O., Szakos D., Kasza Gy. (2022): Country of origin and place of provenance related food labelling regulation in the European Union (Élelmiszerek származási országának és eredethelyének jelölési szabályozása az Európai Unióban) Magyar Állatorvosok Lapja 144: (1) pp. 45-58

[5] 74/2012. (VII. 25.) VM rendelet egyes önkéntes megkülönböztető megjelölések élelmiszereken történő használatáról Hozzáférés/Acquired: 12.07.2022

[6] Szegedyné Fricz Á., Dömölki, M., Kuti, B., Izsó, T., Szakos, D., Bognár, L., Kasza, Gy., (2016): Minőségi magyar termékek nyomában – a Magyar Élelmiszerkönyv működése (Searching for quality Hungarian products-the operation of the Hungarian Food Codex). Élelmiszer Vizsgálati Közlemények 62:(4) pp. 1338-1351.

[7] Az Európai Parlament és a Tanács 1169/2011/EU rendelete a fogyasztók élelmiszerekkel kapcsolatos tájékoztatásáról. Hozzáférés/Acquired: 17.07.2022

[8] European Commission, Nutrition labelling. Hozzáférés/Acquired: 17.07.2022

DOI

Received: November 2022 – Accepted: December 2022

¹ University of Szeged, Faculty of Engineering, Institute of Food Engineering

Keywords

plant-based meat alternatives, meat analogues, terminology, imitation meat

2. Introduction

Plant-based diets are becoming increasingly important for both health-conscious and environmentally conscious consumers and are even contributing to a sustainable food supply chain.

In the last few years, plant-based foods and prepared meals, which are traditionally known as meat-based dishes, have been increasingly available in increasing quantities and variety on the international and domestic markets. This is one of the biggest food innovations of our time and one of the most popular trends.

Burger alternatives and other new innovative products, alternative meat substitutes (so-called ‘plant-based meat alternatives’) such as ‘vegan burgers’, ‘soy steaks’, ‘tofu sausages’ and the like are emerging.

The consumption of plant-based foods is growing at an unprecedented rate [1]. Such foods are increasingly available to vegetarians and even more strict vegans, as well as to flexitarians who occasionally choose a meat-free diet.

After the first plant-based meat analogue, the ImpossibleTM Burger [2] and Beyond Meat [3], a succession of plant-based foods mimicking the colour, texture and taste of meat and meat products have appeared commercially and in fast food chains.

Flexitarians have become the largest dietary group after omnivores (carnivores) and have a major role to play in effectively reducing the consumption of meat and other animal products and thus in combating climate change [4].

Taking into account all those who actively reduce or completely abandon at least some animal products, including vegetarians, pescatarians and flexitarians, this group represents 30.8% of the total population: 10-30% of Europeans no longer consider themselves to be fully carnivorous.

Like the terms, ‘vegetarian’ and ‘flexitarian’, the terms ‘plant-based diet’ have no official definition in the EU. Nor has the meaning of the term ‘plant-based meat alternatives’, which is most commonly used in the English literature and press, been defined. On the one hand, there has been a heated debate in the European Union about the designation of meat substitutes. Questions have arisen as to whether terms such as ‘vegetarian hamburger’ or ‘soya sausage’ are misleading to the consumer. The European Parliament has decided that ‘meat’ can be plant-based, but only milk of animal origin can be called milk. Plant-based products cannot be called milk, cream, butter, cheese or yoghurt because the legislation states that they can only be used to describe food of animal origin [5, 6].

On the other hand, there is no uniform terminology to describe this group of products, as the terms “meat analogues” and “meat substitutes” are used in international communication alongside “plant-based meat alternatives”. In addition, “plant-based meat substitutes” and “plant-based meat alternatives” have started to be translated into Hungarian as “meat imitates” and other terms.

Obviously, simple, understandable terms should be used for consumers, but how acceptable are these to the professional public?

3. Objective

As the content of the Hungarian terms occasionally used for the so-called “plant-based meat alternatives” is unclear and they are not clearly synonymous, and some of them have a pejorative connotation (e.g., “imitation”), it was deemed necessary to develop a professional consensus on the subject in order to provide objective information.

Our aim was to achieve a broader acceptance of the terms, which would be easily understood by consumers.

4. Method

A survey was conducted among Hungarian experts (food science and technology, nutrition academia and industry) on the acceptable Hungarian names for foods made from plant-based ingredients and appearing to be meat products.

The questionnaire was sent to experts in person at conferences organised by the Hungarian Nutrition Society and the BSE Platform in June and October 2022, and by direct mail between June and October 2022. Lecturers and researchers from three major universities in Hungary, as well as middle and senior managers of food processing companies and employees of official food control institutions participated in the questionnaire based on a personal interview.

The first question of the questionnaire was a spontaneous question, asking respondents to identify the name of a product based on a picture of a product that looked like a hamburger but was described as being made exclusively from vegetable ingredients (Figure 1). At this point in the face-to-face interview, the respondent could not yet see the possible names offered.

The second set of questions offered possible different descriptions for the same food shown in the picture. From these, the respondent had to choose

1. which one is “not appropriate”,
2. which is “acceptable” and
3. which was completely appropriate (“completely agree”).

The third set of questions generally offered different options for the general description of plant-based foods that appear to be meat/meat products. These were to be judged according to the same three categories. Although we have tried to list as many possible answers in Hungarian as possible, we have tried to keep the questionnaire short, quick to fill in and to the point. Thus, the substantive questions were only one page, followed by a few demographic questions. The questionnaire is presented in Table 1.

5. Demographic data

The questionnaire was completed by 58 respondents in the form of a personal interview. Even for the demographic questions, our questionnaire only covered the strictly necessary facts, such as the gender of the experts, their professional experience (0-5; 6-15; 16+ years) and the sector in which they work (food processing; academic sector, i.e., university or research institute; official control and others). We also asked whether the respondent was a vegetarian or not.

6. Results

6.1. Spontaneous product name

In response to the first spontaneous question on how the respondent would name the product made exclusively from vegetable ingredients shown in Figure 1, the vast majority of respondents suggested the term “veggie burger”. A total of 77 possible names were collected from 58 respondents, of which 37 different names could be distinguished. One person could not suggest a Hungarian name. Twenty respondents gave two different answers.

Of the 77 responses, 23 gave the name “vegan burger” (29.87%) and 6 gave the name “vegan burger”. So the terms “vegan burger” or “vegan burger” were used in a total of 37.66% of the 77 responses. Furthermore, 6 people chose the name “hamburger”.

Below is a list in alphabetical order of all possible names that were mentioned by only one respondent:

organic burger fake burger, sustainable, yummy.yummy green burger, burger substitute, burger-like product, herba sandwich, meatless burger, fake burger, fake burger, non-burger, plant-based food, plant-based burger, plant-based burger,

Some respondents used the following terms to emphasise the vegetable nature of the product:

plant burger, plant burger, plant buri, plant patty, plant based food, plant based burger, vegetable burger, vegetable meatloaf, plant based burger, plant patty, herba sandwich, plant burger, plantby/plantby, vegetarian sandwich, veggie hambi, vegan protein hambi, veggie burger.

Others have suggested a sustainable name for plant-based products: green burger, green buri, green patty, green burger, sustainable.

And a group of respondents highlighted the origin of the product from non-animal meat, not “real” meat: fake burger, fake burger, fake hamburger, meatless hamburger, hamburger substitute, hamburger-like product, not burger.

There was also a reference to the innovative nature of the product: trendy burger.

The terms ‘organic burger’, ‘yummy-yummy’ and ‘sandwich’ did not fit into any of these categories.

6.2. Preferred terms

Based on the same picture (Figure 1), we asked targeted questions about possible Hungarian names for the product group. Sixteen different names were given (Table 1). These included the terms slice, scone (as meat patty), burger and hamburger, which were varied with vegetable, vegetable and vegetarian. Each respondent was asked to decide whether, as an expert, they fully agreed with the term, whether the term was acceptable to them or not acceptable at all. We deliberately did not give the answer option “I don’t know (decide)”.

The term “meat pie” was unanimously (98.25%) rejected by respondents. The term “vegetable meatballs” was also not liked by the experts, being rejected by 73%. Two thirds of experts (66.66%) rejected the term “hamburger”. The terms “vegetable schnitzel”, “vegetable schnitzel” and “vegetarian schnitzel” were unanimously (60%) rejected.

The least objection was to the term “veggie burger”, which was rejected by only 10%. The terms “vegetarian burger” and “veggie burger” were judged in the same way by the experts, with 50% in full agreement and 36% finding them acceptable.

The experts’ opinions on all the terms studied are presented in Figure 2.

6.3. General description of the product group

Foodstuffs made exclusively from vegetable raw materials, which are intended to replace or substitute foodstuffs made from meat of animal origin and which also resemble meat products in appearance, are gaining ground.

For the next question, we wanted to know what terms could be used in general for meat substitutes (often referred to as plant-based meat alternatives or meat analogues).

More general terms such as “meat substitute”, “meat substitute”, “meat analogue”, “meat-like”, “alternative” or “meat imitation” were given.

While in the English literature the terms plant-based meat alternatives or plant-based meat analogues are most commonly used, Hungarian experts have rejected the names described as meat alternatives or meat analogues by mirror translation.

The term “plant-based meat” was rejected by 74% of the experts, and the terms “meat-like” (72.7%), “meat alternative” (59%) and “meat analogue” (52.7%) were not considered appropriate. The term “imitation meat” was considered inappropriate by half of the respondents (51%).

The lowest level of disagreement (16%) and the highest level of agreement (41%) was found for “meat substitute (food) of plant origin”. This was followed by the term “meat substitute (food)” with 35% support and 19.6% disagreement. In both cases the word “... substitute” was used. The term “meat substitute (food)” was considered acceptable by more than half of the respondents (50.8%). A summary of the results is shown in Figure 3.

So, based on the experts’ opinions and the results of our survey, we recommend the use of the terms “substitute” and “replacement”, as opposed to “kind”, “alternative” and “analogue”, which were generally rejected.

When completing our questionnaire, we also gave respondents the opportunity to suggest other generic terms, and 17 respondents took up this option. Some of the relevant suggestions are: “do not include the word meat at all”, “it is better to use the name of the raw material (e.g., mushroom) instead of ‘vegetable’ or ‘plant’” and “vegetable protein preparation”.

The adjective structure ‘vegetable’ is generally more acceptable than the adjective structure ‘vegetable’. For example, for both “veggie burger” vs “veggie burger” and “veggie hamburger” vs “veggie hamburger”, the “veggie” product name was more acceptable.

Taking the “completely agree” and “agreeable” responses together, the support for the terms “vegetable burger” (76%) and “vegetable burger” (89%) was higher in both cases compared to “vegetable burger” (61%) and “vegetable burger” (78%).

6.4. Presentation of demographic data

A significant number of respondents were women (68.4%).

When asked if the professional completing the questionnaire declared themselves to be a vegetarian, a low proportion of respondents answered yes, only 3.5%, which is below the number of vegetarians in the national population, estimated to be around 5%. Since the respondents belonged to a narrower segment, the difference between the percentage obtained and the expected percentage can be considered to be within the margin of error.

Based on the answers to the questions on professional experience, the majority have been in the field for more than 16 years and are therefore considered to be senior experts.

There were four possible answers based on the field of work experience and two options were possible, as it is realistic that experts may have additional activities or spent a significant part of their career in another field.

Almost one third of respondents (28.6%) work in the food processing sector.

Unsurprisingly for the authors, there is a predominance of respondents from the academic sector (57.9%), but it is important to stress that this includes universities, research institutes and all other publicly or privately funded research organisations. But, as mentioned above, there were several possible answers.

Only 8.8% of respondents work in the field of public control.

Thus, a total of 95.3% of respondents work in food processing, academia and official control. This means that almost five percent of respondents (4.7%) work in other fields. In our direct experience, this includes, for example, consultants and those working in the government sector.

7. Acknowledgements

We would also like to thank the food experts working in universities, research institutes, food industry and regulatory control organisations who helped us with our questionnaire by providing their opinions during the face-to-face interviews.

8. Annex

You are looking at a picture of a food made exclusively from plant ingredients

How would you name the product?

Answer:

„Which of the following names do you consider appropriate?”

“In general, which term do you consider appropriate to describe foods made exclusively from vegetable ingredients?”

Other, namely:

Demographic data (used for statistical analysis purposes only):

Gender:

Vegetarian

You age:

Professional experience in food science, technology or nutrition:

Employed/worked in academia:

Worked/worked in the field of official control:

Worked in other fields:

If you would like to comment in more detail on this topic or would like to receive the results, please provide your name and contact details.

For the research at the Institute of Food Engineering at the University of Szeged, please allow about 6-7 minutes. Your answers will be used anonymously.

You are looking at a picture of a food made exclusively from plant-based ingredients:

9. References

[1] ADM (2020): Top Five Global Trends that will Shape the Food Industry in 2021. Nutraceuticals Now. Wednesday, 28 October, 2020. Hozzáférés: 2022.10. 07.

[2] Y. Jin et al. (2018): Evaluating Potential Risks of Food Allergy and Toxicity of Soy Leghemoglobin Expressed in Pichia pastoris. Mol Nutr Food Res. 2018 Jan; 62(1): 1700297. Published online 2017 Oct 17. DOI

[3] Impossible (2020) Hozzáférés: 2022.10. 06.

[4] Beyond Buzz (2020) Hozzáférés: 2022.10. 07.

[5] Bánáti D. (2020): Flexitarianism – the sustainable food consumption? (Flexitariánus étrend – a fenntartható táplálkozás?) Journal of Food Investigation. Vol. 68, No. 3., (Élelmiszervizsgálati közlemények – 2022. LXVIII. évf. 3. szám) pp: 4058-4091 DOI

[6] Bánáti D. (2020): Veggie burgers, vegan meats? The ruling of the European Parliament paved the way for meat substitutes with meat denominations. (Vega hamburgerek, vegán húsok? Az Európai Parlament döntése a növényi alapú húspótló élelmiszerek elnevezéséről.) Journal of Food Investigation. Vol. 66. No. 4. / LXVI. évf. 4. szám, pp.: 3159-3174.

¹ Hungarian Standards Institution

Published national standards from July 2022 to November 2022

07.100.30 Food microbiology

MSZ EN ISO 4833-1:2013/A1:2022 Microbiology of the food chain. Horizontal method for the enumeration of microorganisms. Part 1: Colony count at 30 °C by the pour plate technique. Amendment 1: Clarification of scope (ISO 4833-1:2013/Amd 1:2022) – which is amendment of MSZ EN ISO 4833-1:2014 –

MSZ EN ISO 11133:2014/A2:2020 Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media. Amendment 2 (ISO 11133:2014/Amd 2:2020) – which is amendment of MSZ EN ISO 4833-1:2014 –

MSZ EN 15634-1:2020 Foodstuffs. Detection of food allergens by molecular biological methods.
Part 1: General considerations – which has withdrawn the MSZ EN 15933:2013 –

MSZ EN 15634-2:2020 Foodstuffs. Detection of food allergens by molecular biological methods.
Part 2: Celery (Apium graveolens). Detection of a specific DNA sequence in cooked sausages by real-time PCR

MSZ EN ISO 23418:2022 Microbiology of the food chain. Whole genome sequencing for typing and genomic characterization of bacteria. General requirements and guidance (ISO 23418:2022)

13.020.55 Biobased products

MSZ EN 17605:2022 Algae and algae products. Methods of sampling and analysis. Sample treatment

65 Agriculture

65.120 Animal feeding stuffs

MSZ EN 17504:2022 Animal feeding stuffs: Methods of sampling and analysis. Determination of gossypol in cotton seed and feeding stuff by LC-MS/MS

67 Food technology

67.050 General methods of tests and analysis for food products

MSZ EN 1787:2022 Foodstuff. Detection of irradiated foodstuff containing cellulose by ESR spectroscopy – which has withdrawn the MSZ EN 1787:2001–

MSZ EN 13708:2022 Foodstuffs. Detection of irradiated foodstuff containing crystalline sugar by ESR spectroscopy – which has withdrawn the MSZ EN 13708:2002 –

67.100 Milk and milk products

MSZ EN ISO 23319:2022 Cheese and processed cheese products, caseins and caseinates. Determination of fat content. Gravimetric method (ISO 23319:2022) – which has withdrawn the MSZ EN ISO 1735:2004 –

67.140 Tea. Coffee. Cocoa

MSZ ISO 11287:2022 Green tea. Definition and basic requirements

67.200 Edible oils and fats. Oilseeds

MSZ EN 14111:2022 Fat and oil derivatives. Fatty Acid Methyl Esters (FAME). Determination of iodine value – which has withdrawn the MSZ EN 14111:2004 –

For further information please contact Ms Anna Szalay, sector manager on food and agriculture, e-mail: a.szalay@mszt.hu