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LITHUANIAN UNIVERSITY OF AGRICULTURE          Ingrida Kraujutienė      AN INFLUENCE OF CULTIVAR AND GROWING TECHNOLOGY UPON CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITY OF SPROUTED WHEAT       Summary of doctoral dissertation Biomedical sciences, agronomy (06B)                 Akademija, 2007
Dissertation was prepared at Lithuanian University of Agriculture in the year 2003-2007.  Scientific advisor: assoc. prof. dr. Elvyra Jarienė University of Agriculture, biomedical sciences, (Lithuanian agronomy - 06B).  Consultant: assoc. prof. dr. Honorata Danilčenko (Lithuanian University of Agriculture, biomedical sciences, agronomy - 06B).  Dissertation is being defended at the Council of Agronomy of the Lithuanian University of Agriculture:  Chairman: prof. habil. dr. Rimantas Velička (Lithuanian University of Agriculture, biomedical sciences, agronomy - 06B).  Members: prof. habil. dr. Pavelas Duchovskis (Lithuanian Institute of Horticulture, biomedical sciences, agronomy - 06B); assoc. prof. dr. Vytautas Liakas (Lithuanian University of Agriculture, biomedical sciences, agronomy - 06B); habil. dr. Leonida Novickienė(Institute of Botany, biomedical sciences, botany - 04B); prof. habil. dr. Algirdas Sliesaravičius (Lithuanian University of Agriculture, biomedical sciences, agronomy - 06B).  Official opponents: assoc. prof. dr. Simas Glioeris (Lithuanian University of Agriculture, biomedical sciences, agronomy - 06B); dr. Pranas Vikelis (Lithuanian Institute of Horticulture, biomedical sciences, agronomy -06B.  Defence of dissertation will take place at the public meeting of the Council of Agronomy on 30that 11:00 a. m. in room No. 261, Central buildingof November, 2007, of the Lithuanian University of Agriculture   Address: Lithuanian University of Agriculture Studentu str. 11, LT - 53361 Akademija, Kaunas district, Lithuania Tel. (8 - 37) 75 22 54, fax. (8 - 37) 39 75 00, e - mail:  Summary of doctoral dissertation was distributed on 30thof October, 2007. Doctoral dissertation is available in the library of the Lithuanian University of Agriculture and Lithuanian Institute of Agriculture.
Daktaro disertacijos santrauka Biomedicinos mokslai, agronomija (06B)    
         Akademija, 2007
Disertacija rengta 2003  2007 metais Lietuvos emėsūkio universitete.  Mokslinėvadovė: doc. dr. Elvyra Jarienė em (Lietuvosėsūkio universitetas, biomedicinos mokslai, agronomija  06B).  Mokslinė snokatlutnė: doc. dr. Honorata Danilčenko (Lietuvos emėsūkio universitetas, biomedicinos mokslai, agronomija  06B).  Disertacija ginama Lietuvos emėsūkio universiteto Agronomijos mokslo krypties taryboje:  Pirmininkas: prof. habil. dr. Rimantas Velička (Lietuvos emėsūkio universitetas, biomedicinos mokslai, agronomija  06B).  Nariai: prof. habil. dr. Pavelas Duchovskis (Lietuvos sodininkystės ir darininkystės institutas, biomedicinos mokslai, agronomija  06B); doc. dr. Vytautas Liakas (Lietuvos emėsūkio universitetas, biomedicinos mokslai, agronomija  06B); habil. dr. Leonida Novickienėinstitutas, biomedicinos mokslai, botanika  04B);(Botanikos prof. habil. dr. Algirdas Sliesaravičius (Lietuvos emėsūkio universitetas, biomedicinos mokslai, agronomija  06B). . Oficialieji oponentai: doc. dr. Simas Glioeris (Lietuvos emėsūkio universitetas, biomedicinos mokslai, agronomija  06B); dr. Pranas Vikelis (Lietuvos sodininkystės ir darininkystės institutas, biomedicinos mokslai, agronomija  06B)   Disertacija bus ginama vieame Agronomijos mokslo krypties tarybos posėdyje 2007 m. lapkričio 30 d. 11 val. Lietuvos emėsūkio universiteto centrinių rūmų 261 auditorijoje.  Adresas: Lietuvos emėsūkio universitetas Studentųg. 11, LT  53361 Akademija, Kauno raj., Lietuva Tel. (8  37) 75 22 54, faks. (8  37) 39 75 00, el. patas  Disertacijos santrauka isiuntinėta 2007 spalio 30 dieną. Disertaciją galima periūrėti Lietuvos emėsūkio universiteto ir Lietuvos emdirbystės instituto bibliotekose.   
INTRODUCTION  At present germination is more and more widely being used not only for improve-ment of nutritional grain quality but also as a raw material for healthy food production (Lo-renz, 1980; Price, 1988; Lintschinger et al., 1997; Yang et al., 2001). The amount of dry substances and starch in germinated grain reduces. However, the amounts of amino acids compositions, polyunsaturated fatty acids, B group vitamins, sugar increases and the content of anti nutritional substances reduces (Finney, 1978; Chavan, Ka-dam, 1989). Germinated grain contains more essential amino acids (lysine, methionine etc.), which take part in protein production in a human body (Tkachuk, 1979; Hartmuth-Hoene, 1988; Jahn-Deesbach, Schipper, 1991; Schöne et al., 1997). Besides, dietary fiber in grain bran is not lost (Seibold, 1990). There has been established that the longer is the period of germination, the higher amount of vitamin C, beta carotene and other antioxidants (Hart-muth-Hoene et al., 1987; Heinonen et al., 1989; Cole et al., 1983; Yang et al., 2001). The content of bioactive substances - ferments significantly increases in germinated grain (Lo-renz, 1980; Edwards et al., 1989; Kolodziejczyk, Michniewicz, 2004). It has been measured that germination ferments stimulate the processes of anti-oxidation biosynthesis (Maillard, Berset, 1995). Nowadays healthy food becomes more and more popular. Firstly, it must not be con-taminated with harmful for human body chemical substances. Organical farming is one of the solutions to reduce damaging anthropogenic influence to biota and produce free of chemicals raw material. The research results of many scientists proved that organical prod-ucts contain significantly less amounts of nitrates (Woese et al., 1997; Alföldi et al., 2001; Worthington, 2001), pesticides (Baker et al., 2002) and heavy metals (Kurfürst, Beck, 1995). However, the mineral substances in organically grown grain increase slightly (Huber, 2003; Worthington, 1998) and the amount of proteins reduces (Starling, Richards, 1990; Eltun, 1996). Accordingly, one of the methods to improve the quality of organically grown wheat could be germination. Further, scientific literature provides very poor research data of or-ganically grown germinated wheat nutritional qualities. Thus, it is inevitably important to define the alternation of nutritional qualities in organically grown wheat during germination. Aim of research.To evaluate a quality of germinated wheat grain and apply them to enrich fermented milk products. Tasks of research: 1. of different growing technologies (organical and conven-To estimate an influence tional), separate cultivars of wheat ('irvinta 1' and 'Zentos') and germinating time upon wheat grain nutritive composition, activity of enzymes, quantity of amino acids and vita-mins, value of energy and mycological pollution. 2. To enrich fermented milk products with germinated wheat grain (yogurt and curd) estimating microbiological pollution and ensuring the security of new product. Scientific novelty of the work.There is the first time when there have been com-pared an activity of enzymes such asα- andβ-amylase, maltase, xylanase, cellulase, catalase and protease, the quantity of vitamins C and E and alterable and inalterable amino acids and the value of energy of separate wheat grain cultivars 'irvinta 1' and 'Zentos' grown up fol-lowing separate technologies and thereafter sprouted. Germinated wheat grain have been used for producing new fermented milk products when biochemical composition and microbiological pollution have been investigated.   
 Practical importance of the work.Germinated wheat 'irvinta 1' grain have been used to enrich such milk products as yogurt and curd when nutritive value and biochemical composition have been researched. There have been evaluated quality of sprouted grain, yo-gurt, curd and their compounds. After one day there have been investigated mycological pollution, activity of yeast and coliform bacteria germ and active acidity. There was task to ensure a partial security of fermented milk products by using effective and conditionally harmless for human being preservatives such as sorbic acid and calcium propionate. The re-sults are valuable to expand an assortment of fermented milk products, to improve their quality and security and for innovation aims at recast industry. Approbation of work rezults. Research results presented at the scientific conferences: 1.KraujutienėI., Danilčenko H., JarienėE., Tarasevičienė. The influence of legume like additives for wheat bread quality // International conference "Safe and quality plant food-stuff at EU context", Kaunas. 2003. 2.Kraujutienė I., Korduienė Savickien S.,ėSvirskis A. Alternative plant additives in  J., bread // International scientific - pratical conference Innovation development trends of food products, Jelgava. 2004. 3. Steponavičienė A.,Kraujutienė I., Steponavičius D. Spread of micromycetes in grain during its harvesting and storing // International scientific conference of graduate students "Youth is heading for progress", LŪU. 2005. 4.Kraujutienė I., JarienėE., Danilčenko H., Trečiokas K., Petrauskas V., Steponavičienė A., Schöne F. und Kinast C. Vergleich von Enzymaktivitäten in ungekeimten und gekeimten Weizensamen aus ökologischem und konventionellem Anbau // Wissenschaftliche Tagung. Jena, 2006. 5.Kraujutienė Icomparison of wheat grains grown in organical and conven-. Nutritive tional systems // 12-th international scientific-practical conference "Human and nature Safety", LŪU. 2006. 6. KorduienėS.,KraujutienėI.Research of hazardous for health pollutions in germinated seeds for foodstuff // KMU Respublical conference of students of public health sciences, KMU. 2006. 7.Kraujutienė I., Jarienė Danil E.,čenko H., lapakauskas V., Venskutonienė E., Korduienė An influence of growing technology to enzyme activity in germinated for S. food wheat grains // 3rd international conference on quality and safety in food production chain. Wroclaw, 2007.   Volume of the work   The doctoral dissertation is written in Lithuanian. It consists of the introduction, litera-ture review, materials and methods, results and discussion, conclusions, list of scientific publications dealing with dissertation theme and references. Total volume of the work is 110 pages. Research data are being presented in 35 tables and 39 figures.
 METHODOLOGY  Research object.Two cultivars of winter wheat grain (Triticum aestivumL. Emend. Fiori et Poal) 'Zentos' and 'irvinta 1' there have been studied. Wheat have been grown at Experiment station of Lithuanian University of Agriculture and the Agroecological center following conventional and organical technologies. Mentioned wheat cultivars have been chosen due to high and constant harvest and their ability to stand the winter freeze. Organically grown both wheat cultivars have been fertilized with organical fertilizers norm of 30 t·ha-1 nor pesticides nor growth regulators for care appliedand there have been of plants. Conventionally grown both wheat cultivars have been fertilized with nitrogen norm of 200 kg·ha-1(N60wheat vegetation revives. On June the wheat have) at springtime when been fertilized through the leaves by spraying nitrogen fertilizers norm of 30 kg·ha-1(N15). At that time the roots are weak. The grain have been stained before sowing with agent Maksim norm of 1 kg·ha-1. The wheat crop have been sprinkled with fungicide Folkonnorm of 0.5 l·ha-1at their pubescence time. Growth regulator Cicocel has been sprinkled with norm of 1.0 l·ha-1at wheat corn period. Sprouting. The wheat being soaked for first 12 hours at 18oC temperature water with ratio 1:4. Further the process is being continued at multistorey sprouting vessel and dark room with temperature 18÷20oC. The grain being washed with flowing water periodically every 12 hours. There have been investigated an activity ofα- irβ-amylase every 12 hours. The activity of non germinated wheat grainα- irβ-amylase has been chosen as control measure to compare the results to. The maximum germination time is 48 hours, i.e. while length of sprout reaches 3-5 mm. This time choose many investigators. Germinated and non-germinated wheat grain quality indexes.Nutritive composi-tion of both germinated and non-germinated wheat grain have been estimated at Warmian-Masurian university at Olsztyn (Poland) according Lithuanian standards now being in force: dry substance - LST 1530-98, proteins - LST 1497:97, starch - ICC 123/1:1994, mineral substance - LST ISO 2171:2000, fat - ISO 7302:1982, nutritive fibre - LST EN ISO 6865:2001.  Amount of vitamin E (DL-αtokopherol)  hydrolized with ethanol potassium hydroxide solution and vitamin EA sample being being extracted using petrolether. The solvent being evaporated out and the rest being dis-solved at methanol and diluted until needful concentration if needed. An amount of vitamin E being estimated by method of chromatography of inverse phase high effect liquids (RP-HPLC) using fluorescentic or UV detector (ISO 6867:2002).  Amount of vitamin C  10 ml of homogenized sample being mixed with 10 ml n-butanol solution for 1 min-ute. Then being centrifugated at rotation of 48400 rev·min-1at temperature 4oC. Crystal frac-tion being filtrated and diluted until required concentration. An amount of vitamin C being estimated by method of chromatography of inverse phase high effect liquids (RP-HPLC) using fluorescentic or UV detector (Omaye et. al., 1979; Vanderslice, Higgs, 1993).  Energetical value  This method is based on calculationBeing used a calorific method called a bomb. of released caloric energy (ISO 9831:1998).   
 Amount of amino acids  Released amino acids being extracted using diluted hydrochloric acid. At the same time there being extracted macromolecules in possession of nitrogen being precipitated us-ing sulphosalicylic acid and separated by filtration. Cysteine and methionine must be oxi-dized until cystine acid and methionine sulphone before hydrolysis. Tyrosine must be esti-mated at non oxidized hydrolysis solutions.  Oxidation being executed using compound of formic acid and phenol at temperature 0oAn excess of oxidation reagent being analysed using chloride disulphide. Both oxi-C. dized and non oxidized samples being hydrolysed using hydrochloric acid for 23 hours (c=6 mol·l-1). Amino acids being separated by chromatography of ion interchange and being estimated by photometric method at wave range of 570 nm (for prolyne 440 nm) when reac-tion being executed with ninhydrine (technical regulation for estimation of amino acids and olacvyndox amounts at feedstuffs).  Amount of sacharyde  1000 µl of sample being centrifugated. Then the liquid given being heated at 100oC temperature water bath for 10 minutes. Proteins denatured being centrifugated again. Then 400 µl of liquid given being mixed with distilled water in ratio 1:2. Solution given is being cetrifugated for 15 minutes. Then solution being assayed by ESC method at special bottles (Navarro-Alvarez W., 1994).  Activity of enzymes  There have been investigated an activity of enzymes in both germinated and non-germinated wheat grain. Activity ofα- andβ-amylase have been estimated every 12 hours from the beginning of sprouting process until it reached 48 hours. An activity of the rest en-zymes - catalase, xylanase, cellulase, maltase and protease have been investigated just 48 hours after beginning of germination process.  An activity of xylanase, cellulase, maltase, α- andβ-amylase have been estimated by DNS method (3,5-dinitrosalicylic acid) (Bailey et al. 1992). An estimation of catalase activ-ity is based on hydrogene peroxide analysis into water and oxygen (Aebi, 1984) and prote-ase activity is being estimated according Tomarellis methods et al. (1949).  Milk quality  estimated sensually. Another data have been got atInitial milk quality indexes being accredited laboratory at State Enterprise Pieno tyrimai according available milk buying up rules. These data are following: milk composition - amount of fat, lactose and proteins (%), quality indexes - overall bacterial pollution (thousands·cm-3), number of somatic cells (thousands·cm-3are being detected inhibitors and point) and amount of urine (mg %). There of milk congelation to control adulteration. Methodology of yogurt and curd fermentation. Yogurtmilk with fat amount of 4.85 % ac-has been made at laboratory of natural cording following technology scheme: heat-treatment at 90-92oC for 3-4 min. cooling until fermentation temperature and fermentation at 42oCfermentation until pH 4.5-4.6 at temperature 42oCmixing and fast cooling until 20oCadding of sprouted wheat grain (5 % of milk amount) and mixing of compound repeatedly cooling up until 6-8oC and keeping at temperature not higher as 6oC (Mačionienėir kt., 2005).  There has been used dry ferment YC-X16 (DVS) CHR made by company HANSEN (Denmark). That ferment includes obligatory yogurt cultureLactobacillus delbrueckiisubsp. Bulgaricus, Streptococcus tehermophilus. The samples have been fermented by adding 0.03 % of this culture.
Curd  has been produced at laboratory of the same milk according following technol-ogy scheme: milk fermentation proceeded at temperature 24-30oC (suitable for mesophylic bacteria)curdle milk mixed with fresh one in ratio 2:1 and heated up until 60oCcurdle milk poured into cloth and left for compression by own weight (formated curdle separation must be proceeded at temperature not lower 16oC) curd cooled until 6-8oC pressing (Mačionienė, 2002).  Samples of yogurt and curd have been produced at laboratory at Lithuanian Food In-stitute and later repeatedly produced at Warmian-Masurian university at Olsztyn (Poland). Microbiological quality estimation of germinated and non-germinated wheat grain and milk products:fresh yogurt and curd samples have been estimated, i.e. their keeping time did not exceed one day. There have been estimated samples with 7 days of keeping time also. These samples have been kept at temperature 6-8oC. Estimation indexes were such as overall bacterial pollution, number of yeast and mildew fungus, number of coliform bacteria and pH.  Total number of microorganisms has been evaluated by sowing into plates at tight nourishment breeding - ground (Plate Count Scim milk agar)according LST EN ISO 4833. Estimation of yeast and mildew fungus number is based on product sample and its solutions sowing into breeding - ground of yeast extract, glucose and chloramphenicol agar according LST ISO 661. Number of coliform bacteria being evaluated using the method of most reli-able figure according LST ISO 5541-2. There have been executed research of micromycetes springs and their sorts separa-tion in both germinated and non-germinated wheat grain. Method of micromycete sorts separation and detection of their springs is based on product and its solution sowing into agar breeding - ground of beer mash. Rising micromycetes colonies have been studied on third, fifth and seventh days of their development accordingly to references of LST ISO 7954. There are being separated monocultures from grown fungus colonies by lighting mi-croscopy. Then there are being estimated their cultural and morfological features and further these monocultures being identified accordingly published references Ellis (1971), Domsch and other (1980), Ramirez (1982), Nelson and other (1983), Samson (1988), Lugauskas and other (2002). Additionally there has been determined total number of microorganisms and yeast in germinated grain used for production of milk products.  There have been applied harmless for human being preservatives on purpose to avoid pollution of germinated wheat grain with microscopic fungus springs and that way prolong expiry date to use grain as foodstuff. These preservatives are against untimely moulding of products and it is calcium propionate GmbH, Germany) and sorbic acid (Jiali In-(Rietmann ternational Corporational Corporation, China) (Люк,Ягер, 2000). Concentration used is 1.5 and 2 % of sprouted wheat grain weight accordingly references of Lithuanian hygiene norm (HN 53:2003).  Microbiological study has been executed at Warmian-Masurian university at Olsztyn (Poland).  Sensual estimation of yogurt and curd.Sensually milk products have been evalu-ated by international board of 7 tasters. Samples for that purpose have been chosen accord-ing LST ISO 707 and kept at room temperature for half an hour before tasting. Average value of quality indexes have been determined using scale of 5 points (least value character-ises worst product quality and highest value-best one).  Statistical estimation of research results.Research have been repeated three times. Data given have been processed using integrated data management and analysis program Statistica(2000 m.). Student and Fisher tests have been applied to compare average values.
Method of dispersive analysis have been used to estimate research results and determine data arithmetic means, reliance intervals and standart deviations. Research data correlation has been calculated using programTable curve 2Dversion5.01.Given data have been ap-proximated to linear relation estimating correlation coefficient, standart value errors, reli-ance intervals, process describing equation and its coefficients when stature level is 0.05.  RESEARCH RESULTS AND DISCUSSION   Estimation of quality indexes of both sprouted and non-sprouted wheat grain grown following different growing technologies.Chemical composition is the most im-portant index indicating grain suitability for nutrition purposes. Both cultivar and growing conditions have an influence upon chemical grain composition (Lukow, McVetty, 1991; Seibel, Steeart, 1997; Evers, Millar, 2002). Sprouting conditions have an influence upon chemical composition of sprouted grain.  An amount of dry substance at germinated grain is less about 5.8 % to compare to non-germinated grain irrespective of growing technology (Table 1). The same trend has been determined with nutritive fibres (11.6 %) and starch (23.3 %).   Table 1.Estimation of wheat grain quality indexes (%) (LUA, 2003-2007) Non-germinated wheat grain Nutritive substances Organical growing Conventional growing 'irvinta 1' 'Zentos' 'irvinta 1' 'Zentos' Dry substances 86.61±0.18 86.47±0.21 86.65±0.34 86.92±0.28 Mineral substances 1.51±0.03 1.59±0.03 1.48±0.01 1.56±0.05 Proteins 10.59±0.08 10.49±0.12 9.76±0.13 13.15±0.11 Fat 1.33±0.05 1.68±0.03 1.47±0.09 1.59±0.05 Nutritive fibres 3.06±0.04 3.47±0.04 3.23±0.02 2.94±0.13 Starch 68.34±0.05 68.73±0.26 69.16±0.17 66.07±0.07  Germinated wheat grain Dry substances 82.23±0.28 81.45±0.22 82.29±0.10 81.91±0.21 Mineral substances 1.79±0.04 1.74±0.02 1.75±0.01 1.76±0.01 Proteins 11.48±0.20 11.15±0.08 10.83±0.09 14.04±0.26 Fat 1.62±0.03 1.60±0.04 1.63±0.01 1.58±0.04 Nutritive fibres 3.03±0.06 2.74±0.04 2.90±0.02 2.71±0.05 Starch 55.48±0.25 56.01±0.16 56.05±0.11 53.29±0.04   The results given are coincident with conclusions of other scientists which maintain that germination process reduces an amount of dry substances in grain (Lorenz, 1980; Hartmuth-Hoene et al., 1987). It is due to decrease of carbohydrates during germination. Small amount of soluble ones being leached during soaking and largest amount of carbohy-drates being used during grain intensive breathing. There has been determined that play of two factors such as cultivar and germination process has an essential influence upon amount of mineral substances (Table 2). Calculation shows that non-germinated wheat grain 'Zentos' has more mineral substances 5.4 % and germinated ones has less 1.4 % comparing to wheat cultivar 'irvinta 1'. Sprouted wheat grain has more mineral substances 14.7 % if to compare to non-sprouted ones. This result does not mind conclusions of another scientists (Lorenz, 1980).    
Table 2.Statistical values of nutritive substances depending on play of cultivar (V), growing technology (A) and germination (D) factors Monofactor play Multifactor play z s Culti- Growing Germination En yme var technology A×V D×V D×A D×A×V V A D Dry substances 0.114 0.125<0.001 0.985 0.203 0.051 0.960 Mineral sub- 0.068 0.202<0.001 0.2 stances 700.0040.586 0.371 Proteins 0.104 0.0600.005 <0.001 0.849 0.974 0.846 Fat<0.0010.728 1.5680.039 <0.0010.542 0.096 Nutritive fibres 0.079 <0.001 0.023 0.005 0.088 0.358 <0.001 Starch0.010 0.035 <0.001 <0.001 0.800 0.871 0.919 Note-statistically important differences are marked in bold.   There has been determined during our research that genetics of wheat cultivar studied along with growing technology has an essential influence on synthesis of proteins. Research data show that amount of proteins in both germinated and non-germinated wheat grain 'irvinta 1' is higher 2 % when grown organically while wheat grain 'Zentos' has even 32.1 % higher amount of proteins when grown conventionally. In summary we can say the cultivar 'irvinta' is more adapted to grow organically bearing in mind grain ability to accu-mulate proteins. In that way there being partially denied data of another researchers (Har-muth-Hoene, 1988). If to compare sprouted and non-sprouted wheat grain the first have more proteins approximately 8 %.  There has been determined higher amount of fat in non-germinated grain 'Zentos' comparing to germinated ones round 2.8 % and cultivar 'irvinta 1' grain gives contra result demonstrating 16.1 % higher amount of fat when organically grown corn being germinated (Table 1). Evaluating growing technologies cultivar 'Zentos' is better of 'irvinta 1'. Cultivar has an essential influence on amount of mineral substances just under play of two factors (Table 2). Results show that organically grown grain 'Zentos' accumulated 2 % more nutri-tive fibre while grain 'irvinta 1' did it 8.9 % more when grown conventionally. In both sprouted and non-sprouted wheat grain 'irvinta 1' grown conventionally an amount of starch is round 0.6 % higher comparing to cultivar 'Zentos'. While organical technology ap-plied gave different result - grain 'Zentos' accumulated 4.7 % larger amount of starch than 'irvinta 1'.  enzymes activity in germinated and non-germinated wheat grain.Comparison of Grain biochemical composition and enzyme activity depends on many factors: weather con-ditions during growth time and harvest time, grain stocking conditions, fractionation when milling, etc. (Fox, 1982). Ezyme activity is displayed in table 3.