Analele UniversităŃii din Oradea Fascicula: Ecotoxicologie, Zootehnie şi Tehnologii de Industrie Alimentară, 2010 CHANGES IN WHEAT DURING STORAGE AT THREE DIFERENT TEMPERATURES 1219 Ruska L.*, Timar A. V.* *University of Oradea, Faculty of Environmental Protection, 26 Gen. Magheru St., 410048 Oradea, Romania, e-mail: atimar@uoradea.ro Abstract Biochemical changes in wheat grains stored at 10, 25 and 45 C for six months were studied. A significant decrease in ph and an increase in titratable acidity were observed during storage of wheat grains at 25 C and 45 C. Moisture contents of wheat grains decreased by 15% at 25 C and 26% at 45 C during six months of storage. A significant decrease in water soluble amylase (20 28%) along with an increase in insoluble amylase contents (7.6 17%) were observed during storage at 25 and 45 C. Amylase activity of the samples showed a decrease as the storage progressed. Key words: Biochemical changes, Storage temperature, Wheat grains. INTRODUCTION Wheat is one of the major sources of protein and energy for the human population throughout the world. In Pakistan, wheat is stored in jute bags and earthen pots under adverse temperature and moisture conditions. It has been observed by many workers that cereal and legume grains undergo pronounced biochemical changes during storage. The moisture content of the grains or humidity and the storage temperature have been shown to exert dramatic changes in the acidity, ph, free amino nitrogen, crude protein, and reduced protein quality. Significant changes in soluble sugars and amylase contents of the grains have also been reported during storage at elevated temperature. Ebersdobler and Tsao et al. found that Maillard cross linkages were formed in food stored under adverse conditions of temperature and moisture with consequent reduction in protein digestibility. The association between the physical changes and the changes in the chemical 110 composition of food has made the biochemical and nutritional quality control of the stored products increasingly essential. The aim of this study was to evaluate the effect of storage temperature on the stability of wheat with respect to some biochemical parameters and protein digestibility. MATERIALS AND METHODS Freshly harvested wheat was obtained from Agriculture Research Institute, SCAZ (ORADEA) and stored at 10, 25 and 45 C for a period of
six months. All wheat samples were free from insect infestation and no chemicals were used for preservation. About 100 g of each sample with about 14% moisture were placed in screw cap plastic bottles of uniform size. Three bottles of each treatment were randomly selected at the end of each month; contents were pooled and thoroughly mixed together. The samples were analyzed for ph, titratable acidity, moisture, amylase, amylase, total available lysine, soluble sugars and protein digestibility. The ph was determined on a filtrate of a 2 g ground sample (80 mesh size) in 20 ml distilled water using a glass electrode ph meter. The titratable acidity was expressed as sodium hydroxide required neutralizing the acids in a 100 g sample using phenolphthalein as an indicator. Moisture was determined using the standard method of AOAC. Amylase activity in wheat was measured by the method of Bernfeld after extraction of the enzyme with sodium acetate buffer. Total amylase and water soluble amylase contents in stored wheat samples were determined using the Sowbhagya & Bhattacharya method; the insoluble amylase contents were calculated by difference. Total soluble sugars were estimated by the phenol-sulphuric acid method of Dubois et al. using sucrose as the standard. Dye binding method was used for the estimation of total available lysine in wheat sample after hydrolyzing with 6N HCl. The principle of the method is a quantitative binding of the azodye acid orange 12 by the basic groups of proteins. Lysine concentration was determined by a difference method in which measurements were made on a spectrophotometer (Hitachi 220S) at 475 nm before and after blocking the lysine with propionic anhydride. In vitro protein digestibility (IVPD) was measured after digestion with pepsin-hcl solution at 37.5 C for 24 hours. All determinations were carried out in triplicate and standard deviations (SD) were calculated according to the method of Steel & Torrie. Duncans multiple range tests were used to determine significant differences (p < 0:05). RESULTS AND DISCUSSION Biochemical changes in wheat grains occurred to various extents during storage at different temperatures. The range of storage temperature included in this study (i.e. 10 45 C) covered the atmospheric temperatures that the wheat grains would encounter in Romania, Oradea. It is apparent from Table 1 data that there were no changes at all in ph and titratable acidity of wheat grains kept at 10 C for six months. However, significant (p < 0:05) changes in ph and titratable acidity occurred on storing wheat grains at 25 and 45 C for different time periods. 1220
A decrease in ph and an increase in titratable acidity started appearing just after one month of storage at 25 and 45 C. The mean titratable acidity of the stored wheat grains was 4.00 mg NaOH/100 g at 25 C and 4.97 mg NaOH/100 g at 45 C after 5 months, while it was 3.12 mg NaOH/100 g for freshly harvested wheat grains. However, ph of the freshly harvested wheat grains was 6.52 whereas ph values of the wheat grains at 25 and 45 C were 5.92 and 4.98, respectively, after 5 months of storage. No further changes in ph and acidity were observed at 25 C after 5 months. On the other hand, ph and titratable acidity of the stored wheat grains were further changed to some extent at 45 C after 5 months. These results are consistent with the findings of Onigbinde & Akinyele who reported an increase in acidity during storage of corn grains and flour. The increase in the acidity in stored grains could be attributed to the increasing concentration of the free fatty acid and phosphates which resulted from increased grain deterioration. The binding of the amino group of amino acids, short chain peptides and protein, leaving the carboxylic ends free and the presence of acid byproducts of advanced Maillard reactions are other possible causes of the increased acidity in the samples stored at elevated temperatures. Table 1. ph, titratable acidity and moisture contents of wheat grains during storage at three temperatures (mean SD, triplicate samples) Storage 10 C time Moisture ph (months) % 1221 Acidity 0 14.29 ± 0.4 6.52 ± 0.1 3.12 ± 0.2 1 14.21 ± 0.3 6.50 ± 0.1 3.12 ± 0.2 2 14.17 ± 0.2 6.50 ± 0.2 3.16 ± 0.1 3 14.11 ± 0.4 6.47 ± 0.2 3.16 ± 0.1 4 14.02 ± 0.4 6.44 ± 0.1 3.20 ± 0.3 5 13.97 ± 0.3 6.42 ± 0.1 3.21 ± 0.2 6 13.96 ± 0.4 6.41 ± 0.1 3.21 ± 0.1 Storage 25 C time Moisture ph (months) % Acidity 0 14.29 ± 0.5 6.52 ± 0.2 3.12 ± 0.1 1 13.90 ± 0.4 6.45 ± 0.3 3.46 ± 0.2 2 13.62 ± 0.5 6.30 ± 0.1 3.62 ± 0.1 3 13.25 ± 0.5 6.20 ± 0.2 3.78 ± 0.1 4 12.87 ± 0.4 6.00 ± 0.3 3.92 ± 0.2 5 12.22 ± 0.3 5.92 ± 0.1 4.00 ± 0.3 6 12.22 ± 0.5 5.85 ± 0.2 4.08 ± 0.3 Storage 45 C time Moisture ph (months) % Acidity
0 14.29 ± 0.4 6.52 ± 0.3 3.12 ± 0.1 1 13.73 ± 0.6 6.23 ± 0.2 3.72 ± 0.2 2 13.55 ± 0.5 5.89 ± 0.2 3.98 ± 0.1 3 13.06 ± 0.4 5.40 ± 0..3 4.27 ± 0.3 4 12.03 ± 0.5 5.00 ± 0.1 4.67 ± 0.3 5 11.26 ± 0.6 4.98 ± 0.2 4.97 ± 0.3 6 10.57 ± 0.4 4.63 ± 0.3 5.24 ± 0.2 A gradual decline in the level of moisture took place at 25 and 45 C while there was no change in moisture content during storage of wheat grains at 10 C for six months (Table 1). The decrease in moisture became significant at 25 C after three months and 45 C after one month of storage (p < 0:05). Moisture contents of wheat grains decreased by 15% at 25 C and 26% at 45 C during six months of storage. Similar observations were also made by other workers during storage of corn at 55 C. About 28% loss in moisture content was also observed by Rehman et al. during storage of rice at 45 C for six months. It is apparent from Table 2 data that amylase activity in wheat grains decreased to various extents during storage at different temperatures. Decrease in amylase activity was extremely slow at 10 C whereas it was comparatively higher at 45 C than at 25 C during six months of storage. Table 2. Amylase activity and amylase contents in wheat grains during storage at three temperatures (mean SD, triplicate samples) Storage 10 C time (months) Amylase activity (I.U.) Water soluble Insoluble amylase 0 12.12 ±0.4 12.00 ±0.5 8.75 ±0.4 8.70 ±0.3 23.25±0.6 23.30±0.4 Storage 25 C time (months) Amylase activity (I.U.) Water soluble Insoluble amylase 0 12.12 ±0.3 11.85 ±0.5 8.75 ±0.3 8.65 ±0.3 23.25±0.7 23.35±0.6 Storage 45 C time (months) Amylase activity (I.U.) Water soluble Insoluble amylase 0 12.12 ±0.2 11.75 ±0.4 8.75 ±0.4 8.25 ±0.6 23.25 ±0.6 23.75 ±0.5 *Significantly different from the corresponding level before storage (p < 0.05). The amylase 112 activity of the freshly harvested wheat grains was 12.12 units/g which became 9.50 units/g at 25 C and 8.0 units/g at 45 C after 6 months. Total amylase content (32.0%) in the wheat grain remained unchanged during storage (Table 2) which was consistent with the findings of Swamy et al. A slow but steady decrease in water soluble amylase content, along with an increase in insoluble amylase at 25 and 45 C, was observed during 6 months of storage. In freshly harvested wheat grains, the concentration of soluble and insoluble amylase contents was 8.75 and 23.25%, respectively. 1222
These values were found to be 7.0 and 25.0% at 25 C and 6.25 and 25.75% at 45 C after 6 months. It is apparent from these results that the decrease in soluble amylase was accompanied by an increase in the insoluble amylase content as has already been observed during storage of rice. Increase in insoluble amylase may be due to crystallization of straight chain compounds of glucose (water soluble amylase) into branched chain compounds (insoluble amylase) containing hundreds of glucose molecules. Changes, in soluble and insoluble amylase contents during storage, were found to be significant (p < 0:05) at all storage temperatures. Total available lysine in freshly harvested wheat grains was 2.92% which was decreased to various extents with storage (Table 3). About 10% decrease in total available lysine was observed at 25 C after 3 months and at 45 C after 2 months storage of wheat grains. However, 18.0% and 22.6% decreases in total available lysine were also observed at 25 and 45 C, respectively, after 6 months of storage. On the other hand, only a 6.5% total available lysine was decreased at 10 C during storage. Decrease in total available lysine at 25 and 45 C was significant (p < 0:05) while it was found to be non significant at 10 C during 6 months of storage. The decrease in total available lysine during storage at different temperatures could be the result of some structural changes which inhibited proteolysis and amino acid solubility. Table 3 data show significant (p < 0:05) changes in total soluble sugars during the 6 months storage of wheat grains. About a 37% decrease at 45 C and a 9 to 12% increase in total soluble sugars were observed at 10 and 25 C during 6 months of storage. Table 3. Total available lysine, total soluble sugars and in vitro protein digestibility (IVPD) of wheat grains during storage at three temperatures (mean SD, triplicate samples) Storage 10 C time Total Total 1 IVPD (months) available soluble % lysine ' fo sugars ' % 0 2.92 ± 0.1 3.44 ± 0.2 74.68 ± 0.8 1 2.89 ± 0.1 3.46 ± 0.1 74.63 ± 0.8 2 2.85 ± 0.2 3.53 ± 0.1 74.66 ± 0.7 3 2.82 ± 0.2 3.58 ± 0.2 74.61 ± 0.6 4 2.80 ± 0.1 3.68 ± 0.3 74.60 ± 0.7 5 2.77 ± 0.3 3.72 ± 0.3 74.43 ± 0.8 6 2.73 ± 0.3 3.76 ± 0.2 74.40 ± 0.8 Storage 25 C time Total Total 1 IVPD (months) available soluble % lysine ' fo sugars ' % 1223
0 2.92 ± 0.1 3.44 ± 0.2 74.68 ± 0.9 1 2.80 ± 0.2 3.48 ± 0.1 73.81 ± 0.7 2 2.71 ± 0.3 3.55 ± 0.2 73.60 ± 0.8 3 2.63 ± 0.1* 3.60 ± 0.1* 73.57 ± 0.6 4 2.51 ± 0.1 3.72 ± 0.1 72.26 ± 0.8 5 2.46 ± 0.1 3.78 ± 0.2 71.00 ± 0.9 6 2.39 ± 0.1 3.85 ± 0.2 71.00 ± 0.9 Storage 45 C time Total 1 Total 1 IVPD (months) available soluble % lysine ' fo sugars ' % 0 2.92± 0.2 3.44 ± 0.2 74.68 ± 0.8 1 2.72± 0.1 3.40 ± 0.1 72.51 ± 0.9 2 2.61± 0.1* 3.12 ± 0.2* 72.00 ± 0.7 3 2.48± 0.2 2.83 ± 0.2 71.77 ± 0.8* 4 2.40± 0.2 2.63 ± 0.2 71.50 ± 0.7 5 2.29± 0.3 2.33 ± 0.2 69.21 ± 0.8 6 2.26± 0.3 2.16 ± 0.1 67.00 ± 0.9 *Significantly different from the corresponding level before storage (p < 0.05). The increase in the soluble sugars could be the result of amylolytic activity of the endogenous amylases whereas the decrease in soluble sugars at 45 C might be due to their involvement in Maillard reactions. In vitro protein digestibility (IVPD) of wheat grains was significantly affected at 45 C (p < 0:05) whereas it remained unchanged under 10 C storage (Table 3). Initially, IVPD of wheat grains was 74.68% which became 71% (5% decrease) and 67% (10.28% decreases) at 25 and 45 C, respectively, after 6 months of storage. These results are consistent with the findings of other workers who reported a distinct decrease in protein digestibility of 115 wheat, corn and cowpeas during storage. Decrease in protein digestibility could be the result of Maillard reaction, during which free amino groups of protein and carbonyl groups of reducing sugars form complex intermediate compounds by interacting with each other during storage of food materials. These complex compounds might have inhibited proteolysis and caused reduction in amino acid solubility to some extent at elevated temperatures. CONCLUSIONS Nutritional quality of wheat was adversely affected as a result of storage at elevated temperatures. In vitro protein digestibility of wheat decreased by 5% and 10% during six months of storage at 25 and 45 C, respectively. 1224
Significant losses in lysine occurred on storage of wheat at 25 and 45 C. Losses in soluble sugars were also found in the case of wheat stored at 45 C for six months. Significant changes in other parameters were also observed during storage of wheat. In view of these facts, it is suggested that wheat should not be stored above 25 C in order to minimize nutrient losses during storage. ACKNOWLEDGMENTS The researches were carried out in the Exploratory research project code 690/2009 The study of influences of some technological elements upon the wheat yield quality in the conditions of the North-Western part of Romania. We want to thank C.N.C.S.I.S. for founding and supporting the researchs. REFERENCES 1. Kumar R, R Singh, 1984, Levels of free sugars, intermediate metabolites and enzymes of sucrose starch conversion in developing wheat grains. J Agric Fd Chem 32: 806 808. 2. Onigbinde AO, IO Akinyele, 1988, Biochemical and nutritional changes in corn (Zeamays) during storage at three temperatures. J Food Sci 53: 117 120. 3. South JB, WR Morrison, OE Nelson, 1991, A relationship between the amylase and lipid contents of starches from various mutants for amylase contents in maize. J Cereal Sci 14: 267 278. 4. Swamy IYM, CM Sowbhagya, KR Bhattacharya, 1978, Changes in the physicochemical properties of rice during aging. J Sci Fd Agric 29: 627 629. 5. Onayemi O, OA Osibogun, O Obembe, 1986, Effect of different storage and cooking methods on some biochemical, nutritional and sensory characteristics of cowpeas (V. unguiculata L. Walp). J Food Sci 51: 153 156. 6. Rehman ZU, M Yasin, WH Shah, AFM Ehtheshamuddin, 1995, Biochemical changes in rice during storage at three different temperatures. Pak J Sci Ind Res 38: 98 100. 117 7. Honseney RC., 1986, Principles of Cereal Science and Technology. American Association of Cereal Chemists Inc, St. Paul. Minnesota. 1225