Chemical composition and allergic activity of bread Relationship to non-yeast microorganisms and baking temperatures-

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1 Chemical composition and allergic activity of bread Relationship to non-yeast microorganisms and baking temperatures- M. Shimoyamada a, M. Hori b, S. Kasuya c, T. Suzuki d and H. Nagano e a School of Food, Agricultural and Environmental Sciences, Miyagi University, Sendai, Japan (shimoyam@myu.ac.jp) b Department of Food and Nutrition, Gifu City Women's College, Gifu, Japan c Faculty of Regional Studies, Gifu University, Gifu, Japan d United Graduate School of Agricultural Science, Gifu University, Gifu, Japan e Faculty of Education, Gifu University, Gifu, Japan (nagano@gifu-u.ac.jp) ABSTRACT Pieces of bread were collected from some regions of the world, and various microorganisms belonging to bacteria as well as yeast were found in the bread samples collected by an optical microscopic observation. SDS-PAGE showed that some of the bread contained mainly partially hydrolyzed proteins, suggesting bacterial proteases hydrolyzing protein and lowering of bread allergenicity. Bread baked in a stone oven at various temperatures was analyzed for protein, free amino acid and carbohydrates. Extractable protein, free amino acid and maltose contents were decreased with increase in baking temperature till 270 C, whereas glucose and maltotriose contents were very low irrespective of baking temperature. Allergen levels were also decreased with increase in the temperature. The effect of baking temperature was considered to be more than that of oven types. Keywords: bread making, microbes, stone oven, chemical composition, allergenicity INTRODUCTION Bread, which has been eaten since an ancient period, is heated by dry air, steam and oil, using ovens, stone ovens, tandoors, etc. Many investigators reported on heat transfer in baking of bread [1]. Most of breads usually consumed are made from wheat flour, so allergy derived from breads is an unavoidable problem, which should be solved right now. Some workers reported that some enzymes are effective for hypoallergnicity [2,3]. Although yeast has generally been used for the fermentation for bread making, there are many kinds of microorganisms co-existing in dough [4]. Enzymes derived from microorganisms are expected to decrease allergenicity in bread dough [5]. In first step of this study, pieces of bread were collected from some regions of the world, and traces of microorganisms, which had already sterilized by baking, were observed under an optical microscope to discuss enzymes possibly existed in bread dough, some of which were responsible for some important roles such as decrease in allergenicity. In second step, breads were baked in a stone oven at various temperatures. The resulting breads were analyzed for protein, free amino acids, carbohydrates and allergens. Effect of baking temperatures and type of oven were discussed especially on allergen decreasing. MATERIALS & METHODS Collection of fragments of bread in some regions of the world Bread samples were purchased at local markets in Egypt, Turkey, Italy and China. The sample was dir-dried, packed in a plastic bag and transported to our laboratory. Baking of bread For bread baking, strong wheat flour harvested in Japan was purchased from Ebetsu flour milling Co., Ltd. (Ebetsu, Japan). The flour was mixed with NaCl (1.5% to flour weight), yeast (6%, Hoshino Natural leaven (Hoshino Tennen Koubo Pandane, Inc.)) and water (60%) for dough preparation. Fermented dough was baked in a stone oven at 240, 270 or 300 C. For control, other fermented dough was baked in a common oven at 255 C.

2 Extraction of protein and amino acid fractions from bread sample Bread sample baked in a stone oven was lyophilized and milled. Smaller fraction (< 0.45 mm) of the resulting milled powder was collected to afford measuring sample (freeze-dried sample). Each sample was mixed with 10 volume of 0.5 M NaCl and incubated in a refrigerator overnight. The resulting mixture was centrifuged at 11,850 g, 4 C for 60 min, and the supernatant was used for protein determination. The bread sample was separately extracted with 30 mm Tris-HCl (ph 8.0, containing 1% SDS and 20 mm 2-mercaptoethanol). The supernatant was applied to SDS-PAGE. The extract (Tris-HCl containing SDS and 2-mercaptoethanol) was also applied to protein determination. Determination of protein Protein concentration in each extract was measured colorimetrically according to Lowry et al. [6]. Amino acid analysis For amino acid analysis, an automated amino acid analysis HPLC system (L-2130, Hitachi Co., Japan) was used. A column used was #2619PH ( I.D., Hitachi Co, Japan). Eluent was reacted at 45 C after mixing with o-phthalaldehyde (OPA) reagent, and then monitored by fluorescence (Ex. 340 nm, Em. 450nm). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was done by according to Laemmli [7] with 12.5% of polyacrylamide gel and the gel was stained by CBB R250 (Nacalai Tesque, Inc., Kyoto Japan). Enzyme-linked immunosorbent assay To estimate allergen in bread samples baked in a stone oven or a common oven, FASTKIT ELISA ver. II (Nippon Meat Packers Inc., Osaka, Japan) was used. Extracts from lyophilized bread samples were applied to the kit. Extraction and analysis of carbohydrate from bread Bread sample was mixed with 20 volume of distilled water and incubated at room temperature for 10 min, and the supernatant was concentrated to dryness by an evaporator, and solubilized with 2 ml of distilled water. The solubilized sample was subjected to HPLC. A column used was an Asahipak NH2P-50 4E (4.6 mm id 250 mm) (Shodex, Tokyo, Japan) and mobile phase was acetonitrile- water (75:25), flow rate was 1.0 ml/min. Detection was carried out with a Refractive index detector (L3350, Hitachi Co., Tokyo, Japan). RESULTS & DISCUSSION yeast bacteria Japan Egypt China Figure 1. Optical microscope images of bread samples collected in some countries

3 kda M A B M C Figure 2. SDS-PAGE patterns of protein fraction from bread collected in China and Egypt. M, molecular marker; A, wheat flour; B, China; C, Egypt Observation of pieces of bread by a microscope, which were collected in some region of the world, and protein and amino acid composition Pieces of breads were collected in some region of the world. The pieces of bread were observed by an optical microscope (Figure 1). Some microorganisms morphologically belonging to Bacillus, Lactobacillus, etc. were observed as well as yeast. It is possible that these microorganisms produce some enzymes like proteases, which may decompose allergenic proteins. Proteins extracted from the bread samples collected in Egypt showed almost intact protein patterns, indicating that little proteins were decomposed. However, those collected in China showed that almost of proteins were hydrolyzed to low molecular weight constituents (Figure 2). These data may suggest that the bread in China has potentially low allergenic activity. As for amino acid composition, there also were much Table 1. Contents of protein and carbohydrate in bread baked at various temperatures. Protein and Amino acid Carbohydrate Conditions protein FAA glucose maltose maltotriose Wheat flour Stone oven at Tr at at Tr Common oven at Tr FAA, free amino acid Tr, Trace amount

4 differences between the bread made in China and Egypt. Methods for bread making, for example, oven type, formula, baking temperature, etc. were considered to be different between China and Egypt, and the differences in microorganisms grown in dough for bread making may also affect composition of proteins and amino acids in the bread. Chemical composition of bread baked in a stone oven Protein contents in the breads (Table 1) were about fourth of that in wheat flour. Protein content decreased with increase in baking temperature till 270 C and the content baked at 300 C was almost equal to the value at 270 C. On the other hand, the bread baked in a common oven at 255 C, showed medium protein content between 240 and 270 C by a stone oven. From these data, baking temperature may be more effective for protein content rather than oven types. The decreasing effect of baking was saturated over 270 C. These decreases in protein were thought to be mainly caused from insolubilization of protein in the bread during baking. Total free amino acid content in the bread was low. It was also decreased by baking until the baking temperature increased to 270 C (Table 1). The contents in the breads baked at 270 and 300 C were almost equal, suggesting that the effect of baking temperature plateaus at 270 C. These decreases in amino acid contents were thought to be responsible for thermal decomposition of the amino acids and/or adsorption to insolubilized proteins. The total amino acid content in the bread was also more influenced by baking temperature than by oven types as well as the protein content. In all free amino acids detected, content of γ aminobutyric acid (GABA), which was reported to lower blood pressure, was much higher than other amino acids, especially glutamic acid, which caused umami taste. The free amino acid contents in the bread were low and decreased with increase in baking temperature. Differing from protein, many of amino acids decreased with increase in temperature till 300 C. Carbohydrates were also analyzed by HPLC (Table 1). In the bread baked in this experiment, glucose contents were constantly very low. On the other hand, maltose, which was formed by α-amylase from wheat flour, was decreased with increase in baking temperature till 270 C. These data also showed that maltose formed was thermally decomposed by baking but the temperature effect was saturated over 270 C. Only trace amount of maltotriose was detected in the samples baked at all temperatures used. Composition of protein fraction extracted from the baked breads and allergen activity against allergic patient sera Protein fractions extracted from lyophilized bread samples were applied to SDS-PAGE (Figure 3). Each protein band, especially ranging from 30 to 37 kda, was paled with increase in baking temperatures, but there (kda) M A B C D M Figure 3. SDS-polyacrylamide gel electrophoresis patterns on wheat flour and bread of baking conditions. Lane M, molecular weight makers; A, Oven baking (255 ºC); B, Stone oven baking (240 ºC); C, Stone oven (270 ºC); D, Stone oven (300 ºC).

5 Figure 4. Quantity of wheat antigen that remains after baking at various temperatures. After baking, each sample was milled into powder, extracted with 0.5M NaCl. were little differences in protein composition among any samples. There was little difference in protein constituents in breads between baked in a stone oven and in a common oven. These decrease in allergen protein content, especially, albumin (ca kda) and prolamin (ca. 31 kda), suggested decrease in allergen activity [8]. Measuring the allergen activity of the protein fraction by ELISA (Figure 4), baking decreased the allergen level of the breads with increase in temperature until 270 C. Allergen level of the bread baked at 270 C was almost half of that of wheat flour. On the other hand, the allergen level of the bread baked at 300 C was almost equivalent to that baked at 270 C, similar to the result for total protein and free amino acid contents. Considering these data, insolubilization of protein was related to decrease in allergen level as well as denaturation and decomposition of allergen. CONCLUSION Some bacteria were found in bread which was collected in some region of the world by an optical microscope. From SDS-PAGE patterns, some of the bread samples showed partially hydrolyzed protein compositions, especially from China, indicating hypoallergenicity. As for bread baked in a stone oven at various temperatures, extractable protein was decreased with increase in the baking temperature till 270 C. Free amino acid and maltose content were also decreased. On the other hand, glucose and maltotriose contents were very low irrespective of baking temperatures. ELISA showed that allergen levels of the bread samples were also decreased with increase in the temperature. The effect of baking temperature was considered to be more than that of oven types. REFERENCES [1] Therdthai, N., & Zhou, W. (2003). Recent advances in the studies of bread baking process and their impact on the brad baking technology. Food Science and Technology Research, 9, [2] Watanabe, M., Suzuki, T., Ikezawa, Z., & Arai, S. (1994). Controlled enzymatic treatment of wheat proteins for production of hypoallergenic flour, Bioscience, Biotechnology and Biochemistry, 58, [3] Tanabe, S., Arai, S., & Watanabe, M. (1996). Modification of wheat flour with bromelain and baking hypoallergenic bread with added ingredients. Bioscience, Biotechnology and Biochemistry, 60, [4] Liu, C., & Nagano, H. (2008). Purification and Characterization of a Proteolytic Enzyme from Bacillus subtilis M2-4. Journal of Home Economics of Japan, 59,

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