微生物法生产脱苦大豆功能性多肽的研究
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摘要
大豆多肽是指将大豆蛋白水解成3～6个氨基酸的多肽，它能成为一种很有前途的功能性食品。但是许多大豆蛋白水解后往往产生一些苦味成分，影响了制品的风味，而目前的脱苦方法步骤繁琐，且成本较高。
利用食品级的枯草芽孢杆菌直接水解大豆蛋白，一步法完成水解和脱苦，降低了生产成本，加速了功能性多肽的产业化进程。因此，枯草芽孢杆菌生产脱苦大豆功能性多肽是一种具有广泛应用前景的方法，此方法在国内外尚未有相关报道。本论文进行了以下四方面的研究：(1)脱苦大豆功能性多肽产生菌的筛选；(2)液体发酵条件的优化研究；(3)吸附法固定枯草芽孢杆菌液体发酵产大豆多肽的研究；(4)枯草芽孢杆菌发酵产大豆多肽溶液的加工功能特性研究。研究结果如下：
(1) 对本实验室保藏的八株枯草芽孢杆菌(Bacillus subtilis)进行摇瓶试验，采用苦味值和10％TCA酸溶性多肽含量两项指标进行筛选，得到一株适合于水解豆粕粉的菌株B. subtilis石河子Ⅲ。液态发酵过程测定结果表明，该菌株的产蛋白酶类型为同步合成型。对其48小时发酵液进行氨基酸产物的分析结果表明，该菌株产生的羧肽酶能特异性地作用于酪氨酸、苯丙氨酸这两种疏水性氨基酸。
(2) 研究了接种量对多肽产量影响，结果表明，B. subtilis石河子Ⅲ菌株发酵产多肽的最佳接种量为5％。同时，对影响多肽产量的豆粕粉浓度、发酵液初始pH值、发酵温度、时间四因素用正交试验方法进行优化。由正交试验结果可知，该菌株发酵产多肽的最佳条件为：豆粕粉3g，水50ml，pH 6.5(自然pH)，温度30℃，发酵时间48hr。优化后该菌株发酵豆粕粉后的多肽溶液酸溶性多肽含量能维持在1000μg／ml以上。
(3) 研究了甲壳素浓度、面积、种子液pH值等因素对吸附B. subtilis菌体效
果的影响。此外，还测定了不同吸附批次的多肽产量、平均肽链长度等质
量指标。结果表明，甲壳素吸附菌体最佳条件为：0．一％左右的整条甲壳
素（面积约为4cm’），种子液PH值为6。不同发酵批次的稳定性较好，每
一批次的 4 8 h r发酵液中酸溶性多肽含量维持在 10 0 0p g／ml以上，平均链
长为3．5左右，苦味值为2．0。可见，甲壳素不仅对菌体的生长有促进作
用，还能成为一种有效的固定化细胞载体。
（4）研究了大豆多肽溶液的加工功能特性，例上。可溶性氮指数 NS、浊度、乳
化性等。结果表明，不同发酵时间的多肽溶液有着不同的加工特性。水解
度m随着发酵时间的延长而上升；可溶性氮指数（NSI）与发酵时间和训
值均有关，总体上发酵时间越长，NSI越大，中性叩值T NSI较高，酸
性和碱性叩值下 NS较低；相同发酵时间的多肽溶液，PH值越低，浊度
越高，36hr的多肽溶液在nH3．0－7．0范围内，浊度较低；多肽溶液均有
着较好的乳化性，但乳化稳定性较差。
Soybean peptide, which can be a kind of potential function food, is produced by hydrolyzing soybean protein into peptide with 3-6 amino acid residues. However, some bitter components, affecting the flavour of products, will be produced after hydrolyzation of soy protein.
The methods of debittering existing now are not only laborious but of high cost. It cuts down production cost, and accelerates the industrialzation of functional peptide to produce peptide which combines hydrolyzing soy protein and debittering in one step with food-grade Bacillus sub tills. Thus the production of debittered functional soybean peptide with Bacillus subtilis will have widely-use prospect. There are no relative internal and external reports so far. This thesis focused on the following four aspects: (1)Screening of strain producing debittered functional soybean peptide; (2)Optimizing its liquid fermentation conditions; (3)Study of the soybean peptide production by stabilization of Bacillus subtilis: (4)Processing properties of soybean peptide produced by Bacillus subtilis. The results were as follows: (1)According to two indexes, bitterness value and content of 10%TCA soluable peptide, a strain coded Bacillus subtilis Shi HeziIII which was fit for hydrolyzing defatted soybean was screened out fr
om eight Bacillus subtilis strains stored in the lab. The results of determination during liquid fermentation showed that the production of protease was synchronous with the growth of strain in liquid fermentation. The amino acids analysis of 48hr fermented liquid showed that this strain secreted some kinds of exopeptidase, which can specially hydrolyze three hydroxy amino acids-Tyr. Phe, and His.
(2) After the study of effect of inoculation volume on peptide yield, four factors(defatted sovbean concentration, initial pH of fermented culture, fermentation
temperature and time) affecting peptide yield were optimized through orthogonal experiment. The results showed that 5% of inoculation is fittest for this strain to produce peptide. The best fermentation conditions of this strain were: defatted soybean 3g, H2O 50ml, pH natural, temperature 30 , time 48hr. The acid-soluable peptide content of the peptide solution produced by this strain after optimization could be over 1000 g/ml.
(3) The effects of factors, including chitin concentration, area, and pH value, on absorption of Bacillus subtilis Shi HeziIII was studied. Besides, three quality indexes (acid-soluable peptide content, the average length of peptide, bitter value) of different fermentation batches were determined. It showed that the best absorbent condition for chitin to absorb the strain was whole piece of chitin 0.2g, pH 6.0. The stability of various batches was ideal. Three indexes of every batch of 48hr fermentation culture were as follows: acid-soluble peptide content was over 1 000 g/ml, average length of peptide 3.5, and bitter value 2. It could be concluded that chitin could not only promote the growth of the strain but be an effective bioreactor.
(4) The processing specific properties of soybean peptide, such as nitrogen soluable index(NSI), turbidity, emulsifying characteristics, were studied. The result showed that peptide solutions with various fermentation time had different processing properties. I . Degree of hydrolyzation increased with fermentation time. II. NSI was related to both fermentation time and pH value, the longer time was, the higher NSI was, NSI was relatively high in neutral pH. III. The lower pH was, the higher turbidity was. The turbidity of 36hr peptide solution was low from pH3.0 to 7.0. IV. All the peptide solutions had high emulsifying active index(EAI) but low emulsifying stability index(ESI).
利用食品级的枯草芽孢杆菌直接水解大豆蛋白，一步法完成水解和脱苦，降低了生产成本，加速了功能性多肽的产业化进程。因此，枯草芽孢杆菌生产脱苦大豆功能性多肽是一种具有广泛应用前景的方法，此方法在国内外尚未有相关报道。本论文进行了以下四方面的研究：(1)脱苦大豆功能性多肽产生菌的筛选；(2)液体发酵条件的优化研究；(3)吸附法固定枯草芽孢杆菌液体发酵产大豆多肽的研究；(4)枯草芽孢杆菌发酵产大豆多肽溶液的加工功能特性研究。研究结果如下：
(1) 对本实验室保藏的八株枯草芽孢杆菌(Bacillus subtilis)进行摇瓶试验，采用苦味值和10％TCA酸溶性多肽含量两项指标进行筛选，得到一株适合于水解豆粕粉的菌株B. subtilis石河子Ⅲ。液态发酵过程测定结果表明，该菌株的产蛋白酶类型为同步合成型。对其48小时发酵液进行氨基酸产物的分析结果表明，该菌株产生的羧肽酶能特异性地作用于酪氨酸、苯丙氨酸这两种疏水性氨基酸。
(2) 研究了接种量对多肽产量影响，结果表明，B. subtilis石河子Ⅲ菌株发酵产多肽的最佳接种量为5％。同时，对影响多肽产量的豆粕粉浓度、发酵液初始pH值、发酵温度、时间四因素用正交试验方法进行优化。由正交试验结果可知，该菌株发酵产多肽的最佳条件为：豆粕粉3g，水50ml，pH 6.5(自然pH)，温度30℃，发酵时间48hr。优化后该菌株发酵豆粕粉后的多肽溶液酸溶性多肽含量能维持在1000μg／ml以上。
(3) 研究了甲壳素浓度、面积、种子液pH值等因素对吸附B. subtilis菌体效
果的影响。此外，还测定了不同吸附批次的多肽产量、平均肽链长度等质
量指标。结果表明，甲壳素吸附菌体最佳条件为：0．一％左右的整条甲壳
素（面积约为4cm’），种子液PH值为6。不同发酵批次的稳定性较好，每
一批次的 4 8 h r发酵液中酸溶性多肽含量维持在 10 0 0p g／ml以上，平均链
长为3．5左右，苦味值为2．0。可见，甲壳素不仅对菌体的生长有促进作
用，还能成为一种有效的固定化细胞载体。
（4）研究了大豆多肽溶液的加工功能特性，例上。可溶性氮指数 NS、浊度、乳
化性等。结果表明，不同发酵时间的多肽溶液有着不同的加工特性。水解
度m随着发酵时间的延长而上升；可溶性氮指数（NSI）与发酵时间和训
值均有关，总体上发酵时间越长，NSI越大，中性叩值T NSI较高，酸
性和碱性叩值下 NS较低；相同发酵时间的多肽溶液，PH值越低，浊度
越高，36hr的多肽溶液在nH3．0－7．0范围内，浊度较低；多肽溶液均有
着较好的乳化性，但乳化稳定性较差。
Soybean peptide, which can be a kind of potential function food, is produced by hydrolyzing soybean protein into peptide with 3-6 amino acid residues. However, some bitter components, affecting the flavour of products, will be produced after hydrolyzation of soy protein.
The methods of debittering existing now are not only laborious but of high cost. It cuts down production cost, and accelerates the industrialzation of functional peptide to produce peptide which combines hydrolyzing soy protein and debittering in one step with food-grade Bacillus sub tills. Thus the production of debittered functional soybean peptide with Bacillus subtilis will have widely-use prospect. There are no relative internal and external reports so far. This thesis focused on the following four aspects: (1)Screening of strain producing debittered functional soybean peptide; (2)Optimizing its liquid fermentation conditions; (3)Study of the soybean peptide production by stabilization of Bacillus subtilis: (4)Processing properties of soybean peptide produced by Bacillus subtilis. The results were as follows: (1)According to two indexes, bitterness value and content of 10%TCA soluable peptide, a strain coded Bacillus subtilis Shi HeziIII which was fit for hydrolyzing defatted soybean was screened out fr
om eight Bacillus subtilis strains stored in the lab. The results of determination during liquid fermentation showed that the production of protease was synchronous with the growth of strain in liquid fermentation. The amino acids analysis of 48hr fermented liquid showed that this strain secreted some kinds of exopeptidase, which can specially hydrolyze three hydroxy amino acids-Tyr. Phe, and His.
(2) After the study of effect of inoculation volume on peptide yield, four factors(defatted sovbean concentration, initial pH of fermented culture, fermentation
temperature and time) affecting peptide yield were optimized through orthogonal experiment. The results showed that 5% of inoculation is fittest for this strain to produce peptide. The best fermentation conditions of this strain were: defatted soybean 3g, H2O 50ml, pH natural, temperature 30 , time 48hr. The acid-soluable peptide content of the peptide solution produced by this strain after optimization could be over 1000 g/ml.
(3) The effects of factors, including chitin concentration, area, and pH value, on absorption of Bacillus subtilis Shi HeziIII was studied. Besides, three quality indexes (acid-soluable peptide content, the average length of peptide, bitter value) of different fermentation batches were determined. It showed that the best absorbent condition for chitin to absorb the strain was whole piece of chitin 0.2g, pH 6.0. The stability of various batches was ideal. Three indexes of every batch of 48hr fermentation culture were as follows: acid-soluble peptide content was over 1 000 g/ml, average length of peptide 3.5, and bitter value 2. It could be concluded that chitin could not only promote the growth of the strain but be an effective bioreactor.
(4) The processing specific properties of soybean peptide, such as nitrogen soluable index(NSI), turbidity, emulsifying characteristics, were studied. The result showed that peptide solutions with various fermentation time had different processing properties. I . Degree of hydrolyzation increased with fermentation time. II. NSI was related to both fermentation time and pH value, the longer time was, the higher NSI was, NSI was relatively high in neutral pH. III. The lower pH was, the higher turbidity was. The turbidity of 36hr peptide solution was low from pH3.0 to 7.0. IV. All the peptide solutions had high emulsifying active index(EAI) but low emulsifying stability index(ESI).
引文
1) 丁纯孝，新大豆蛋白食品，中国粮油食品，1985，1：31-33
2) 邓勇 吴煜欢，大豆多肽研究与开发：现状·问题-建议，中国农业大学学报，1994，4(4)：89-93
3) Sook Yk., Functional Properties of protelytic Enzyme Modified Soy Protein Isolates, J Agric Food Chem, 1990,38(3):651-656
4) 严栋林，周静，大豆多肽的功能和生产，粮食加工新技术，中日食品新科技研讨会论文集。
5) 唐传和，彭志英，大豆蛋白水解物的苦肽以及脱苦方法进展，2000，25(6)：167-173
6) Norio Ishibashi., Yasuhiro Arita., Hidenori et al., Bitterness of L-leucine-containing Peptides, Agric. Biol. Chem, 1987, 51(9): 2389-2394
7) Ney KH., Bitterness of Peptides:amino acid composition and chain length, Washington (DC): American Chemical Society, 1979, 149-173
8) Ney KH, Gisela R., A computer program predicting the bitterness of peptides esp. in protein hydrolyzates, based on amino acid composition and chain length(computer Q), Dev Food Sci, 1986, 12:543-550
9) Ishibashi N., Bitterness of Leucine- containing peptides, Agric. Biol. Chem, 1987, 51 (9):2389-2394
10) Ishibashi N., Bitterness of Phenylalanine and tyrosine-containing peptides, Agric. Biol. Chem, 1987, 51(2):3309-3313
11) Ishibashi N., Taste of proline-containing peptides, Agric. Biol. Chem, 1988, 52
(1) 95-98
12) Tanford C., Contribution of hydrophobic interactions to the stability of the globular conformation of proteins[J]. J. Am. Chem. Soc, 1962, 84:4240-4247
13) Ishibashi N., A mechanism for bitter taste sensibility in peptides, Agric. Biol. Chem, 1988, 52(3): 819-827
14) David, E. S., Diana, J.O., Keith,R.M., Roger, A.S. Protease-catalyzed condensation of peptides as a potential means to reduce the bitter taste of hydrophobic peptides found in protein hydrolysates. Enzyme and Microbial Technology. 1998,22,100-110
15) Pederson.B., Removing Bitterness from protein hydrolysate. Food Technology,1994,10:96-98
16) Andre D．Siemensma，肽链长度对婴儿抗过敏性代乳品的意义，中国乳品工业，1994，22(6)：273-275
17) Minagawa E.,et al. Debittering mechanics in bitter peptides of enzymic hydrolysates from milk casein by aminopeptidase. J.Food Sci., 1989,54(5):1125-1129
18) H.Sumi., Accumulation of Vatamin K(menaquinone-7)in plasma after ingestion of Natto and Natto Bacilli (B.subtilis Natto). http:www.tenprint.com.jp/dnlenglish.htm
19) Matsuoka H, Fuke Y, et al., Purification and Debittering effect of aminopeptidase Ⅱ from Penicillium caseicolum, Agric Food Chem, 1991, 39: 1392-1395
20) 许永红，蛋白酶法水解物苦味的控制，食品工业科技，1997(3)：1-3
21) 吴建平，日本生理活性肽的市场动态。食品工业，1998，(3)：8～9
22) 易维学，大豆低聚肽的功能性质及其应用，食品配料专辑，2001，8(2)：36-38
23) 庞广昌，陈庆森等，生物活性肽的研究进展理论基础与展望，食品科学，2001，22(2)：80-83
24) Andre D．Siemensma，肽链长度对婴儿抗过敏性代乳品的意义，中国乳品工
业，1994，22(6)：273-275
25) M.Fukutake. M.Takahashi et al., Quantification of genstein and genistin in soybeans and soybean products., Food and Chemical Toxiology, 1996, 34: 457-461
26) 盛国华，大豆多肽的功能及应用，食品工业科技，1993(6)：21-26
27) 葛文光，大豆多肽的生理功能及作用效果，无锡轻工大学学报，1996，(3)：272-27
28) 潘进权，刘耘，大豆多肽研究概况，中国调味品，2003(2)：6-10
29) Hiroatsu Matsuoka. Purification and Debittering effect of Aminopeptidase Ⅱ from Penicilium caseicolum. J. Agric. Food Chem. 1991, (39):1392～1395
30) Hironori Umetsu, Hiroatu Matsuoka., and Eiji Ichishima. Debitter Peptides from Milk Casein by Wheat Carboxypeptidase. J Agric.Food Chem..1983,(31):50～53
2) 邓勇 吴煜欢，大豆多肽研究与开发：现状·问题-建议，中国农业大学学报，1994，4(4)：89-93
3) Sook Yk., Functional Properties of protelytic Enzyme Modified Soy Protein Isolates, J Agric Food Chem, 1990,38(3):651-656
4) 严栋林，周静，大豆多肽的功能和生产，粮食加工新技术，中日食品新科技研讨会论文集。
5) 唐传和，彭志英，大豆蛋白水解物的苦肽以及脱苦方法进展，2000，25(6)：167-173
6) Norio Ishibashi., Yasuhiro Arita., Hidenori et al., Bitterness of L-leucine-containing Peptides, Agric. Biol. Chem, 1987, 51(9): 2389-2394
7) Ney KH., Bitterness of Peptides:amino acid composition and chain length, Washington (DC): American Chemical Society, 1979, 149-173
8) Ney KH, Gisela R., A computer program predicting the bitterness of peptides esp. in protein hydrolyzates, based on amino acid composition and chain length(computer Q), Dev Food Sci, 1986, 12:543-550
9) Ishibashi N., Bitterness of Leucine- containing peptides, Agric. Biol. Chem, 1987, 51 (9):2389-2394
10) Ishibashi N., Bitterness of Phenylalanine and tyrosine-containing peptides, Agric. Biol. Chem, 1987, 51(2):3309-3313
11) Ishibashi N., Taste of proline-containing peptides, Agric. Biol. Chem, 1988, 52
(1) 95-98
12) Tanford C., Contribution of hydrophobic interactions to the stability of the globular conformation of proteins[J]. J. Am. Chem. Soc, 1962, 84:4240-4247
13) Ishibashi N., A mechanism for bitter taste sensibility in peptides, Agric. Biol. Chem, 1988, 52(3): 819-827
14) David, E. S., Diana, J.O., Keith,R.M., Roger, A.S. Protease-catalyzed condensation of peptides as a potential means to reduce the bitter taste of hydrophobic peptides found in protein hydrolysates. Enzyme and Microbial Technology. 1998,22,100-110
15) Pederson.B., Removing Bitterness from protein hydrolysate. Food Technology,1994,10:96-98
16) Andre D．Siemensma，肽链长度对婴儿抗过敏性代乳品的意义，中国乳品工业，1994，22(6)：273-275
17) Minagawa E.,et al. Debittering mechanics in bitter peptides of enzymic hydrolysates from milk casein by aminopeptidase. J.Food Sci., 1989,54(5):1125-1129
18) H.Sumi., Accumulation of Vatamin K(menaquinone-7)in plasma after ingestion of Natto and Natto Bacilli (B.subtilis Natto). http:www.tenprint.com.jp/dnlenglish.htm
19) Matsuoka H, Fuke Y, et al., Purification and Debittering effect of aminopeptidase Ⅱ from Penicillium caseicolum, Agric Food Chem, 1991, 39: 1392-1395
20) 许永红，蛋白酶法水解物苦味的控制，食品工业科技，1997(3)：1-3
21) 吴建平，日本生理活性肽的市场动态。食品工业，1998，(3)：8～9
22) 易维学，大豆低聚肽的功能性质及其应用，食品配料专辑，2001，8(2)：36-38
23) 庞广昌，陈庆森等，生物活性肽的研究进展理论基础与展望，食品科学，2001，22(2)：80-83
24) Andre D．Siemensma，肽链长度对婴儿抗过敏性代乳品的意义，中国乳品工
业，1994，22(6)：273-275
25) M.Fukutake. M.Takahashi et al., Quantification of genstein and genistin in soybeans and soybean products., Food and Chemical Toxiology, 1996, 34: 457-461
26) 盛国华，大豆多肽的功能及应用，食品工业科技，1993(6)：21-26
27) 葛文光，大豆多肽的生理功能及作用效果，无锡轻工大学学报，1996，(3)：272-27
28) 潘进权，刘耘，大豆多肽研究概况，中国调味品，2003(2)：6-10
29) Hiroatsu Matsuoka. Purification and Debittering effect of Aminopeptidase Ⅱ from Penicilium caseicolum. J. Agric. Food Chem. 1991, (39):1392～1395
30) Hironori Umetsu, Hiroatu Matsuoka., and Eiji Ichishima. Debitter Peptides from Milk Casein by Wheat Carboxypeptidase. J Agric.Food Chem..1983,(31):50～53