亚基缺失特异大豆品种的筛选及β亚基对大豆加工特性的影响
|
摘要
大豆蛋白质是优质的植物蛋白源，其主要组分为11S(glycinin)和7S(β-conglycinin)。11S(glycinin)是由酸性亚基A和碱性亚基B构成的，A和B形成五种复合体：Ⅰ(A_(2a)B_2，A_(1b)B_(1b)，A_2B_(1a))和Ⅱ(A_5A_4B_3、A_3B_4)；7S(β-conglycinin)是由α’，α和β三个亚基构成的。
本研究以1624份大豆品种为材料，用SDS-PAGE电泳和western-blotting电泳的方法，对蛋白亚基的缺失情况进行了分析和鉴定。并进一步以缺少或缺失β亚基的大豆为原料，研究了β亚基对大豆加工特性的影响。
研究结果表明，我国的大豆品种有许多A_3、A_5、γ、β亚基缺少或缺失的天然变异的材料，其中我们首次发现了缺失β亚基的特异大豆品种。经western-blotting分析结果证实该品种大豆确实缺失β亚基。对其氨基酸分析结果表明，该品种含硫氨基酸含量非常丰富。
以β亚基含量不同的大豆为原料调制豆乳时，对其豆乳中蛋白质溶出状态的研究结果表明，β亚基缺失品种的豆乳中蛋白质粒子的相对含量明显高于普通大豆调制的豆乳，而且对Ca~(2+)引起的凝聚反应较敏感；然而当β亚基含量增加时，豆乳中粒子含量逐渐减少。SDS-PAGE电泳分析β亚基含量不同的豆乳粒子构成表明，随着豆乳中β亚基含量的增加，粒子组成中β亚基的含量也增加，但碱性亚基的含量却减少。当蛋白浓度一定时，用富含β亚基的大豆蛋白调整豆乳中β亚基的含量，制成豆乳凝胶，然后分析其质构特性，结果表明，随着β亚基含量的增加，豆腐凝胶的硬度、弹性等质构特性均降低。对不同品种大豆分离蛋白的功能特性的研究结果表明，缺失β亚基的品种凝胶质构特性较差，乳化稳定性也较低。以上结果表明β亚基在大豆的加工过程中对蛋白质存在形态和凝胶网络结构的形成以及大豆分离蛋白的功能特性起着重要的作用。
Soybean is a good dietary protein resource.11S (glycinin) and 7S (p-conglycinin) are major components in soybean. Glycinin is consisted of acidic subunits (A) and basic subunits (B), Acidic and basic subunits formed five complexes: group I (A1aB2, A1bB1b,A2B1a) and II(A5A4B3, A3B4), P-conglycinin is consisted of a', a, and p subunits.
The soybean protein subunit compositions of more than 1624 varieties were analyzed by SDS-PAGE and western-blotting methods in this study, and the lacking extent of the p subunits was analyzed and identified. The effect of p subunits on soybean process properties were used the low or lacking p subunits soybean as materials.
The results, showed that there were many native aberrant materials which having low content of or lacking A3, A, γ,β subunits among the soybean varieties in China, and the special soy variety lacking p subunits was found for the first time. The results of the western-blotting analysis confirmed this result. The result of amino acid analysis of the variety lacking the p subunits showed that the suit-ammo acid content was high in this variety.
The extraction states of protein in soymilk prepared with the varieties of different p subunit content were investigated. The results showed that the relative content of the protein particles in soymilks prepared with lacking p subunit was higher than the common obviously, and the protein particles were more sensitive to the coagulate. Reaction caused by the calciulmion and hydrogenion. However, as the increase of the content of p subunit, the protein particles in soymilk decreased gradually. The constitutes of protein particles in soymilks prepared with varieties containing different p subunit content were analyzed by SDS-PAGE, the results showed that the more p subunit in soymilks, the more p subunit content in protein particles, and the less basic subunit content. Keeping the protein concentration constantly, the p subunit content in soymilk was adjusted by soy protein riched p subunit, and the textural characteristics of tofu gel prepared with adjusted soymilk were analyzed, the results showed that the hardness and springiness of tofu gel decreased with p subunit content increasing, the whole texture characteristics such as the hardness and the flexibility of the tofu gel decrease. The functional properties of various SPI were investigated, the results indicated that the variety lacking p subunits showed the lower gel textural propeties and emulsified stability. As the results above, the p subunits have important effect on exist state of the protein and the gel network during the soybean process.
本研究以1624份大豆品种为材料，用SDS-PAGE电泳和western-blotting电泳的方法，对蛋白亚基的缺失情况进行了分析和鉴定。并进一步以缺少或缺失β亚基的大豆为原料，研究了β亚基对大豆加工特性的影响。
研究结果表明，我国的大豆品种有许多A_3、A_5、γ、β亚基缺少或缺失的天然变异的材料，其中我们首次发现了缺失β亚基的特异大豆品种。经western-blotting分析结果证实该品种大豆确实缺失β亚基。对其氨基酸分析结果表明，该品种含硫氨基酸含量非常丰富。
以β亚基含量不同的大豆为原料调制豆乳时，对其豆乳中蛋白质溶出状态的研究结果表明，β亚基缺失品种的豆乳中蛋白质粒子的相对含量明显高于普通大豆调制的豆乳，而且对Ca~(2+)引起的凝聚反应较敏感；然而当β亚基含量增加时，豆乳中粒子含量逐渐减少。SDS-PAGE电泳分析β亚基含量不同的豆乳粒子构成表明，随着豆乳中β亚基含量的增加，粒子组成中β亚基的含量也增加，但碱性亚基的含量却减少。当蛋白浓度一定时，用富含β亚基的大豆蛋白调整豆乳中β亚基的含量，制成豆乳凝胶，然后分析其质构特性，结果表明，随着β亚基含量的增加，豆腐凝胶的硬度、弹性等质构特性均降低。对不同品种大豆分离蛋白的功能特性的研究结果表明，缺失β亚基的品种凝胶质构特性较差，乳化稳定性也较低。以上结果表明β亚基在大豆的加工过程中对蛋白质存在形态和凝胶网络结构的形成以及大豆分离蛋白的功能特性起着重要的作用。
Soybean is a good dietary protein resource.11S (glycinin) and 7S (p-conglycinin) are major components in soybean. Glycinin is consisted of acidic subunits (A) and basic subunits (B), Acidic and basic subunits formed five complexes: group I (A1aB2, A1bB1b,A2B1a) and II(A5A4B3, A3B4), P-conglycinin is consisted of a', a, and p subunits.
The soybean protein subunit compositions of more than 1624 varieties were analyzed by SDS-PAGE and western-blotting methods in this study, and the lacking extent of the p subunits was analyzed and identified. The effect of p subunits on soybean process properties were used the low or lacking p subunits soybean as materials.
The results, showed that there were many native aberrant materials which having low content of or lacking A3, A, γ,β subunits among the soybean varieties in China, and the special soy variety lacking p subunits was found for the first time. The results of the western-blotting analysis confirmed this result. The result of amino acid analysis of the variety lacking the p subunits showed that the suit-ammo acid content was high in this variety.
The extraction states of protein in soymilk prepared with the varieties of different p subunit content were investigated. The results showed that the relative content of the protein particles in soymilks prepared with lacking p subunit was higher than the common obviously, and the protein particles were more sensitive to the coagulate. Reaction caused by the calciulmion and hydrogenion. However, as the increase of the content of p subunit, the protein particles in soymilk decreased gradually. The constitutes of protein particles in soymilks prepared with varieties containing different p subunit content were analyzed by SDS-PAGE, the results showed that the more p subunit in soymilks, the more p subunit content in protein particles, and the less basic subunit content. Keeping the protein concentration constantly, the p subunit content in soymilk was adjusted by soy protein riched p subunit, and the textural characteristics of tofu gel prepared with adjusted soymilk were analyzed, the results showed that the hardness and springiness of tofu gel decreased with p subunit content increasing, the whole texture characteristics such as the hardness and the flexibility of the tofu gel decrease. The functional properties of various SPI were investigated, the results indicated that the variety lacking p subunits showed the lower gel textural propeties and emulsified stability. As the results above, the p subunits have important effect on exist state of the protein and the gel network during the soybean process.
引文
1. Nishinari K, Kohyama K, Zhang Y, et al. Rheological study on the Effect of the A_5 Subunit on the Gelation Characteristics of Soybean proteins. Agric biol.chem. 1991,55(2):351-355
2．菊池彰夫，酒井真次，岡部昭典．子実成分遗伝的变异豆腐加工適性．东北农业研究，1989，42：111-112
3．内海成．种子质高品质化关食品化学遗伝子工学的研究．Nippon Nogeikagaku Kaishi，1989，63(9)：1471-1478
4．喜多村 啓介．栄养性及加工適性改良育种．日本醸造协会志，1994，12：926-931
5．羽鹿牧太，高桥将一，酒井真次等，新品种育成特性，2002年，第40号，79-94
6．陈海敏，华欲飞．品种差异对犬豆蛋白质功能性的影响．中国油脂，2000，6：178-180
7．中国大豆品质区划，农业部种植管理司编著，中国农业出版社2003
8．赵文述，孟祥勋，顾其敏等．中国大豆种子蛋白质SBTi-A2电泳谱带新类型的遗传研究．大豆科学，1995，14(1)，36-39
9．麻浩，官春云．大豆脂肪氧化酶生理作用研究进展．大豆科学，1999，18(1)：62-66
10．麻浩，官春云．大豆种子脂肪氧化酶与豆制品产生腥味关系的研究进展，中国油料作物学报，1999，21(2)，73-76
11．第七届全国大豆学术讨论会论文摘要集，2001，5月，48-49，41-42
12．徐豹等，野生大豆种子贮藏蛋白组分11S／7S的研究．作物学报，1990，3，235-241
13．松本幸雄．食品物性 日本：朝仓书店，1982第8集
14．山内文男，大久保一良．大豆的科学 日本：朝仓书店，1992，27-53
15．山内文男．大豆蛋白质耩造食品特性．Nippon shokuhin Kogyo Gakkaishi, 1994, 41(3)：233-240
16. Paul E.Staswick, Mark A.Hermodson, Niels C.Nielsen, at al. Identification of the Acidic and Basic Subunit Complexes of Glycinin. The Jouranl of Biological Chemistry, 1981,256(16): 8752-8755
17. Silvana P, Maria C. Soy protein Isoiate Components and Their Interactions. J. of Agric.Food Chem, 1995,43:1762-1767
18．张红城，彭至英，赵谋明等．大豆蛋白及其制品的研究．粮食与饲料工业，1998，6，36-38
19．内海成．质构造机能探．Nippon Shokuhin Kagaku Kogaku Kaishi，1988．48．15-16
20. Gatriona M, Lakemond M, Harmen H ea al. Differences in Denaturation of Genetic Variants of Soy Glycinin. J.Agric Food Chem, 2002, 50: 4275-4281
21. Maruyama N,Sato R, Wada Y, et al. Structure-Physicochemical Function Relationships of Soybean β-Conglycinin Constituent Subunits. J.Agric.Food Chem,1999,47:5278-5284
22. Hirano H, Kagawa H, Kamata Y, et al. structural homology among the major 7S globulin subunits of soybean seed storage proteins, 1987,26(1):41-45
23．石彦国，任丽．大豆制品工艺学北京：中国轻工业出版社，2001，70-71
24. Muruyama N., Katsube T., Wada Y., et al.The roles of the N-linked glycans and extension regions of soybean β-conglycinin folding, assembly and structural features. Eur. J. Biochem, 1998,258,854-862
25. Fukushim D. Structures of plant storage proteins and their functions. Food Reviews International, 1991,7(3):353-379
26．森友彦．大豆蛋白质形成机构．Nippon Nogeikagaku Kaishi，1988，62，5，882-885
27．周瑞宝，7S和11S蛋白的结构与功能．中国粮油学报，1998，13(6)：39-42
28．陈海敏，华欲飞编著．大豆蛋白组成与功能关系研究．西部粮油科技，2001，26(3)，36-38
29．胡超，黄丽华，李文哲．大豆球蛋白11S／7S比值对大豆蛋白功能性的影响．中国粮油学报，2004，19(1)：40-42
30. Patricia a.Murphy, Hui-ping Chen, Catherine c.Hauck, et al. Soybean Protein Composition and Tofu Quality, Food Technology. 1997,51 (3):86-88
31．小野伴忠．大豆质凝固反应基本-凝固机构要因．大豆技术，1995，64-68
32. Guo shun-tang, T Ono. M, Mikami, Interaction between Protein and Lipid in Soybean Milk at Elevated Temperature. J.Agric.Food and Chem., 1997,45(12):4601-4605
33. Takahashi K, Takada Y, Kikuchi A,shimada,H.;Adachi,T.;Tabuchi,K,Tohoku. Agric.Res., 1998,51:71-72
34. Nishinari k, k kohyama, Y Zhang, et al. Rheological study on the effect of the A5 subunit on the geltaion characteristics of soybeen prote.in. Agric.Biol.Chem, 1991,55,351-355
35. Ono T, Choi M.R, Ikeda A, et al. Changes in the Composition and Size Distribution of Soymilk protein particles by Heating. Agric.Biol.Chem., 1991,55:2291
36. Tezuka M, Taira H, Igarashi Y, et al. Properties of Tofus and Soy Milks Prepared from Soybeans Having Different Subunits of Glycinin. J.Agric.Food Chem, 2000,48:1111-1117
37．钱虎君，盖钧镒，吉东风等．豆腐产量、品质与籽粒营养成分及加工性状的关系．中国粮油学报，1999，14(5)：35-39
38．武天龙，杨庆凯，孟庆喜．大豆不同品种豆腐产量和质量的研究分析．大豆科学，1986，5(3)：189-195
39．金俊培，盖钧镒．大豆地方品种豆腐产量、品质及有关加工性状的相关．中国农业科学，1996，29，(2)：28-33
40．许显滨，陈霞，赵乃新等．大豆品种11SA_5端球蛋白与豆腐加工特性的研究．大豆科学，1991，10(3)：250-251
41. Laemmli U. K. Cleavage of structrural proteins during the sembly of the head of bacteriophage T4. Nature, 1970,227:680-685
42．落合(广黄)牍，神公明，垣原得彰等．糖质回收抗原特性．生化学，1987，59(10)：1164-1167
43．铃木春男．质溶液硫酸简单除去法．生化学，1989，61(2)：109-111
44．前岛正羲，中川强，旭正．电気泳动质分离回收植物贮藏质膜酵素例．生化学，1987，59：1040-1042
45．赵永芳编著．生物化学技术原理及其应用．武汉：武汉大学出版社，1994
46．刘平黄 利用PRRS病毒重组N蛋白建立ELISA诊断试剂盒的研究学位论文 北京：中国农业大学 2001
47．奥村宣明．电气泳动法，2002，26-31
48．郭尧君编著．蛋白质电泳实验技术．北京：科学出版社，1999
49．沈关心，龚非力等译，抗体技术实验指南，北京：科学出版社，2003，161-184
50．王浩丹 周申 主编 生物医学标记示踪技术 北京：人民卫生出版社 1995
51．李建武，余瑞元，袁明秀等合编，生物化学实验原理和方法，北京：北京大学出版社．2000，160-163
52. Kevin N Pearce, John E Kinsella. Emulsifying Proterties of Proteins: Evaluation of a Turbidimetric Technique. J. Agric.Food Chem, 1978,26(3):
53. Bernard e chore, Alistair s grand,son, Michael j lewis. Emulsifying properties of soy Protein isolates obteained by microfiltration. J Sci Food Agric, 2002,(82):267-272
54．山岸辰则，山内文男．食品物性，第12集，128-129
55．陈复生，李里特．分子力对大豆蛋白透明凝胶作用机理研究．食品科学，2001，22(10)：27-32
56．钱虎君，盖钧镒，喻德跃．大豆豆乳产量、品质及加工性状德遗传变异和遗传规律研究．作物学报，2001，6(11)：880-885
57．钱虎君，盖钧镒，喻德跃．大豆豆乳性状的遗传育种研究进展．中国油料作物学报，2003，25：97-100
58. Guo st, C.tsukamoto, K.takahasi, et al. Incorporation of Soymilk Lipid into Soy protein Coagulum by the Addition of Calcium Chloride. J.Food Science, 2002, 67(9): 3215～3219
59. Bradford M. M. A rapid and sensitive method for quantitation of protein utilizing the principle of protein-dye-binding. Analytical Biochemistry, 1978,72:248-258
60．小野伴忠．豆乳中成分存在形态豆腐形成．大豆技术，1992，14：28-33
61．陈炳卿主编．营养与食品卫生学第三版，北京：人民卫生出版社，1999，79
62. Saio K, Koyama E, Yamazaki S. Protein-Calcium-Phytic Acid relationships in soybean(Ⅲ): Effect of Phytic acid on coagulative reaction in tofu-making. Agri Biol Chem, 1969,33,36-42
63. Schaeffer M J, Love J. Relationships between soybean components and tofu texture. J of Food Qualiti,1992,15,53-58
64. Wang H L, Swain E W, Kwolek W F. Effect of soybean varieties on the yield and quality of tofu. Cereal Chemistry,1983,60(3):245-248
2．菊池彰夫，酒井真次，岡部昭典．子実成分遗伝的变异豆腐加工適性．东北农业研究，1989，42：111-112
3．内海成．种子质高品质化关食品化学遗伝子工学的研究．Nippon Nogeikagaku Kaishi，1989，63(9)：1471-1478
4．喜多村 啓介．栄养性及加工適性改良育种．日本醸造协会志，1994，12：926-931
5．羽鹿牧太，高桥将一，酒井真次等，新品种育成特性，2002年，第40号，79-94
6．陈海敏，华欲飞．品种差异对犬豆蛋白质功能性的影响．中国油脂，2000，6：178-180
7．中国大豆品质区划，农业部种植管理司编著，中国农业出版社2003
8．赵文述，孟祥勋，顾其敏等．中国大豆种子蛋白质SBTi-A2电泳谱带新类型的遗传研究．大豆科学，1995，14(1)，36-39
9．麻浩，官春云．大豆脂肪氧化酶生理作用研究进展．大豆科学，1999，18(1)：62-66
10．麻浩，官春云．大豆种子脂肪氧化酶与豆制品产生腥味关系的研究进展，中国油料作物学报，1999，21(2)，73-76
11．第七届全国大豆学术讨论会论文摘要集，2001，5月，48-49，41-42
12．徐豹等，野生大豆种子贮藏蛋白组分11S／7S的研究．作物学报，1990，3，235-241
13．松本幸雄．食品物性 日本：朝仓书店，1982第8集
14．山内文男，大久保一良．大豆的科学 日本：朝仓书店，1992，27-53
15．山内文男．大豆蛋白质耩造食品特性．Nippon shokuhin Kogyo Gakkaishi, 1994, 41(3)：233-240
16. Paul E.Staswick, Mark A.Hermodson, Niels C.Nielsen, at al. Identification of the Acidic and Basic Subunit Complexes of Glycinin. The Jouranl of Biological Chemistry, 1981,256(16): 8752-8755
17. Silvana P, Maria C. Soy protein Isoiate Components and Their Interactions. J. of Agric.Food Chem, 1995,43:1762-1767
18．张红城，彭至英，赵谋明等．大豆蛋白及其制品的研究．粮食与饲料工业，1998，6，36-38
19．内海成．质构造机能探．Nippon Shokuhin Kagaku Kogaku Kaishi，1988．48．15-16
20. Gatriona M, Lakemond M, Harmen H ea al. Differences in Denaturation of Genetic Variants of Soy Glycinin. J.Agric Food Chem, 2002, 50: 4275-4281
21. Maruyama N,Sato R, Wada Y, et al. Structure-Physicochemical Function Relationships of Soybean β-Conglycinin Constituent Subunits. J.Agric.Food Chem,1999,47:5278-5284
22. Hirano H, Kagawa H, Kamata Y, et al. structural homology among the major 7S globulin subunits of soybean seed storage proteins, 1987,26(1):41-45
23．石彦国，任丽．大豆制品工艺学北京：中国轻工业出版社，2001，70-71
24. Muruyama N., Katsube T., Wada Y., et al.The roles of the N-linked glycans and extension regions of soybean β-conglycinin folding, assembly and structural features. Eur. J. Biochem, 1998,258,854-862
25. Fukushim D. Structures of plant storage proteins and their functions. Food Reviews International, 1991,7(3):353-379
26．森友彦．大豆蛋白质形成机构．Nippon Nogeikagaku Kaishi，1988，62，5，882-885
27．周瑞宝，7S和11S蛋白的结构与功能．中国粮油学报，1998，13(6)：39-42
28．陈海敏，华欲飞编著．大豆蛋白组成与功能关系研究．西部粮油科技，2001，26(3)，36-38
29．胡超，黄丽华，李文哲．大豆球蛋白11S／7S比值对大豆蛋白功能性的影响．中国粮油学报，2004，19(1)：40-42
30. Patricia a.Murphy, Hui-ping Chen, Catherine c.Hauck, et al. Soybean Protein Composition and Tofu Quality, Food Technology. 1997,51 (3):86-88
31．小野伴忠．大豆质凝固反应基本-凝固机构要因．大豆技术，1995，64-68
32. Guo shun-tang, T Ono. M, Mikami, Interaction between Protein and Lipid in Soybean Milk at Elevated Temperature. J.Agric.Food and Chem., 1997,45(12):4601-4605
33. Takahashi K, Takada Y, Kikuchi A,shimada,H.;Adachi,T.;Tabuchi,K,Tohoku. Agric.Res., 1998,51:71-72
34. Nishinari k, k kohyama, Y Zhang, et al. Rheological study on the effect of the A5 subunit on the geltaion characteristics of soybeen prote.in. Agric.Biol.Chem, 1991,55,351-355
35. Ono T, Choi M.R, Ikeda A, et al. Changes in the Composition and Size Distribution of Soymilk protein particles by Heating. Agric.Biol.Chem., 1991,55:2291
36. Tezuka M, Taira H, Igarashi Y, et al. Properties of Tofus and Soy Milks Prepared from Soybeans Having Different Subunits of Glycinin. J.Agric.Food Chem, 2000,48:1111-1117
37．钱虎君，盖钧镒，吉东风等．豆腐产量、品质与籽粒营养成分及加工性状的关系．中国粮油学报，1999，14(5)：35-39
38．武天龙，杨庆凯，孟庆喜．大豆不同品种豆腐产量和质量的研究分析．大豆科学，1986，5(3)：189-195
39．金俊培，盖钧镒．大豆地方品种豆腐产量、品质及有关加工性状的相关．中国农业科学，1996，29，(2)：28-33
40．许显滨，陈霞，赵乃新等．大豆品种11SA_5端球蛋白与豆腐加工特性的研究．大豆科学，1991，10(3)：250-251
41. Laemmli U. K. Cleavage of structrural proteins during the sembly of the head of bacteriophage T4. Nature, 1970,227:680-685
42．落合(广黄)牍，神公明，垣原得彰等．糖质回收抗原特性．生化学，1987，59(10)：1164-1167
43．铃木春男．质溶液硫酸简单除去法．生化学，1989，61(2)：109-111
44．前岛正羲，中川强，旭正．电気泳动质分离回收植物贮藏质膜酵素例．生化学，1987，59：1040-1042
45．赵永芳编著．生物化学技术原理及其应用．武汉：武汉大学出版社，1994
46．刘平黄 利用PRRS病毒重组N蛋白建立ELISA诊断试剂盒的研究学位论文 北京：中国农业大学 2001
47．奥村宣明．电气泳动法，2002，26-31
48．郭尧君编著．蛋白质电泳实验技术．北京：科学出版社，1999
49．沈关心，龚非力等译，抗体技术实验指南，北京：科学出版社，2003，161-184
50．王浩丹 周申 主编 生物医学标记示踪技术 北京：人民卫生出版社 1995
51．李建武，余瑞元，袁明秀等合编，生物化学实验原理和方法，北京：北京大学出版社．2000，160-163
52. Kevin N Pearce, John E Kinsella. Emulsifying Proterties of Proteins: Evaluation of a Turbidimetric Technique. J. Agric.Food Chem, 1978,26(3):
53. Bernard e chore, Alistair s grand,son, Michael j lewis. Emulsifying properties of soy Protein isolates obteained by microfiltration. J Sci Food Agric, 2002,(82):267-272
54．山岸辰则，山内文男．食品物性，第12集，128-129
55．陈复生，李里特．分子力对大豆蛋白透明凝胶作用机理研究．食品科学，2001，22(10)：27-32
56．钱虎君，盖钧镒，喻德跃．大豆豆乳产量、品质及加工性状德遗传变异和遗传规律研究．作物学报，2001，6(11)：880-885
57．钱虎君，盖钧镒，喻德跃．大豆豆乳性状的遗传育种研究进展．中国油料作物学报，2003，25：97-100
58. Guo st, C.tsukamoto, K.takahasi, et al. Incorporation of Soymilk Lipid into Soy protein Coagulum by the Addition of Calcium Chloride. J.Food Science, 2002, 67(9): 3215～3219
59. Bradford M. M. A rapid and sensitive method for quantitation of protein utilizing the principle of protein-dye-binding. Analytical Biochemistry, 1978,72:248-258
60．小野伴忠．豆乳中成分存在形态豆腐形成．大豆技术，1992，14：28-33
61．陈炳卿主编．营养与食品卫生学第三版，北京：人民卫生出版社，1999，79
62. Saio K, Koyama E, Yamazaki S. Protein-Calcium-Phytic Acid relationships in soybean(Ⅲ): Effect of Phytic acid on coagulative reaction in tofu-making. Agri Biol Chem, 1969,33,36-42
63. Schaeffer M J, Love J. Relationships between soybean components and tofu texture. J of Food Qualiti,1992,15,53-58
64. Wang H L, Swain E W, Kwolek W F. Effect of soybean varieties on the yield and quality of tofu. Cereal Chemistry,1983,60(3):245-248