植物乳杆菌对猪肉蛋白提取物的分解能力及在发酵香肠中的应用
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摘要
论文包括三方面的内容：(1)植物乳杆菌(Lactobacillus plantarum6003，简称Lp6003)的全细胞悬浮液、细胞裂解液(cell extracts，简称CE)、全细胞悬浮液和细胞裂解液(CE)(1：1)的混合物对猪肌浆蛋白提取物和肌原纤维蛋白提取物分解能力的初步研究；(2)发酵香肠使用的常规腌制剂(NaCl、NANO_2、抗坏血酸)和温度对Lp6003的产酸性能及对Lp6003全细胞悬浮液分解猪肉蛋白提取物的能力的影响；(3)接种Lp6003的发酵香肠、接种Lp6003并添加0.1％木瓜蛋白酶(papain)的香肠、自然发酵香肠在成熟过程中的微生物和理化特性的变化。本文的创新点在于初步探讨了国内发酵香肠生产中常用的乳酸菌发酵剂—植物乳杆菌6003在细胞水平上的蛋白水解酶系统及其影响因素。
第一部分：研究了Lp6003的全细胞悬浮液、细胞裂解液、全细胞悬浮液和细胞裂解液等比例混合物对猪肉的肌浆蛋白提取物肌原纤维蛋白提取物的分解能力。结果表明，发酵96小时后，Lp6003全细胞悬浮液对肌浆蛋白提取物具有明显的分解作用，比对肌原纤维蛋白提取物的分解作用强；而单独的CE对两种蛋白提取物均没有明显的分解作用；全细胞悬浮液和CE等比例混合物比单独全细胞悬浮液对两种蛋白提取物的分解作用强，这说明CE加强了Lp6003全细胞对猪肉蛋白提取物的分解作用，并提高了Lp6003细胞的存活力。添加Lp6003全细胞悬浮液的肌浆蛋白提取物中大多数游离氨基酸含量明显下降，肌原纤维蛋白提取物中游离氨基酸含量有所增加；添加全细胞悬浮液和CE等比例混合物增加了肌浆蛋白提取物中Glu、Ala的含量和肌原纤维蛋白提取物中Ala、Lys的含量。
第二部分：研究了发酵香肠生产过程中使用的常规腌制剂和温度对Lp6003产酸性能及Lp6003全细胞分解猪肉中两种蛋白提取物的能力的影响。结果表明，25℃、3％NaCl或150ppmNaNO_2时Lp6003在模拟肉汤培养基中表现出比较适合生产发酵香肠的产酸量，高浓度的NaCl和NaNO_2明显降低了Lp6003的产酸量。常规的发酵温度(25℃)不能促进Lp6003全细胞对肌浆蛋白提取物、肌原纤维蛋白提取物的分解作用，温度是影响Lp6003全细胞蛋白酶活性的重要因素；添加抗坏血酸明显增强了Lp6003全细胞对肌浆蛋白提取物的分解作用；添加NaCl明显增强了Lp6003全细胞对肌原纤维蛋白提取物的分解作用，NaNO_2和抗坏血酸对Lp6003全细胞于此蛋白的分解能力没有影响。
中 文摘 要
第三部分：研究了 Lp删、接种 Lp测并添加 0．l％papain、自然发酵三种
发酵香肠在成熟过程中的微生物和理化特性的变化情况。结果表明，接种LP6003香
肠的乳酸菌在成熟过程中始终处于绝对优势菌状态，保证了产品的安全性和保藏
性，有助于产生乳酸菌特有的发酵特性并能抑制腐败微生物的生长繁殖；接种 LP6003
香肠的非蛋白氮州PN）含量高于自然发酵香肠，PH值、TVB个值和亚硝酸钠残留量均
明显低于自然发酵香肠（P＜0．05）；接种 LP6003香肠中乳酸菌数、细菌总数、PH
值、挥发性盐基氮OVBwX 亚硝酸钠残留量在添加papain和不添加papain组之间
均没有显著差异河＞0．05X L天后加值明显高于不加 papain香肠u＜0．05）。
The thesis includes three parts:(1)The hydrolysis of whole cells ,cell extracts(CE) and the combination of both(1:1)from Lactobacillus plantarum6003 against pork sarcoplasmic and myofibrillar protein extracts were studied primarily; (2)The effect of the common curing condition(NaCl, NaNO2, Ascorbic acid and temperature)used during the manufacture of fermented sausage on the Acid production and proteolytic activity of Lp6003 against pork proteins extracts was primarily studied;(3)The changes of physicochemical characteristics and microorganism during the fermentation and ripening of the sausage which inoculated Lp6003, Lp6003with 1% papain and non-inoculated were studied. The innovation was that the proteinase systems and the factor of cell level of Lp6003 was predicted primarily. The results in detail are following :
(1)This work focuses on the hydrolysis of whole cells, cell extracts(CE) and the combination of both (1:1) from Lactobacillus plantarum6003 on pork sarcoplasmic and myofibrillar protein extracts. The results showed whole cells of Lp6003 initated the more intense hydrolysis of sarcoplasmic proteins after 96h remarkably than those of myofibrillar proteins, and CE almost did not initated the hydrolysis of pork protein extracts. The hydrolysis of the combination of both was more evident than that of the addition of cell only, which proved that CE intensified the proteolysis of cell on pork protein extracts and increased the livability of cells. The action of whole cells deseased the concentration of almost all armino acid from sarcoplasmic proteins remarkbaly, while increased those from myofibrillar proteins.The action of both enzymatic sources on sarcoplasmic proteins caused maximal increases in Glutamic, Alanine, while the action of those on myofibrillar proteins caused increases in Alanine, Lysine.
(2)The effect of the common curing ingredient and temperature used during the manufacture of fermented sausage on the acid production of Lp6003 and proteolytic activity of Lp6003 whole cells against pork proteins extracts was studied. The results showed that Lp6003 exhibited a appropriate quantity of acidification at 25 ,3%NaCl or 150ppmNaNO2 incubating in broth; The quantity of acidification of Lp6003 dropped remarkably when using high concentration of NaCl or NaNO2 in broth. Common temperature during
fermentation did not exert a stimulation against pork protein extracts hydrolysis of Lp6003 whole cells, and temperature was the main factor affected the proteinases activity of Lp6003 whole cells; The addition of ascorbic acid favoured the hydrolysis of Lp6003 whole cells to sarcoplasmic protein extract; the addition of NaCl favoured the hydrolysis of Lp6003 whole cells to myofibrillar proteins extract, while no effect was recorded when the addition of NaNO2 and ascorbic acid on the hydrolytic activity of Lp6003 whole cells against the proteins extracts.
(3) The changes of microorganism and physicochemical characteristics during the ripening of the sausage which inoculated Lp6003, Lp6003 with 1% papain and non-inoculated were studied. The results showed that Lactobacillus absolutely preponderant microorganism during the ripening of the sausages inoculated Lp6003;which were responsible for the control of bad microorganisms and characteristic of lactobacillus and the security and preservation of the products; The NPN of sausages inoculated Lp6003 (with papain or without papain) have been more than those of the non-inoculated; The pH, TVB-N and NaNO2 concentration of sausages inoculated Lp6003 have been lower than those of non-inoculated. No obvious difference of the quantities of Lactobacillus counts , bacteria counts , pH, TVB-N and rudimental NaNO2 have been observed between the sausages with papain and without papain , while obvious difference of the Aw has been observed since the 5th day between the two kinds sausages.
第一部分：研究了Lp6003的全细胞悬浮液、细胞裂解液、全细胞悬浮液和细胞裂解液等比例混合物对猪肉的肌浆蛋白提取物肌原纤维蛋白提取物的分解能力。结果表明，发酵96小时后，Lp6003全细胞悬浮液对肌浆蛋白提取物具有明显的分解作用，比对肌原纤维蛋白提取物的分解作用强；而单独的CE对两种蛋白提取物均没有明显的分解作用；全细胞悬浮液和CE等比例混合物比单独全细胞悬浮液对两种蛋白提取物的分解作用强，这说明CE加强了Lp6003全细胞对猪肉蛋白提取物的分解作用，并提高了Lp6003细胞的存活力。添加Lp6003全细胞悬浮液的肌浆蛋白提取物中大多数游离氨基酸含量明显下降，肌原纤维蛋白提取物中游离氨基酸含量有所增加；添加全细胞悬浮液和CE等比例混合物增加了肌浆蛋白提取物中Glu、Ala的含量和肌原纤维蛋白提取物中Ala、Lys的含量。
第二部分：研究了发酵香肠生产过程中使用的常规腌制剂和温度对Lp6003产酸性能及Lp6003全细胞分解猪肉中两种蛋白提取物的能力的影响。结果表明，25℃、3％NaCl或150ppmNaNO_2时Lp6003在模拟肉汤培养基中表现出比较适合生产发酵香肠的产酸量，高浓度的NaCl和NaNO_2明显降低了Lp6003的产酸量。常规的发酵温度(25℃)不能促进Lp6003全细胞对肌浆蛋白提取物、肌原纤维蛋白提取物的分解作用，温度是影响Lp6003全细胞蛋白酶活性的重要因素；添加抗坏血酸明显增强了Lp6003全细胞对肌浆蛋白提取物的分解作用；添加NaCl明显增强了Lp6003全细胞对肌原纤维蛋白提取物的分解作用，NaNO_2和抗坏血酸对Lp6003全细胞于此蛋白的分解能力没有影响。
中 文摘 要
第三部分：研究了 Lp删、接种 Lp测并添加 0．l％papain、自然发酵三种
发酵香肠在成熟过程中的微生物和理化特性的变化情况。结果表明，接种LP6003香
肠的乳酸菌在成熟过程中始终处于绝对优势菌状态，保证了产品的安全性和保藏
性，有助于产生乳酸菌特有的发酵特性并能抑制腐败微生物的生长繁殖；接种 LP6003
香肠的非蛋白氮州PN）含量高于自然发酵香肠，PH值、TVB个值和亚硝酸钠残留量均
明显低于自然发酵香肠（P＜0．05）；接种 LP6003香肠中乳酸菌数、细菌总数、PH
值、挥发性盐基氮OVBwX 亚硝酸钠残留量在添加papain和不添加papain组之间
均没有显著差异河＞0．05X L天后加值明显高于不加 papain香肠u＜0．05）。
The thesis includes three parts:(1)The hydrolysis of whole cells ,cell extracts(CE) and the combination of both(1:1)from Lactobacillus plantarum6003 against pork sarcoplasmic and myofibrillar protein extracts were studied primarily; (2)The effect of the common curing condition(NaCl, NaNO2, Ascorbic acid and temperature)used during the manufacture of fermented sausage on the Acid production and proteolytic activity of Lp6003 against pork proteins extracts was primarily studied;(3)The changes of physicochemical characteristics and microorganism during the fermentation and ripening of the sausage which inoculated Lp6003, Lp6003with 1% papain and non-inoculated were studied. The innovation was that the proteinase systems and the factor of cell level of Lp6003 was predicted primarily. The results in detail are following :
(1)This work focuses on the hydrolysis of whole cells, cell extracts(CE) and the combination of both (1:1) from Lactobacillus plantarum6003 on pork sarcoplasmic and myofibrillar protein extracts. The results showed whole cells of Lp6003 initated the more intense hydrolysis of sarcoplasmic proteins after 96h remarkably than those of myofibrillar proteins, and CE almost did not initated the hydrolysis of pork protein extracts. The hydrolysis of the combination of both was more evident than that of the addition of cell only, which proved that CE intensified the proteolysis of cell on pork protein extracts and increased the livability of cells. The action of whole cells deseased the concentration of almost all armino acid from sarcoplasmic proteins remarkbaly, while increased those from myofibrillar proteins.The action of both enzymatic sources on sarcoplasmic proteins caused maximal increases in Glutamic, Alanine, while the action of those on myofibrillar proteins caused increases in Alanine, Lysine.
(2)The effect of the common curing ingredient and temperature used during the manufacture of fermented sausage on the acid production of Lp6003 and proteolytic activity of Lp6003 whole cells against pork proteins extracts was studied. The results showed that Lp6003 exhibited a appropriate quantity of acidification at 25 ,3%NaCl or 150ppmNaNO2 incubating in broth; The quantity of acidification of Lp6003 dropped remarkably when using high concentration of NaCl or NaNO2 in broth. Common temperature during
fermentation did not exert a stimulation against pork protein extracts hydrolysis of Lp6003 whole cells, and temperature was the main factor affected the proteinases activity of Lp6003 whole cells; The addition of ascorbic acid favoured the hydrolysis of Lp6003 whole cells to sarcoplasmic protein extract; the addition of NaCl favoured the hydrolysis of Lp6003 whole cells to myofibrillar proteins extract, while no effect was recorded when the addition of NaNO2 and ascorbic acid on the hydrolytic activity of Lp6003 whole cells against the proteins extracts.
(3) The changes of microorganism and physicochemical characteristics during the ripening of the sausage which inoculated Lp6003, Lp6003 with 1% papain and non-inoculated were studied. The results showed that Lactobacillus absolutely preponderant microorganism during the ripening of the sausages inoculated Lp6003;which were responsible for the control of bad microorganisms and characteristic of lactobacillus and the security and preservation of the products; The NPN of sausages inoculated Lp6003 (with papain or without papain) have been more than those of the non-inoculated; The pH, TVB-N and NaNO2 concentration of sausages inoculated Lp6003 have been lower than those of non-inoculated. No obvious difference of the quantities of Lactobacillus counts , bacteria counts , pH, TVB-N and rudimental NaNO2 have been observed between the sausages with papain and without papain , while obvious difference of the Aw has been observed since the 5th day between the two kinds sausages.
引文
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104. Toldra F, Aristoy M.Availability of essential amino acids in dry-cured ham.Internation J of Food Sci and Nutrition, 1993,44:215～219
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89. Sanz Y,Toldra F.Purification and characterization of an aminopepdase from Lactobacillus sake.J of Agri Food Chemi, 1997a,45:1552～1558
90. Sanz Y, Toldra F.Aminopepdase activities from Lactobacillus sake in models of curing ingredients and processing condition for dry sausages. J Food Sci,1997b,62,1211～1213,1234
91. Sanz Y, Mulholland,Toldra F et al.Purification and characterization of a tripeptidase from Lactobacillus sake.J Agri Food Chem, 1998,38:331～352
92. Sanz Y,Toldra F.Aminopepdase activities from Lactobacillus sake affected by amines in dry sausages,J Food Sci, 1998a,63,894～896
93. Sanz Y,Toldra F.Myoglobin as an inhibitor of exopeptidases from Lactobacillus sake.Applied and Environmental Micro, 1998b,64,2313～2314
94. Sanz Y ,Fadda S ,Vignolo G et al.Hydrolytic action of lactobacillus casei CRL705 on pork muscle sarcoplasmic and myofibrillar protein.Journal of Agricultural Food Chem, 1999,47:3441～3448
95. Sanz Y, Vignolo G, Aristoy M. Hydrolysis of muscle myofibrillar proteins by Lactobacillus curvatus and Lactobacillus sake.International Journal of Food Microbiology, 1999,53(2): 115～125
96. Selgas M,Garcia M,GarciaD,Fernando G.Lipolytic and proteolytic activity of micrococci isolated from dry fermented sausages.Fleischwirtsch,1993,73,1164～1166
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101. Toldra F,Mirailes M,Flores J.Protein extractability in dry-cured ham. Food Chemi, 1992,44:391～394
102. Toldra F,Aristoy M CPart C et al.Muscle and adipose tissue aminopeptidase activities in raw and dry cured ham.J Food Sci,1992a,57:816～818
103. Toldra F, RicoE.Activities of pork muscle proteases in model cured meat systems. Biochimie,1992b,74:291～296
104. Toldra F, Aristoy M.Availability of essential amino acids in dry-cured ham.Internation J of Food Sci and Nutrition, 1993,44:215～219
105. Toldra F, Rico E,Flores J.Cathepsin B.D.H.L activities in the processing of dry～cured ham.J Sci Food Agric, 1993 a,62:157～161
106. Toldra F, Cervero C,Part C et al.Porcine aminopeptidase activity as affercted by curing agents.J Food Sci,1993b,58:724～726
107. Toldra F, Flores M ,Aristoy M et al.Pattern of muscle proteolytic and lipolytic enzymes from light and heavy pigs.J Sci Food Agric,1996,71:124～128
108. Toldra F, Flores M,Sanz Y.Dry-cured ham flavor:enzymatic generation influence.Food Chem,1997,59:523～530
109. Toldra F.The role of muscle protase and lipases in flavour development during the processing of dry-cured ham.Food Sc, 1998,38:331～352
110. Verplaetse Abosschere M.Proteolysis during dry sausages ripening .In proceedings 35th International Congress of Meat Science and Technology. Copenhagen. Denmark. 1989,815～819
111. Verplaetse A.Influence of raw meat properties and processing technology on aroma quality of raw fermented meat products.In processing of the 40th In congress of Meat Sci and Technol, 1994, 45～65
112. Vignolo G.Acid production and proteolytic activity of lactobacillus strains isolated from dry sausages.J Food Pro, 1988,51:481～484
113. Visser S.Proteolytic enzymes and their relation to cheese repening and flavour.J of Dairy Sci, 1993,76:329～350
114. Wardlaw F, Skelley G.Changes in meat components during fermentation heat processing and drying of a summer sausages.J of Food Science, 1998,38:1228～1231
115. Wirth F.Curing:colour formation and colour retention in frank-type sausages. Fleischwitrschaft, 1986,66:354～358
116. W Hammes,C Herter.Developments in meat starter culturer.Meat Science,1998,49:125～136.
117. Zalacain Z,Zapelena M.Dry Fermented Sausages Elaborated with Lipase from Candida cylindracea. Comparison with Traditional Formulations, Meat Sci, 1995,40:55～61
118. Zalacain Z,Zapelena M.Use of Lipase from Rhizomucor miehei in Dry Fermented Sausages Elaboration:Microbial,Chemical and Sensory Analysis, Meat Sci,1997,45:99～105