非发酵性豆制品（豆腐丝）主要腐败细菌的分离鉴定及其防腐研究
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摘要
本文以非发酵性豆制品(豆腐丝)为试验材料，从其腐败过程中分离纯化得到4株主要腐败细菌：df1、df2、df3、df4，对其形态和菌落特征、生理生化特性进行了系统的研究，并进行了分类鉴定。结果表明引发非发酵性豆制品腐败变质的4株主要细菌为：成团肠杆菌(E.agglomerans)、短小芽孢杆菌(B.pumilus)、巨大芽孢杆菌(B.megaterium)和溶酪葡萄球菌(S.caseolyticus)。
测定了尼泊金酯、山梨酸钾、复合防腐剂和天然防腐剂蜂胶对4株主要腐败细菌的抑制作用，结果表明：尼泊金酯防腐剂在国标规定的最大用量1‰内不能抑制全部4株细菌；山梨酸钾在最大限量1‰内也不能抑制4株腐败菌；复合防腐剂能够抑制df1、df3、df4，而不能抑制df2；而蜂胶仅用0.4‰浓度即可全部抑制4株腐败细菌，蜂胶对4株细菌的最小抑菌浓度(MIC)均为0.4‰，因此选用0.4‰的蜂胶浓度对非发酵型豆制品进行防腐试验。
测定了培养基中添加0.1‰蜂胶后4株主要腐败细菌生长情况，并与这4株细菌的生长曲线相比较，结果表明：蜂胶对4株菌的抑制作用主要表现在抑制菌体延迟期和对数期的生长，从而抑制菌数的增长。
本文采用菌落总数、有效酸度(pH值)、挥发性盐基氮含量(TVB-N)三个指标衡量非发酵性豆制品(豆腐丝)的防腐效果。未经处理的豆腐丝在22℃贮存30d，菌落总数由最初5.7×10~4cfu/ml，变为4.8×10~9cfu/ml；其pH值最初为6.71，但随后样品pH值变化趋势为先下降后上升至7.10；而挥发性盐基氮也由最初的4.93mg/100g变化为182.96mg/100。当贮存到菌落总数为1×10~5cfu/ml，即达到国家非发酵性豆制品菌落总数卫生指标的临界值时，TVB-N为24.00mg/100g，因此把TVB-N≤24.00mg/100g作为豆制品的理化指标。设计蜂胶加入浓度、包装方式、贮存温度三因素二水平L_4(2~3)的正交试验，以挥发性盐基氮含量多少作为指标来确定防腐的最佳组合。结果表明在贮存过程中对延长非发酵性豆制品(豆腐丝)保质期的最佳组合为：0.4‰喷涂加入蜂胶+0.02×10~5Pa真空包装+4℃贮存，货架期可延长至35d。按照这种组合处理的样品第35天菌落总数为9.3×10~4cfu/ml，pH值为6.56，挥发性盐基氮为22.00mg/100g，符合国家标准，样品经过感官评定在色泽和风味上与空白无明显差异。
This experiment, using non-ferment soy product for its material, gained four sort of spoilage bacterium: df1, df2, df3, df4 through separation and purification in the spoiling process of non-ferment soy product. In the experiment it carried out systematical research on the properties of configuration, bacterial community and the physiological and biochemical properties. It can be concluded from the final identification that the four leading spoilage bacterium which led to the spoilage of soy product were: E.agglomerans, B.pumilus, B.megaterium.and S.caseolyticus.
The result of testing roles of paraben, potassium sorbate, compound preservatives and natural propolis in inhibiting the four leading spoilage bacterium indicates: Paraben failed to inhibit all the four leading spoilage bacterium at its maximum dosage-1 percent according to international discipline, so did potassium sorbate; Compound preservatives can inhibit df1, df3, df4,but not df2; While propolis can inhibit the four leading spoilage bacterium at a dosage of 0.04 percent. So what was most available in this experiment was: propolis.
Having tested the growth conditions of the four leading spoilage bacterium and compared the growth curves among them, so concluded that propolis inhibited the four leading spoilage bacterium mainly in their growth in delay and logarithm periods so as to inhibit the growth of the number of bacteria .
The total number of bacterial community, the pH in effect and the percentage of volatilization brine nitrogen were applied to measure the antisepsis effect of non-ferment soy product. The total number of bacterial community on the non -ferment soy product changd from 5.7x104cfu/ml at the beginning to 4.8x109cfu/ml after 30-days' storage and the pH rose, fell, then rose, from 6.71 to 7.10 in the long run. the percentage of volatilization brine nitrogen also rose from 4.93mg/100g to 182.96mg/100g. A crossed experiment was also designed with regard to factors affecting it: the concentration of propolis, the packaging mode and the storage temperature, which was carried out to measure the effect of non-ferment soy product in whole process in term of the percentage of volitilization brine nitrogen. The result indicates that the most suitable combination to prolong the time limit of storage non-ferment soy product were: 0.4%opropolis + 0.02x105Pa vacuum packaging +
storaging temperature under 4C,which cart prolong the shelf life to 35-days,making the total number of bacterial cornmuity reached 9.3X 104cfu/ml,pH 6.56 and the percentage of volatilization butyl nitrogen 22.00mg/100g,there was no difference between blank and sample.
测定了尼泊金酯、山梨酸钾、复合防腐剂和天然防腐剂蜂胶对4株主要腐败细菌的抑制作用，结果表明：尼泊金酯防腐剂在国标规定的最大用量1‰内不能抑制全部4株细菌；山梨酸钾在最大限量1‰内也不能抑制4株腐败菌；复合防腐剂能够抑制df1、df3、df4，而不能抑制df2；而蜂胶仅用0.4‰浓度即可全部抑制4株腐败细菌，蜂胶对4株细菌的最小抑菌浓度(MIC)均为0.4‰，因此选用0.4‰的蜂胶浓度对非发酵型豆制品进行防腐试验。
测定了培养基中添加0.1‰蜂胶后4株主要腐败细菌生长情况，并与这4株细菌的生长曲线相比较，结果表明：蜂胶对4株菌的抑制作用主要表现在抑制菌体延迟期和对数期的生长，从而抑制菌数的增长。
本文采用菌落总数、有效酸度(pH值)、挥发性盐基氮含量(TVB-N)三个指标衡量非发酵性豆制品(豆腐丝)的防腐效果。未经处理的豆腐丝在22℃贮存30d，菌落总数由最初5.7×10~4cfu/ml，变为4.8×10~9cfu/ml；其pH值最初为6.71，但随后样品pH值变化趋势为先下降后上升至7.10；而挥发性盐基氮也由最初的4.93mg/100g变化为182.96mg/100。当贮存到菌落总数为1×10~5cfu/ml，即达到国家非发酵性豆制品菌落总数卫生指标的临界值时，TVB-N为24.00mg/100g，因此把TVB-N≤24.00mg/100g作为豆制品的理化指标。设计蜂胶加入浓度、包装方式、贮存温度三因素二水平L_4(2~3)的正交试验，以挥发性盐基氮含量多少作为指标来确定防腐的最佳组合。结果表明在贮存过程中对延长非发酵性豆制品(豆腐丝)保质期的最佳组合为：0.4‰喷涂加入蜂胶+0.02×10~5Pa真空包装+4℃贮存，货架期可延长至35d。按照这种组合处理的样品第35天菌落总数为9.3×10~4cfu/ml，pH值为6.56，挥发性盐基氮为22.00mg/100g，符合国家标准，样品经过感官评定在色泽和风味上与空白无明显差异。
This experiment, using non-ferment soy product for its material, gained four sort of spoilage bacterium: df1, df2, df3, df4 through separation and purification in the spoiling process of non-ferment soy product. In the experiment it carried out systematical research on the properties of configuration, bacterial community and the physiological and biochemical properties. It can be concluded from the final identification that the four leading spoilage bacterium which led to the spoilage of soy product were: E.agglomerans, B.pumilus, B.megaterium.and S.caseolyticus.
The result of testing roles of paraben, potassium sorbate, compound preservatives and natural propolis in inhibiting the four leading spoilage bacterium indicates: Paraben failed to inhibit all the four leading spoilage bacterium at its maximum dosage-1 percent according to international discipline, so did potassium sorbate; Compound preservatives can inhibit df1, df3, df4,but not df2; While propolis can inhibit the four leading spoilage bacterium at a dosage of 0.04 percent. So what was most available in this experiment was: propolis.
Having tested the growth conditions of the four leading spoilage bacterium and compared the growth curves among them, so concluded that propolis inhibited the four leading spoilage bacterium mainly in their growth in delay and logarithm periods so as to inhibit the growth of the number of bacteria .
The total number of bacterial community, the pH in effect and the percentage of volatilization brine nitrogen were applied to measure the antisepsis effect of non-ferment soy product. The total number of bacterial community on the non -ferment soy product changd from 5.7x104cfu/ml at the beginning to 4.8x109cfu/ml after 30-days' storage and the pH rose, fell, then rose, from 6.71 to 7.10 in the long run. the percentage of volatilization brine nitrogen also rose from 4.93mg/100g to 182.96mg/100g. A crossed experiment was also designed with regard to factors affecting it: the concentration of propolis, the packaging mode and the storage temperature, which was carried out to measure the effect of non-ferment soy product in whole process in term of the percentage of volitilization brine nitrogen. The result indicates that the most suitable combination to prolong the time limit of storage non-ferment soy product were: 0.4%opropolis + 0.02x105Pa vacuum packaging +
storaging temperature under 4C,which cart prolong the shelf life to 35-days,making the total number of bacterial cornmuity reached 9.3X 104cfu/ml,pH 6.56 and the percentage of volatilization butyl nitrogen 22.00mg/100g,there was no difference between blank and sample.
引文
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[17] PL Miorin, NC Levy Junior, AR Custodio etal. Antibacterial activity of honey and propolis from Apis mellifera and Tetragonisca angustula against Staphylococcus aureus[J]. J Appl Microbiol,2003,95(5): 913～920.
[18] Hyun Koo,Pedro L. Rosalen ,Jaime A. Cury etal. Effect of compounds in propolis on Streptococcus mutans growth and on glucosyltransferase activity[J].Antimicrobial Agents and Chemotherapy,2002,46(5): 1302～1309.
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[20] 川村贤司．蜂胶在药理作用中的实力和魅力[J]．养蜂科技，2002，5：30～35．
[21] 叶陆星，胡福良．蜂胶中黄酮类化合物的生物学活性[J]．2002,2：9～11．
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[2] Cara L Frankenfeld, Ruth E Patterson, Neilann K Homer et al. Validation of a soy food-frequency questionnaire and evaluation of correlates of plasma isoflavone concentrations in postmenopausal women[J]. American journal of clinical nutrition, 2003, 77(3): 674～680.
[3] Garcia MC,Marina ML,Laborda F eatl. Chemical charicterization of commercial soybean products[J].Food Chemistry, 1998,62(3): 325～331.
[4] 常汝镇．国内外大豆生产动态及研究进展[J]．中国食物与营养，2000，1：13～15．
[5] Dequan Z et al. Survey on research and application of bioflavoids[J]. Food and fermentation Industries,1999,25(5): 52～57.
[6] Johanna W.Lampe. Isoflavinoid and Iignan phytoestrogens as dietary biomarkers [J]. J. Nutr.,2003,133: 956～964.
[7] Yanagihara et al. Atiproliferative effects of isoflavones on human cancer cell line established from the gastrointestinal tract [J]. Cancer Research, 1993,53 (23):5615～5821.
[8] M .Segasothy, P. A.Phillips. Vegetarian diet: panacea for modern lifestyle diseases? [J]. Q . J Med, 1999,92:531～544.
[9] Eisen B, Ungar Yet al.Stability of isoflavones in soy milk stored at elevated and ambient temperatures [J].Journal of Agricultural and Food Chemostry, 2003,51 (8): 2212～2215.
[10] 徐勇，江华，勇强，等．低聚木糖对青春双歧杆菌的增殖[J]．食品科学，2001，22(7)：15～17．
[11] 朱秀清，江连洲，富校轶．国内外大豆加工利用的研究进展[J]．食品科技，2001，6：1～4．
[12] 王若峰，宁正祥，谭龙飞．食品防腐剂研究进展述评[J]．广州食品工业科技，1995，11(1)：5～10．
[13] 孔健，马桂荣．天然食品防腐剂乳酸菌素—Nisin[J]．食品与发酵工业，1995，3：80～82．
[14] 刘稳，马桂荣，朱文淼．乳酸菌的细菌素[J]．中国微生态学杂志，1996,8(2)：54～57．
[15] 李平兰，张篪，江汉湖．产细菌素植物乳杆菌菌株的筛选及其细菌素生物学特征研究[J]．食品发酵工业，1999，25(2)：1～4．
[16] Nikki Horn, Maria I. Martinenez, Jose M. Martinez et al. Enhanced production of pediocin PA-1 and coproduction of nisin and pediocin PA-I by Lactococcus lactis[J].
Applied and environmental microbiology, 1999,65(10): 4443～4450.
[17] PL Miorin, NC Levy Junior, AR Custodio etal. Antibacterial activity of honey and propolis from Apis mellifera and Tetragonisca angustula against Staphylococcus aureus[J]. J Appl Microbiol,2003,95(5): 913～920.
[18] Hyun Koo,Pedro L. Rosalen ,Jaime A. Cury etal. Effect of compounds in propolis on Streptococcus mutans growth and on glucosyltransferase activity[J].Antimicrobial Agents and Chemotherapy,2002,46(5): 1302～1309.
[19] 杨艳彬，朱丽莉，唐明翔．蜂胶抑菌作用的研究[J]．食品科技，1999，6：33～34．
[20] 川村贤司．蜂胶在药理作用中的实力和魅力[J]．养蜂科技，2002，5：30～35．
[21] 叶陆星，胡福良．蜂胶中黄酮类化合物的生物学活性[J]．2002,2：9～11．
[22] 袁芳，许德春，孟丽芬．风味豆制品辐照贮藏保鲜技术研究[J]．核农学报，2001，15(3)：145～148．
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