细菌型豆豉发酵机理及功能性研究
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摘要
豆豉（lobster sauce）是以黑豆或黄豆为原料，利用微生物发酵制成的一种具有独特风味的调味品，既可作调味料又可以直接食用。豆豉含丰富的可溶性氮、可溶性糖、维生素A、维生素B族及维生素E等营养成分及大豆低聚糖、大豆异黄酮、大豆皂苷、褐色色素类、大豆多肽等多种功能因子。
细菌型豆豉大多是利用纳豆枯草杆菌（Bacillus subtilis Natto）等在较高温度下繁殖于蒸熟的大豆上，借助其蛋白酶生产出风味独特的食品。而作为细菌型豆豉的典型代表山东水豆豉，目前尚未见到对其发酵机理及生理功能的系统研究及报道。本文以山东临沂民间自然发酵的自制豆豉为研究对象，对其发酵过程中的微生物群落的变化进行动态分析、益酵菌株的筛选鉴定、发酵过程豆豉成分变化及豆豉生理活性物质进行研究，以期筛选得到符合制曲要求的微生物，为进行纯种发酵提供依据，同时通过对水豆豉的营养和生理功能进行深入了解及发掘，扩大中国豆豉被消费者认同范围及促进市场前景的发展。研究结果和主要结论如下：
（1）细菌型豆豉发酵菌群多样性分析
提取豆豉发酵不同时间的豆豉总DNA，采用对大多细菌16S rDNA基因V3区具有特异性的引物对GC-357F和517R，进行PCR扩增，将长约160bp的扩增产物使用变性剂浓度梯度为30%到60%的聚丙烯酰胺凝胶进行分离，最后对电泳谱图进行分析。同时，采用引物对27F-FAM，1492R对豆豉总DNA进行PCR扩增，将扩增产物进行MspI酶切，对酶切产物进行测序及OUT分析。根据DGGE及T-RFLP分析可知，细菌型豆豉在发酵过程中，多样性指数与丰富度指数较低且比较稳定，优势菌群特征明显。以芽孢杆菌为优势菌群，发酵时间7d为宜。
（2）细菌型豆豉优势菌株的筛选及益酵菌株鉴定
通过常规平板培养方法，将发酵过程豆豉样品中的微生物，进行分离纯化及菌落计数。通过菌体形态、菌落特征观察，筛选得到11株细菌，初步确定B1、B2、B3、B4、B5、B7为芽孢杆菌，NB1、NB2、NB3、NB4为非芽孢杆菌，C1为球菌。通过耐盐性实验及耐热性实验，确定益酵菌株为B1、B2、B3、B4、B5、C6、B7等7株菌。经生理生化及分子生物学鉴定，初步可确定B1为巨大芽孢杆菌（Bacillus megaterium）、B2为凝结芽孢杆菌（B.coagulans）、B3为枯草芽孢杆菌（B.subtilis）、B4为地衣芽孢杆菌（B.licheniformis）、B5为耐热芽孢杆菌（B.kalodurans）、B7为解淀粉芽孢杆菌（B.amyloliquefaciens）、C6为溶酪大球菌（Macrococcus caseolyticus）。
（3）细菌型豆豉发酵过程成分分析
在发酵过程中，对发酵不同时间内的豆豉样品进行成分分析，其中水分、脂肪、蛋白质、总糖、还原糖及粗纤维呈下降趋势；灰分、pH值、总酸，氨基氮、游离氨基酸等呈上升趋势，其中总氨基酸含量变化不大。表明经过发酵豆豉具有较高的营养价值。
（4）细菌型豆豉生理活性物质分析
在发酵过程中，对发酵过程中纤维素酶、蛋白酶的酶活进行测定，结果表明豆豉发酵液中纤维素酶活维持较低水平，而蛋白酶活呈现增长的趋势。将筛选得到的7株菌及豆豉提取液进行纳豆激酶酶活测定，B5、B4等菌株均表现出较高的酶活特性。豆豉提取液因杂质等因素影响，酶活有所降低，但仍比C6、B3、B2、B7、B1等纯菌发酵液的酶活有明显的提高。
随着发酵时间的延长，豆豉中异黄酮染料木素糖苷和大豆苷元糖苷的转化率分别达到69.07%和54.54%，使产品的生理功能得到较大的提高。对豆豉发酵液进行MDA值及SOD值测定，其中全血中MDA值显著下降，血清中SOD活性显著增高。表明豆豉具有较强的抗氧化活性。
Lobster sauce is a unique flavoring made of black bean or soybean by the microbialfermentation, which possesses abundant functional group such as soluble nitrogen, solublesugar, vitamin A, vitamin B family, vitamin E, soybean oligosaccharides, soybean isoflavone,soybean saponin, brown pigment, soybean polypeptide.
Bacterium lobster sauce is a food by the protease from Bacillus Subtilis Natto on cookedsoybean. As a representation of water lobster sauce from bacterium, there is no interrelatedreport as well as the systematic research on its fermentation mechanism and its physiologicalfunction. In this paper, the natural fermented lobster sauce from folk people in Linyi,Shandong province, was studied in detail to discover the dynamic changing on the microbialcommunities as well as the lobster sauce nutrient components during the fermentation process,and the physiological function of lobster sauce were expected to reveal. At the same time, therelated fermentation microbe were screened out and identified. The main results were asfollows:
(1) Diversity analyses of fermentation flora of bacterium lobster sauce
The total DNA of lobster sauce from different fermentation time was extracted and thePCR amplification was carried out by the V3region primers of16S rDNA, named GC-357Fand517R. The160bp amplification products were separated by the denaturing gradient gel of30%to60%polyacrylamide. The PCR amplification was also performed on the total DNA oflobster sauce using the primers of27F-FAM and1492R. The amplification products weredigested by the enzyme Msp I and the digestion products were sequencing and OTU analyzed.The diversity index and abundance index kept steady in a low level, and the characteristics ofdominant flora were obvious in the fermentation process of bacterium lobster sauce byDGGE and T-RFLP analyses. The Bacillus Cohn acted as the dominate flora and thefermentation duration was7d.
(2) Screening and identification of dominant flora in bacterium lobster sauce
The microbe in lobster sauce samples were separated counted on plate medium.11 bacteria were obtained and by thallus shape and colony characters, strain B1, B2, B3, B4, B5and B7were confirmed to be bacillaceae while NB1, NB2, NB3and NB4were non-bacillaceae, and strain C6was cocci. Strain B1, B2, B3, B4, B5, C6and B7were idealfermention bacteria by saline tolerance test and thermal endurance test. The identificationresults according to the physiological and biochemical characteristics as well as the molecularbiology indexes, were as follows: B1was Bacillus megaterium, B2was Bacillus coagulans,B3was Bacillus subtilis, B4was Bacillus licheniformis, B5was Bacillus kalodurans, B7wasBacillus amyloliquefaciens, andC6was Macrococcus caseolyticus.
(3) Component analysis of bacterium lobster sauce in the process of fermentation
The amount of water, fat, protein, total sugar and raw fiber all declined during thefermentation of lobster sauce, while the amount of ash, pH, total acid, amino acid anddissociate amino acid all increased. There was no obvious change on the amount of totalamino acid.
(4) Physiological function analysis of bacterium lobster sauce
The enzyme activity of cellulose maintained a low level in the solution of lobster saucefermentation while the enzyme activity of proteinase showed a tendency of increasing. Withthe fermentation duration, the transformation efficiency of genistein and daidzein, whichplayed a key role in physiological function, reached69.07%,54.54%, respectively. The MDAtest and SOD test showed the MDA value declined in the whole blood while the SOD activityincreased, which meant lobster sauce possessed an excellent anti-oxidation activity.
细菌型豆豉大多是利用纳豆枯草杆菌（Bacillus subtilis Natto）等在较高温度下繁殖于蒸熟的大豆上，借助其蛋白酶生产出风味独特的食品。而作为细菌型豆豉的典型代表山东水豆豉，目前尚未见到对其发酵机理及生理功能的系统研究及报道。本文以山东临沂民间自然发酵的自制豆豉为研究对象，对其发酵过程中的微生物群落的变化进行动态分析、益酵菌株的筛选鉴定、发酵过程豆豉成分变化及豆豉生理活性物质进行研究，以期筛选得到符合制曲要求的微生物，为进行纯种发酵提供依据，同时通过对水豆豉的营养和生理功能进行深入了解及发掘，扩大中国豆豉被消费者认同范围及促进市场前景的发展。研究结果和主要结论如下：
（1）细菌型豆豉发酵菌群多样性分析
提取豆豉发酵不同时间的豆豉总DNA，采用对大多细菌16S rDNA基因V3区具有特异性的引物对GC-357F和517R，进行PCR扩增，将长约160bp的扩增产物使用变性剂浓度梯度为30%到60%的聚丙烯酰胺凝胶进行分离，最后对电泳谱图进行分析。同时，采用引物对27F-FAM，1492R对豆豉总DNA进行PCR扩增，将扩增产物进行MspI酶切，对酶切产物进行测序及OUT分析。根据DGGE及T-RFLP分析可知，细菌型豆豉在发酵过程中，多样性指数与丰富度指数较低且比较稳定，优势菌群特征明显。以芽孢杆菌为优势菌群，发酵时间7d为宜。
（2）细菌型豆豉优势菌株的筛选及益酵菌株鉴定
通过常规平板培养方法，将发酵过程豆豉样品中的微生物，进行分离纯化及菌落计数。通过菌体形态、菌落特征观察，筛选得到11株细菌，初步确定B1、B2、B3、B4、B5、B7为芽孢杆菌，NB1、NB2、NB3、NB4为非芽孢杆菌，C1为球菌。通过耐盐性实验及耐热性实验，确定益酵菌株为B1、B2、B3、B4、B5、C6、B7等7株菌。经生理生化及分子生物学鉴定，初步可确定B1为巨大芽孢杆菌（Bacillus megaterium）、B2为凝结芽孢杆菌（B.coagulans）、B3为枯草芽孢杆菌（B.subtilis）、B4为地衣芽孢杆菌（B.licheniformis）、B5为耐热芽孢杆菌（B.kalodurans）、B7为解淀粉芽孢杆菌（B.amyloliquefaciens）、C6为溶酪大球菌（Macrococcus caseolyticus）。
（3）细菌型豆豉发酵过程成分分析
在发酵过程中，对发酵不同时间内的豆豉样品进行成分分析，其中水分、脂肪、蛋白质、总糖、还原糖及粗纤维呈下降趋势；灰分、pH值、总酸，氨基氮、游离氨基酸等呈上升趋势，其中总氨基酸含量变化不大。表明经过发酵豆豉具有较高的营养价值。
（4）细菌型豆豉生理活性物质分析
在发酵过程中，对发酵过程中纤维素酶、蛋白酶的酶活进行测定，结果表明豆豉发酵液中纤维素酶活维持较低水平，而蛋白酶活呈现增长的趋势。将筛选得到的7株菌及豆豉提取液进行纳豆激酶酶活测定，B5、B4等菌株均表现出较高的酶活特性。豆豉提取液因杂质等因素影响，酶活有所降低，但仍比C6、B3、B2、B7、B1等纯菌发酵液的酶活有明显的提高。
随着发酵时间的延长，豆豉中异黄酮染料木素糖苷和大豆苷元糖苷的转化率分别达到69.07%和54.54%，使产品的生理功能得到较大的提高。对豆豉发酵液进行MDA值及SOD值测定，其中全血中MDA值显著下降，血清中SOD活性显著增高。表明豆豉具有较强的抗氧化活性。
Lobster sauce is a unique flavoring made of black bean or soybean by the microbialfermentation, which possesses abundant functional group such as soluble nitrogen, solublesugar, vitamin A, vitamin B family, vitamin E, soybean oligosaccharides, soybean isoflavone,soybean saponin, brown pigment, soybean polypeptide.
Bacterium lobster sauce is a food by the protease from Bacillus Subtilis Natto on cookedsoybean. As a representation of water lobster sauce from bacterium, there is no interrelatedreport as well as the systematic research on its fermentation mechanism and its physiologicalfunction. In this paper, the natural fermented lobster sauce from folk people in Linyi,Shandong province, was studied in detail to discover the dynamic changing on the microbialcommunities as well as the lobster sauce nutrient components during the fermentation process,and the physiological function of lobster sauce were expected to reveal. At the same time, therelated fermentation microbe were screened out and identified. The main results were asfollows:
(1) Diversity analyses of fermentation flora of bacterium lobster sauce
The total DNA of lobster sauce from different fermentation time was extracted and thePCR amplification was carried out by the V3region primers of16S rDNA, named GC-357Fand517R. The160bp amplification products were separated by the denaturing gradient gel of30%to60%polyacrylamide. The PCR amplification was also performed on the total DNA oflobster sauce using the primers of27F-FAM and1492R. The amplification products weredigested by the enzyme Msp I and the digestion products were sequencing and OTU analyzed.The diversity index and abundance index kept steady in a low level, and the characteristics ofdominant flora were obvious in the fermentation process of bacterium lobster sauce byDGGE and T-RFLP analyses. The Bacillus Cohn acted as the dominate flora and thefermentation duration was7d.
(2) Screening and identification of dominant flora in bacterium lobster sauce
The microbe in lobster sauce samples were separated counted on plate medium.11 bacteria were obtained and by thallus shape and colony characters, strain B1, B2, B3, B4, B5and B7were confirmed to be bacillaceae while NB1, NB2, NB3and NB4were non-bacillaceae, and strain C6was cocci. Strain B1, B2, B3, B4, B5, C6and B7were idealfermention bacteria by saline tolerance test and thermal endurance test. The identificationresults according to the physiological and biochemical characteristics as well as the molecularbiology indexes, were as follows: B1was Bacillus megaterium, B2was Bacillus coagulans,B3was Bacillus subtilis, B4was Bacillus licheniformis, B5was Bacillus kalodurans, B7wasBacillus amyloliquefaciens, andC6was Macrococcus caseolyticus.
(3) Component analysis of bacterium lobster sauce in the process of fermentation
The amount of water, fat, protein, total sugar and raw fiber all declined during thefermentation of lobster sauce, while the amount of ash, pH, total acid, amino acid anddissociate amino acid all increased. There was no obvious change on the amount of totalamino acid.
(4) Physiological function analysis of bacterium lobster sauce
The enzyme activity of cellulose maintained a low level in the solution of lobster saucefermentation while the enzyme activity of proteinase showed a tendency of increasing. Withthe fermentation duration, the transformation efficiency of genistein and daidzein, whichplayed a key role in physiological function, reached69.07%,54.54%, respectively. The MDAtest and SOD test showed the MDA value declined in the whole blood while the SOD activityincreased, which meant lobster sauce possessed an excellent anti-oxidation activity.
引文
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