工业生产黄豆酱乳酸菌和酵母菌的研究
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摘要
黄豆酱是我国传统的发酵豆制品，以大豆和小麦为主要原料。黄豆酱以其特有的色、香、味，丰富的营养价值和良好的保健功能几千年来深受人们的喜爱。在制作黄豆酱的过程中微生物的作用显得尤为的突出，并已经在种曲培养阶段就发现并鉴定的微生物为米曲霉等霉菌。种曲中的霉菌通常利用淀粉、低聚糖、单糖、有机酸及醇类等作为碳源。蛋白质，氨基酸及尿素等作为氮源。而酵母菌和乳酸菌在黄豆酱发酵的过程中对增进风味也起到至关重要的作用。传统制酱工艺通常将大豆与面粉混合并制成块状，经日晒夜露制作种曲，种曲微生物的来源于原料或空气中，其中包括了耐盐性的酵母菌和在厌氧条件下生长良好的乳酸菌。经过相关资料报道这些微生物包括鲁氏酵母、球拟酵母和嗜盐四链球菌。近些年制作黄豆酱更趋于利用纯菌种发酵的方法，不但缩短了发酵周期，而且减少了杂菌的污染。本课题研究旨在对可发酵黄豆酱的鲁氏酵母、球拟酵母和嗜盐四链球菌进行富集培养，并制备成发酵剂，用于黄豆酱的后期发酵，来改善黄豆酱的品质。
1．本文研究了嗜盐四链球菌、鲁氏酵母和球拟酵母菌的耐盐性。研究发现，嗜盐四链球菌可在12％NaCl的培养基中生长良好，鲁氏酵母和球拟酵母可在18％NaCl的培养基中生长良好。
2．嗜盐四链球菌以MRS为培养基，恒定发酵pH值为6.5，控制溶氧量为15％，以3％的接种量接种，搅拌速度120rpm，在34℃下培养28h，嗜盐四链球菌的活菌数可达1.22×10~(12)cfu／mL。富集培养后的嗜盐四链球菌经5000rpm，离心20min，收集菌体，加入10％脱脂乳和5％的甘油作为嗜盐四链球菌细胞悬浮液的冷冻保护介质。经真空冷冻干燥后的嗜盐四链球菌的活细胞数达到5.58×10~(10)cfu／g，细胞存活率为73.28％。
3．在生酱油培养基中，分别以葡萄糖为两株酵母的碳源，以硫酸铵为氮源，向鲁氏酵母培养基中的添加量为0.05％的K_2HPO_4，向球拟酵母培养基中的添加量为0.07％的K_2HPO_4。通过全自动模拟发酵罐进行增菌发酵试验得到，鲁氏酵母最优发酵条件为控制溶氧量在50％，发酵pH值为3.5，以8％接种量接种，搅拌速度120rpm，在25℃下培养32h，鲁氏酵母活菌数最高值可达1.07×10~(11)个／mL。球拟酵母的最优发酵条件为控制溶氧量在60％，发酵pH值为3.5，以6％的接种量，搅拌速度120rpm，在25℃下培养36h，球拟酵母活菌数的最高值可达和1.55×10~(11)个／mL。富集培养后的酵母菌体经5000rpm离心20min，用2.5％的氯化钠溶液洗涤多次，添加司盘60与土温80比例为1：1的混合物作为干燥保护剂，在温度为40℃条件下进行干燥，干燥至水分含量低于8％。干燥的产品经过25倍菌体重量的12Bé麦芽汁在36℃条件下复水15min，降温25℃活化120min。鲁氏酵母和球拟酵母的存活率分别为91.7％和91.07％。
4．将三株菌分别进行单因素发酵试验，确定其单独发酵时的最佳添加时间及添加量。并根据各个菌株单因素发酵的试验结果，采用三因素三水平正交试验设计，研究在混合菌种发酵黄豆酱的方法中嗜盐四链球菌、鲁氏酵母、球拟酵母的最佳配比。得到在酱醪发酵的第11天添加嗜盐四链球菌，添加量为10~6cfu／g酱醪，在酱醪发酵的第14天同时添加鲁氏酵母和球拟酵母，添加量分别为10~5个／g酱醪和10~6个／g酱醪。产品经GC-MS分析，鉴定出24种挥发性风味化合物。并通过对理化指标和风味检测表明，混合菌种发酵的黄豆酱比仅利用米曲霉发酵生产的黄豆酱产品品质更好。
Soybean paste is a traditional fermentation soybean product. Fermented soybean paste madewith such materials as wheat and soybean. Soybean paste has been popular more than one thousandyears because it has pleasure aroma, taste, nutrition and function. Microorganism play veryimportant role in the processing of soybean paste. Microorganism found in koji almost alwaysbelongs to fungi species, Aspergillus oryzae and/or Asp. Sojae. The koji mold has the ability toutilize starch, oligosaccharides, simple sugars, organic acids and alcohols, etc as carbon sources,and protein, amino acids and urea, etc as nitrogen sources. Halophilic yeast and lactic acid bacterialalso play an important role in developing flavour during fermentation of soybean paste, particularlyin the later stage of the fermentation. Traditionally, the mixture of boiled soybean and wheat flourmade cuboid or elliptoid shape and kept in an incubation room to make koji. The organisms comefrom either the previous batch or environment in which they are natural present. The inoculumgenerally contains selected flora of salted tolerant yeasts and bacterial capable of growing underanaerobic conditions. The dominant microorganisms are yeast Zygosaccharomyces Rouxii andTorulopsis sp and certain lactic acid bacteria such as Tetragenococcus Halophilus. Since pureculture of these microorganism speeds up fermentation and reduce the influence of weed yeast andbacteria, its use in commercial soybean paste preparation has been popular in recent years. In thispaper we focus on Tetragenococcus Halophilus, Zygosaccharomyces Rouxii and TorulopsisCandida, to produce commercial used lactic acid bacteria starter and dried yeast that can be appliedin industrial producing soybean paste.
1. Tetragenococcus Halophilus can be grown up at 12% NaCl, and Zygosaccharomyces Rouxiiand Torulopsis Candida can be grown up at 18% NaCl.
2. Tetragenococcus Halophilus is aerobic in MRS in culture medium, with growth optimagenerally at pH 6.5, a temperature of 34℃, a dissoluble oxygen of 15%, a inoculation concentrationof 3%, a stir speed of 120rpm, and the incubation time of 28h. At this optimum cultural condition,the active cell number of Tetragenococcus Halophilus was 1.22×10~(12) cfu/mL. The culture organismwas centrifugalized 20 min at 5000rpm. The bacterial concentrate was mixed with 10% skim milkpowder and 5% glycerin, and was dried by vacuum freezing. The active cell number of driedTetragenococcus Halophilus was 5.58×10~(10) cfu/g., and the survival rate of the dried bacteria was73.28%.
3. The culture media of Zygosaccharomyces Rouxii and Torulopsis Candida contained raw soysauce, glucose, and (NH_4)_2SO_4. It had been proved that 0.05% and 0.07% K_2HPO_4 in the culturemedia benefit the growth of Zygosaccharomyces Rouxii and Torulopsis Candida respectively. Theoptima fermentation condition of Zygosaccharomyces Rouxii was that controlled dissoluble oxygenof 50%, a pH of 3.5, a inoculation concentration of 8%, a stir speed of 120rpm, a temperature of25℃, a incubation time of 32h. The active cells number of Zygosaccharomyces Rouxii was1.07×10~(11)/mL. The optima fermentation condition of Torulopsis Candida was that controlleddissoluble oxygen of 60%, a pH 3.5, a inoculation concentration of 6%, stir speed of 120rpm, atemperature of 25℃, and a incubation time of 36h. The active cells number of Torulopsis Candidawas 1.55×10~(11)/mL. The culture organism was centrifugalized 20 min at 5000rpm. The yeastconcentrates were washed by 2.5% NaCl several times, and the concentrates were mixed withspan60 and tween80 (1:1). The mixture was dried to 8% moisture content at 40℃. The dried yeastpowder was dissolved in 12Béwort at 36℃(15min), and then the yeast solution was incubated at25℃, 120min. The survival rate Zygosaccharomyces Rouxii and Torulopsis Candida were 91.70%and 91.07% respectively.
4. In order to determine the optimal proportion of three strains, the three strains wereinoculated into soybean paste. Based on the effect of single strain on the quality of soybean paste, aL_9(3~3) orthogonal experiment design was conducted to study the proportion of TetragenococcusHalophilus, Zygosaccharomyces Rouxii and Torulopsis Candida. The results were thatTetragenococcus Halophilus was inoculated into soybean paste in the 11th day, and the inoculationconcentration was 10~6cfu/g. Zygosaccharomyces Rouxii and Torulopsis Candida were inoculatedinto soybean paste in the 14th day, however, the inoculation concentration of both was 10~5 cfu/g and10~6cfu/g respectively. 24 volatile flavor compounds were detected from soybean paste by GC-MS.The taste, chemical and physical characteristic and flavors, of that fermented by Aspergillus oryzae,yeast and lactic acid bacteria, were better than that only fermented by single Aspergillus oryzae.
1．本文研究了嗜盐四链球菌、鲁氏酵母和球拟酵母菌的耐盐性。研究发现，嗜盐四链球菌可在12％NaCl的培养基中生长良好，鲁氏酵母和球拟酵母可在18％NaCl的培养基中生长良好。
2．嗜盐四链球菌以MRS为培养基，恒定发酵pH值为6.5，控制溶氧量为15％，以3％的接种量接种，搅拌速度120rpm，在34℃下培养28h，嗜盐四链球菌的活菌数可达1.22×10~(12)cfu／mL。富集培养后的嗜盐四链球菌经5000rpm，离心20min，收集菌体，加入10％脱脂乳和5％的甘油作为嗜盐四链球菌细胞悬浮液的冷冻保护介质。经真空冷冻干燥后的嗜盐四链球菌的活细胞数达到5.58×10~(10)cfu／g，细胞存活率为73.28％。
3．在生酱油培养基中，分别以葡萄糖为两株酵母的碳源，以硫酸铵为氮源，向鲁氏酵母培养基中的添加量为0.05％的K_2HPO_4，向球拟酵母培养基中的添加量为0.07％的K_2HPO_4。通过全自动模拟发酵罐进行增菌发酵试验得到，鲁氏酵母最优发酵条件为控制溶氧量在50％，发酵pH值为3.5，以8％接种量接种，搅拌速度120rpm，在25℃下培养32h，鲁氏酵母活菌数最高值可达1.07×10~(11)个／mL。球拟酵母的最优发酵条件为控制溶氧量在60％，发酵pH值为3.5，以6％的接种量，搅拌速度120rpm，在25℃下培养36h，球拟酵母活菌数的最高值可达和1.55×10~(11)个／mL。富集培养后的酵母菌体经5000rpm离心20min，用2.5％的氯化钠溶液洗涤多次，添加司盘60与土温80比例为1：1的混合物作为干燥保护剂，在温度为40℃条件下进行干燥，干燥至水分含量低于8％。干燥的产品经过25倍菌体重量的12Bé麦芽汁在36℃条件下复水15min，降温25℃活化120min。鲁氏酵母和球拟酵母的存活率分别为91.7％和91.07％。
4．将三株菌分别进行单因素发酵试验，确定其单独发酵时的最佳添加时间及添加量。并根据各个菌株单因素发酵的试验结果，采用三因素三水平正交试验设计，研究在混合菌种发酵黄豆酱的方法中嗜盐四链球菌、鲁氏酵母、球拟酵母的最佳配比。得到在酱醪发酵的第11天添加嗜盐四链球菌，添加量为10~6cfu／g酱醪，在酱醪发酵的第14天同时添加鲁氏酵母和球拟酵母，添加量分别为10~5个／g酱醪和10~6个／g酱醪。产品经GC-MS分析，鉴定出24种挥发性风味化合物。并通过对理化指标和风味检测表明，混合菌种发酵的黄豆酱比仅利用米曲霉发酵生产的黄豆酱产品品质更好。
Soybean paste is a traditional fermentation soybean product. Fermented soybean paste madewith such materials as wheat and soybean. Soybean paste has been popular more than one thousandyears because it has pleasure aroma, taste, nutrition and function. Microorganism play veryimportant role in the processing of soybean paste. Microorganism found in koji almost alwaysbelongs to fungi species, Aspergillus oryzae and/or Asp. Sojae. The koji mold has the ability toutilize starch, oligosaccharides, simple sugars, organic acids and alcohols, etc as carbon sources,and protein, amino acids and urea, etc as nitrogen sources. Halophilic yeast and lactic acid bacterialalso play an important role in developing flavour during fermentation of soybean paste, particularlyin the later stage of the fermentation. Traditionally, the mixture of boiled soybean and wheat flourmade cuboid or elliptoid shape and kept in an incubation room to make koji. The organisms comefrom either the previous batch or environment in which they are natural present. The inoculumgenerally contains selected flora of salted tolerant yeasts and bacterial capable of growing underanaerobic conditions. The dominant microorganisms are yeast Zygosaccharomyces Rouxii andTorulopsis sp and certain lactic acid bacteria such as Tetragenococcus Halophilus. Since pureculture of these microorganism speeds up fermentation and reduce the influence of weed yeast andbacteria, its use in commercial soybean paste preparation has been popular in recent years. In thispaper we focus on Tetragenococcus Halophilus, Zygosaccharomyces Rouxii and TorulopsisCandida, to produce commercial used lactic acid bacteria starter and dried yeast that can be appliedin industrial producing soybean paste.
1. Tetragenococcus Halophilus can be grown up at 12% NaCl, and Zygosaccharomyces Rouxiiand Torulopsis Candida can be grown up at 18% NaCl.
2. Tetragenococcus Halophilus is aerobic in MRS in culture medium, with growth optimagenerally at pH 6.5, a temperature of 34℃, a dissoluble oxygen of 15%, a inoculation concentrationof 3%, a stir speed of 120rpm, and the incubation time of 28h. At this optimum cultural condition,the active cell number of Tetragenococcus Halophilus was 1.22×10~(12) cfu/mL. The culture organismwas centrifugalized 20 min at 5000rpm. The bacterial concentrate was mixed with 10% skim milkpowder and 5% glycerin, and was dried by vacuum freezing. The active cell number of driedTetragenococcus Halophilus was 5.58×10~(10) cfu/g., and the survival rate of the dried bacteria was73.28%.
3. The culture media of Zygosaccharomyces Rouxii and Torulopsis Candida contained raw soysauce, glucose, and (NH_4)_2SO_4. It had been proved that 0.05% and 0.07% K_2HPO_4 in the culturemedia benefit the growth of Zygosaccharomyces Rouxii and Torulopsis Candida respectively. Theoptima fermentation condition of Zygosaccharomyces Rouxii was that controlled dissoluble oxygenof 50%, a pH of 3.5, a inoculation concentration of 8%, a stir speed of 120rpm, a temperature of25℃, a incubation time of 32h. The active cells number of Zygosaccharomyces Rouxii was1.07×10~(11)/mL. The optima fermentation condition of Torulopsis Candida was that controlleddissoluble oxygen of 60%, a pH 3.5, a inoculation concentration of 6%, stir speed of 120rpm, atemperature of 25℃, and a incubation time of 36h. The active cells number of Torulopsis Candidawas 1.55×10~(11)/mL. The culture organism was centrifugalized 20 min at 5000rpm. The yeastconcentrates were washed by 2.5% NaCl several times, and the concentrates were mixed withspan60 and tween80 (1:1). The mixture was dried to 8% moisture content at 40℃. The dried yeastpowder was dissolved in 12Béwort at 36℃(15min), and then the yeast solution was incubated at25℃, 120min. The survival rate Zygosaccharomyces Rouxii and Torulopsis Candida were 91.70%and 91.07% respectively.
4. In order to determine the optimal proportion of three strains, the three strains wereinoculated into soybean paste. Based on the effect of single strain on the quality of soybean paste, aL_9(3~3) orthogonal experiment design was conducted to study the proportion of TetragenococcusHalophilus, Zygosaccharomyces Rouxii and Torulopsis Candida. The results were thatTetragenococcus Halophilus was inoculated into soybean paste in the 11th day, and the inoculationconcentration was 10~6cfu/g. Zygosaccharomyces Rouxii and Torulopsis Candida were inoculatedinto soybean paste in the 14th day, however, the inoculation concentration of both was 10~5 cfu/g and10~6cfu/g respectively. 24 volatile flavor compounds were detected from soybean paste by GC-MS.The taste, chemical and physical characteristic and flavors, of that fermented by Aspergillus oryzae,yeast and lactic acid bacteria, were better than that only fermented by single Aspergillus oryzae.
引文
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