榨菜后熟优势微生物分离鉴定及发酵剂研制
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摘要
榨菜(zhacai)在我国有着悠久的生产历史,其清香味鲜,营养丰富,以其鲜、香、嫩、脆的风味特点在国内外广获殊荣。榨菜与德国酸甜甘蓝、法国酸黄瓜并称世界三大名腌菜。近年来,酱腌菜已朝着“低盐、增酸、低甜味、无防腐剂”的健康方向发展,众多研究人员在生产工艺、产品更新上作了大量的研究工作,使榨菜的生产工艺日趋成熟,产品也随着时代的要求不断创新更新。改进的工艺不但简化了加工过程,而且对产品质量的控制和提高都起到了重要作用。
本研究以涪陵优质榨菜的自然发酵过程为考察对象,通过对传统榨菜自然发酵不同时期微生物的分离、纯化、计数和鉴定,初步探明了传统榨菜发酵不同阶段的菌相组成及微生物菌系的消长变化规律,确定了榨菜发酵体系中的主要微生物及其性能,以及不同发酵阶段的菌种种类及比例。在此基础上,研究了人工接种微生物对榨菜品质及发酵特性的影响,为多菌种榨菜发酵剂的研制提供理论参考。本研究旨在通过对多菌种发酵剂的研究和使用,为提高榨菜品质,简化加工工艺,缩短榨菜加工周期,及榨菜加工的现代化探索路径。试验结论如下:
1.从传统榨菜生产中分离得到细菌包括:乳酸片球菌(Pedicoccusacidilactici)、嗜盐片球菌(Pedicoccus halophilus)、粪链球菌(Streptococcus faecalis)、乳链球菌(Streptococcus lactis)、肠膜明串珠菌(Leuconostoc mesenteriodies)、枯草芽孢杆菌(Bacillus subtilis)、蜡状芽孢杆菌(Bacillus cereus)、球形芽孢杆菌(Bacillus sphaericus)、多粘芽孢杆菌(Bacillus polymyxa)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)、坚强芽孢杆菌(Bacillus firmus)、植物乳杆菌(Lactobacillus plantarum)、发酵乳杆菌(Lactobacillus fermentum)、嗜盐乳杆菌(Lactobacillus.halotolerans)、乳酸乳杆菌(Lactobacillus lactis)等。
2.从传统榨菜生产中分离得到酵母菌包括:鲁氏酵母(Saccharomyces rouxii)、球形酵母(Saccharomyces globosus)、酿酒酵母(Saccharomyces cerevisiae)、小红酵母(Rhodotorula minuta)、易变球拟酵母(Torulopsis versatilis)、汉逊德巴利酵母(Debaryomyces hansenii)、异常汉逊酵母(Hansenula anomala)等。
3.从传统榨菜生产中分离得到的两属霉菌为青霉属(Penicillium)和曲霉属(Aspergillus)。
4.从传统榨菜中分离出的微生物有50%以上属于乳酸菌类,乳酸发酵活跃在榨菜后熟过程中,其中有几种耐高盐的菌种。如嗜盐片球菌,在较高的食盐含量中繁殖良好,与鲁氏酵母共同作用形成特殊酱香成分糖醇。
5.对发酵菌株进行优选,选择出生长速度快、代谢旺盛、产酸能力强、耐盐性和耐温性适宜的三种菌种,分别为植物乳杆菌、肠膜明串珠菌和乳酸乳杆菌。将这三种菌作为发酵菌株对人工接种多菌株采用不同接种量,接种配比,含盐量,含水量及真空度进行正交试验设计并进行感官评价,优化发酵工艺,得到不同因素对榨菜产品品质影响顺序,即:菌种比例＞含盐量＞含水量＞接种量＞真空度。同时得到较为理想的发酵剂组合,即接种量为3%、菌种比例为肠膜明串珠菌:乳酸乳杆菌:植物乳杆菌3:1:1、含水量为80%、含盐量为6%、真空度为0.091MPa。该发酵剂组合生产得榨菜产品与自然发酵榨菜两者在感官评价上无明显差异。对pH值和产酸性能测试表明,人工接种发酵工艺用20天左右,就可达到传统发酵工艺180天左右时的产酸量,风味物质的形成在60天左右完成,使榨菜发酵周期缩短为自然发酵榨菜的1/3。
6.对人工接种发酵榨菜和自然发酵榨菜进行感官鉴评及理化指标分析,以验证人工接种多菌种发酵榨菜的可行性。试验结果表明:
人工接种多种菌种发酵榨菜降低了榨菜生产过程中亚硝酸盐的含量,提高了产品的安全性。
两种方式生产的榨菜在感官品质及理化指标上无显著差异,在发酵过程中,人工接种多菌种发酵榨菜的氨基酸态氮含量和蛋白质含量一直高于自然发酵榨菜,说明人工接种多种菌种加快了榨菜风味物质的形成,缩短了榨菜的发酵周期,在实际生产中是可行的。
This research takes the natural fermentation process of quality zhacai as investgating object, by measuring and analyzing the separation, cultivation, calculation and classification of microorganisms and gross acidity, amino and nitrogen during different fermentation periods, the paper tends to exploit the bacteria form, the changing principle of the bacteria family and the major microorganisms in zhacai ferment system. Based on tendency analysis on traditional zhacai spontaneous fermentation, the paper should study the major microorganisms and their performance in zhacai production, choose bacteria vaccine and its addition, improve preparation and fermentation technique of zhaicai production, thus provide theoretical basis for development of suitable preparatory culture of zhaicai production. The conclusions are as followed:
1. Pedicoccus acidilactici, Pedicoccus halophilus, Streptococcus faecalis, Streptococcus lactis, Leuconostoc mesenteriodies, Bacillus subtilis, Bacillus cereus, Bacillus sphaericus, Bacillus polymyxa, Bacillus stearothermophilus, Bacillus firmus, Lactobacillus plantarum, Lactobacillus.halotolerans, Lactobacillus fermentum, Lactobacillus lactis were selected for production of natural fermented zhacai
2. Saccharomyces rouxii, Saccharomyces globosus, Saccharomyces cerevisiae, Rhodotorula minuta, Torulopsis versatilis, Debaryomyces hansenii, Hansenula anomala were selected for production of natural fermented zhacai
3. Penicillium spp, Aspergillus spp were selected for production of natural fermented zhacai
4. More than half of isolating microorganisms were lactic acid bacteria. Lactic acid bacteria lactic acid fermentation is important during zhcai after ripening and fermentation. Lactic acid bacteria produce lactic acid from hexoses. The zhacai end-products of carbohydrate catabolism by lactic acid bacteria contribute to the flavour, aroma and texture, thereby helping to determine unique product characteristics. For instance, the synergistic effect of Pedicoccus halophilus and Saccharomyces roiai has been investigated in detail due to its pivotal role in allowing growth in the development of flavour and texture in zhacai.
5. The main purpose of this study was to obtain starter cultures for zhacai marking process. Lactic acid bacteria strains were isolated and identified. The metabolic properties (growth rate, acid producing ability, temperature resistance and salinity tolerance) of the selected strains were determined. The strains with best potential as zhacai starter culture were Lactobacillus plantation, Leuconostoc mesenteroides and Lactobacillus lactis. To get a better fermentation technology, the three kinds of bacteria were used as fermentation strains, with different inoculation amount, inoculation for ratio, salinity, water content and vacuity in an orthogonal experimental design and sensory evaluation test. A sequence that indicates the importance of different factors was got, that is, strains ratio > salinity > water content > vacuity. Moreover, a ideal starter culture combination was gained, that is, inoculation amount 3%, the ratio of Lm:La:Lp = 3:1:1, water content 80%, salinity 6% and vacuity 0.091Mpa. In the sensory evaluation test, the quality of zhacai produced with this starter culture has no significant differences with natural fermentation zhacai, as well as their acceptance. Measuring of pH and acid-forming performance results show that artificial inoculation can induced the 180 days fermentation period of traditional fermentation to 20 days, The formation of flavor compounds takes about 60 days, with the same acid-forming amount, and shorten the fermentation period to 1/3 of original fermentation period.
6. The change rules of sensory indexes and physiochemical indexes for artificial inoculation and natural fermentation during zhacai fermentation were studied. The results indicated that artificial inoculation was identical in sensory indexes and physiochemical indexes in relation to natural fermentation, while artificial inoculation was more effective in retaining amino acid nitrogen and protein content of zhacai than natural fermentation. Artificial inoculation also could significantly reduce the nitrite content of zhacai, enhance the safety of zhacai production, artificial inoculation could accelerate the process of zhacai after ripening and fermentation, pick up the development of flavour and texture, shorten the generating period of zhacai production, had little effect on sensory quality. Artificial inoculation is a feasible approach for zhacai actual production.
本研究以涪陵优质榨菜的自然发酵过程为考察对象,通过对传统榨菜自然发酵不同时期微生物的分离、纯化、计数和鉴定,初步探明了传统榨菜发酵不同阶段的菌相组成及微生物菌系的消长变化规律,确定了榨菜发酵体系中的主要微生物及其性能,以及不同发酵阶段的菌种种类及比例。在此基础上,研究了人工接种微生物对榨菜品质及发酵特性的影响,为多菌种榨菜发酵剂的研制提供理论参考。本研究旨在通过对多菌种发酵剂的研究和使用,为提高榨菜品质,简化加工工艺,缩短榨菜加工周期,及榨菜加工的现代化探索路径。试验结论如下:
1.从传统榨菜生产中分离得到细菌包括:乳酸片球菌(Pedicoccusacidilactici)、嗜盐片球菌(Pedicoccus halophilus)、粪链球菌(Streptococcus faecalis)、乳链球菌(Streptococcus lactis)、肠膜明串珠菌(Leuconostoc mesenteriodies)、枯草芽孢杆菌(Bacillus subtilis)、蜡状芽孢杆菌(Bacillus cereus)、球形芽孢杆菌(Bacillus sphaericus)、多粘芽孢杆菌(Bacillus polymyxa)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)、坚强芽孢杆菌(Bacillus firmus)、植物乳杆菌(Lactobacillus plantarum)、发酵乳杆菌(Lactobacillus fermentum)、嗜盐乳杆菌(Lactobacillus.halotolerans)、乳酸乳杆菌(Lactobacillus lactis)等。
2.从传统榨菜生产中分离得到酵母菌包括:鲁氏酵母(Saccharomyces rouxii)、球形酵母(Saccharomyces globosus)、酿酒酵母(Saccharomyces cerevisiae)、小红酵母(Rhodotorula minuta)、易变球拟酵母(Torulopsis versatilis)、汉逊德巴利酵母(Debaryomyces hansenii)、异常汉逊酵母(Hansenula anomala)等。
3.从传统榨菜生产中分离得到的两属霉菌为青霉属(Penicillium)和曲霉属(Aspergillus)。
4.从传统榨菜中分离出的微生物有50%以上属于乳酸菌类,乳酸发酵活跃在榨菜后熟过程中,其中有几种耐高盐的菌种。如嗜盐片球菌,在较高的食盐含量中繁殖良好,与鲁氏酵母共同作用形成特殊酱香成分糖醇。
5.对发酵菌株进行优选,选择出生长速度快、代谢旺盛、产酸能力强、耐盐性和耐温性适宜的三种菌种,分别为植物乳杆菌、肠膜明串珠菌和乳酸乳杆菌。将这三种菌作为发酵菌株对人工接种多菌株采用不同接种量,接种配比,含盐量,含水量及真空度进行正交试验设计并进行感官评价,优化发酵工艺,得到不同因素对榨菜产品品质影响顺序,即:菌种比例＞含盐量＞含水量＞接种量＞真空度。同时得到较为理想的发酵剂组合,即接种量为3%、菌种比例为肠膜明串珠菌:乳酸乳杆菌:植物乳杆菌3:1:1、含水量为80%、含盐量为6%、真空度为0.091MPa。该发酵剂组合生产得榨菜产品与自然发酵榨菜两者在感官评价上无明显差异。对pH值和产酸性能测试表明,人工接种发酵工艺用20天左右,就可达到传统发酵工艺180天左右时的产酸量,风味物质的形成在60天左右完成,使榨菜发酵周期缩短为自然发酵榨菜的1/3。
6.对人工接种发酵榨菜和自然发酵榨菜进行感官鉴评及理化指标分析,以验证人工接种多菌种发酵榨菜的可行性。试验结果表明:
人工接种多种菌种发酵榨菜降低了榨菜生产过程中亚硝酸盐的含量,提高了产品的安全性。
两种方式生产的榨菜在感官品质及理化指标上无显著差异,在发酵过程中,人工接种多菌种发酵榨菜的氨基酸态氮含量和蛋白质含量一直高于自然发酵榨菜,说明人工接种多种菌种加快了榨菜风味物质的形成,缩短了榨菜的发酵周期,在实际生产中是可行的。
This research takes the natural fermentation process of quality zhacai as investgating object, by measuring and analyzing the separation, cultivation, calculation and classification of microorganisms and gross acidity, amino and nitrogen during different fermentation periods, the paper tends to exploit the bacteria form, the changing principle of the bacteria family and the major microorganisms in zhacai ferment system. Based on tendency analysis on traditional zhacai spontaneous fermentation, the paper should study the major microorganisms and their performance in zhacai production, choose bacteria vaccine and its addition, improve preparation and fermentation technique of zhaicai production, thus provide theoretical basis for development of suitable preparatory culture of zhaicai production. The conclusions are as followed:
1. Pedicoccus acidilactici, Pedicoccus halophilus, Streptococcus faecalis, Streptococcus lactis, Leuconostoc mesenteriodies, Bacillus subtilis, Bacillus cereus, Bacillus sphaericus, Bacillus polymyxa, Bacillus stearothermophilus, Bacillus firmus, Lactobacillus plantarum, Lactobacillus.halotolerans, Lactobacillus fermentum, Lactobacillus lactis were selected for production of natural fermented zhacai
2. Saccharomyces rouxii, Saccharomyces globosus, Saccharomyces cerevisiae, Rhodotorula minuta, Torulopsis versatilis, Debaryomyces hansenii, Hansenula anomala were selected for production of natural fermented zhacai
3. Penicillium spp, Aspergillus spp were selected for production of natural fermented zhacai
4. More than half of isolating microorganisms were lactic acid bacteria. Lactic acid bacteria lactic acid fermentation is important during zhcai after ripening and fermentation. Lactic acid bacteria produce lactic acid from hexoses. The zhacai end-products of carbohydrate catabolism by lactic acid bacteria contribute to the flavour, aroma and texture, thereby helping to determine unique product characteristics. For instance, the synergistic effect of Pedicoccus halophilus and Saccharomyces roiai has been investigated in detail due to its pivotal role in allowing growth in the development of flavour and texture in zhacai.
5. The main purpose of this study was to obtain starter cultures for zhacai marking process. Lactic acid bacteria strains were isolated and identified. The metabolic properties (growth rate, acid producing ability, temperature resistance and salinity tolerance) of the selected strains were determined. The strains with best potential as zhacai starter culture were Lactobacillus plantation, Leuconostoc mesenteroides and Lactobacillus lactis. To get a better fermentation technology, the three kinds of bacteria were used as fermentation strains, with different inoculation amount, inoculation for ratio, salinity, water content and vacuity in an orthogonal experimental design and sensory evaluation test. A sequence that indicates the importance of different factors was got, that is, strains ratio > salinity > water content > vacuity. Moreover, a ideal starter culture combination was gained, that is, inoculation amount 3%, the ratio of Lm:La:Lp = 3:1:1, water content 80%, salinity 6% and vacuity 0.091Mpa. In the sensory evaluation test, the quality of zhacai produced with this starter culture has no significant differences with natural fermentation zhacai, as well as their acceptance. Measuring of pH and acid-forming performance results show that artificial inoculation can induced the 180 days fermentation period of traditional fermentation to 20 days, The formation of flavor compounds takes about 60 days, with the same acid-forming amount, and shorten the fermentation period to 1/3 of original fermentation period.
6. The change rules of sensory indexes and physiochemical indexes for artificial inoculation and natural fermentation during zhacai fermentation were studied. The results indicated that artificial inoculation was identical in sensory indexes and physiochemical indexes in relation to natural fermentation, while artificial inoculation was more effective in retaining amino acid nitrogen and protein content of zhacai than natural fermentation. Artificial inoculation also could significantly reduce the nitrite content of zhacai, enhance the safety of zhacai production, artificial inoculation could accelerate the process of zhacai after ripening and fermentation, pick up the development of flavour and texture, shorten the generating period of zhacai production, had little effect on sensory quality. Artificial inoculation is a feasible approach for zhacai actual production.
引文
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