【作者】 武俊瑞；

【导师】 岳喜庆；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2013， 博士

【摘要】 自然发酵酸菜和大酱是我国东北的传统特色食品,因其风味独特,至今仍深受老百姓的喜爱和青睐,在东北人民日常饮食中占据着很大比重。经过长期的自然选择和训化,其中蕴藏着丰富的乳酸菌、酵母菌等有益菌群,是研究和开发有益菌群的天然宝库。然而,由于采用的传统研究方法单一以及存在的缺陷,导致东北酸菜和大酱中的主要微生物类群组成、优势和关键菌群,至今仍不甚明确,也不够准确和客观,进而无法很好地指导研究和生产,至今普通工业化产品仍难以替代。本研究以采自我国东北三省不同地区农户家庭采用传统方法自然发酵的97份酸菜发酵液和43份大酱样品为试验对象,分别通过传统的纯培养技术和16S或26S rDNA序列分析、PCR-DGGE.新一代焦磷酸测序技术等三个不同层面和角度,对东北特色传统发酵酸菜和大酱中的乳酸菌和酵母菌等主要发酵微生物的群落结构及多样性进行系统研究,以期为进一步研究、保护和利用东北传统发酵食品中的有益乳酸菌和酵母菌等主要发酵微生物,提供理论依据。具体研究内容又分为以下七个试验：试验1从东北三省30个地区农户家庭,采集了97份采用传统方法自然发酵的酸菜发酵液样品,通过选择性纯培养技术方法,从中分离筛选出133株乳酸菌疑似菌株,再采用16S rDNA序列同源性分析方法对各菌株属种进行鉴定。结果表明,均为乳酸菌,分属3个属,8个种,呈现出较为丰富的乳酸菌多样性,34株菌归为植物乳杆菌(L.plantarum),占总菌株数的25.56%；23株菌归为棒状乳杆菌(L. coryniformis),占总菌株数的17.29%；21株菌归为弯曲乳杆菌(L. curvatus),占总菌株数的15.79%；18株菌归为清酒乳杆菌(L. sakei),占总菌株数的13.53%；12株菌归为短乳杆菌(L. brevi),占总菌株数的9.02%；13株菌归为屎肠球菌(E. faecium),占总菌株数的9.77%；4株菌归为粪肠球菌(E. faecalis),占总菌株数的3.01%；8株菌归为肠膜明串珠菌(Leu. mesenteroides),占总菌株数的6.02%。以此可以推断,植物乳杆菌、棒状乳杆菌、弯曲乳杆菌和清酒乳杆菌可能是东北自然发酵酸菜发酵液中的优势菌群。试验2从东北三省14个地区农户家庭采集了43份采用传统方法自然发酵的豆酱样品,通过选择性纯培养技术方法,从中分离筛选到了74株乳酸菌疑似菌株,再采用16S rDNA序列分析法对各菌株属种进行鉴定。结果表明,均为乳酸菌,分属8个种,分别是嗜盐四联球菌(T. halopHilus)30株,为样品中分离数量最多的菌种,占总数的40.54%；植物乳杆菌(L. plantarum)14株,占总数的18.92%；清酒乳杆菌(L. sakei)13株,占总数的17.57%；发酵乳杆菌(L.fermentum)9株,占总数12.16%；干酪乳杆菌(L. casei)2株,占总数2.7%；短乳杆菌(L. brevi)2株,占总数2.7%,戊糖片球菌(P. pentosaceus)2株,占总数2.7%,耐久肠球菌(E. durans)2株,占总数2.7%。从乳酸菌地区和样品分布结果可以初步推断出：嗜盐四联球菌、植物乳杆菌和清酒乳杆菌是东北传统发酵豆酱中的优势乳酸菌菌群。试验3通过选择性纯培养技术方法,从43份传统发酵豆酱样品中,分离筛选出93株酵母菌疑似菌株,进一步采用26S rDNAD1/D2区序列分析对各菌株属种进行鉴定。结果表明,均为酵母菌,分属9个种,呈现出较为丰富的多样性。分别是二孢接合酵母(Z. bisporus)28株,占总数的30.11%,该种菌在样品中分离数量最多,且在14个地区采集的样品中均分离到了该菌；近平滑假丝酵母(C. parapsilosis)25株,占总菌数的26.88%,分离数量次之,14个地区采集的样品中均分离到了该菌；汉逊德巴利酵母(D. hansenii)18株,占总数的19.35%,除了齐齐哈尔、延边、辽阳、营口和锦州外,有9个地区样品中都分离到了该种菌；解脂耶氏酵母(Y. lipolytica)8株,占总菌数的8.60%,该菌株仅在大庆市、铁岭市、盘锦市和锦州市采集样品中分离得到,其它地区样品中未分离到；粘质红酵母(R. mucilaginosa)7株,占总数7.53%,该菌种仅在铁岭、沈阳和葫芦岛分离到；涎沫假丝酵母菌(C. zeylanoides)2株,占总数2.15%,该菌株仅在营口地区样品中分离到；浅白隐球酵母(C. albidus)2株,占总数2.15%,仅在大庆地区样品中分离到；C. liquefaciens2株,占总数2.15%,该菌株仅在大庆和铁岭地区样品中分离到；从大庆地区样品中分离到了1株C. sorbosivorans,占总数1.08%。从获得酵母菌在地区和样品分布结果可以初步推断出：二孢接合酵母(Z. bisporus)、汉逊德巴利酵母(D. hansenii)和近平滑假丝酵母(C. parapsilosis)是东北地区传统发酵豆酱中优势酵母菌菌群。试验4从传统发酵酸菜发酵液样品中筛选出30份,直接提取各样品宏基因组,并进行PCR-DGGE,再进一步筛选条带清晰且较为丰富的样品上的条带,进行16S rDNA序列测定和分析比对。结果表明,30份样品均获得了清晰的电泳条带,对筛选出条带较为丰富的11份样品的清晰条带的16S rDNA序列测定,并作分析和比对可知,东北传统发酵酸菜发酵液中蕴藏着丰富的乳酸菌资源,属种多样,其中,植物乳杆菌(L.plantarum)、短乳杆菌(L. brevi)、清酒乳杆菌(L.sakei)、弯曲乳杆菌(L. curvatus)四种菌为传统发酵酸菜发酵液中的优势菌群。试验5从传统发酵大酱样品中筛选出30份,直接提取各样品宏基因组,并进行PCR-DGGE,再进一步筛选条带清晰且较为丰富的样品上的条带,进行16S rDNA序列测定和分析比对。结果表明,30份样品均获得了清晰的电泳条带,对筛选出条带较为丰富的14份样品的清晰条带的16S rDNA序列测定,并作分析和比对可知,东北传统发酵大酱中的细菌属种多样,鉴定出9个属,16个种,乳酸菌从种类和分布上均占优势,而植物乳杆菌(L.plantarum)、明串珠菌(Leuconostoc)、屎肠球菌(E. faecium)和嗜盐四联球菌(T. halopHilus)四种菌又为优势乳酸菌群。试验6对6份酸菜发酵液进行焦磷酸测序和分析,结果表明,样品中细菌以厚壁菌门Firmicutes (41%)和变形菌门Proteobacteria (34%)的细菌为主。乳杆菌目Lactobacillales占厚壁菌门的72%,包括Enterococcaceae、Lactobacillus、Pediococcus、Lactococcus、Leuconostocaceae和Weissella。变形菌门Proteobacteria中Alphaproteobacteria目的产碱菌Alcaligenaceae最多。真菌以子囊孢菌门Ascomycota (87%)中的酵母菌Saccharomycotina亚门(91%)为主。酵母菌亚门Saccharomycotina中的德巴利酵母科Debaryomycetaceae、耶罗威亚酵母科Dipodascaceae、酵母科Saccharomycetaceae和接合酵母Zygosaccharomyces等占多数,而德巴利汉逊酵母Debaryomyces hansenii和酿酒酵母Saccharomyces servazzii为主要发酵酵母。试验7对6份大酱样品进行焦磷酸测序并作分析,结果表明,样品中细菌主要由厚壁菌门Firmicutes (46%)和变形菌门Proteobacteria (30%)为主。厚壁菌门Firmicutes中,主要以芽孢杆菌纲(Bacilli)中的乳杆菌目Lactobacillales (70%)中的Lactobacillus、 Enterococcaceae、Tetragenococcus、Lactococcus、Streptococcus、Leuconostocaceae和Weissella等,以及芽孢杆菌目Bacillales(24%)中的芽孢杆菌科(Bacillaceae)中的地衣芽孢杆菌Bacillus licheniformis为主。变形菌门Proteobacteria中,以Alphaproteobacteria、 Betaproteobacteria和Gammaproteobacteria纲细菌为主。大酱样品中真菌主要为子囊孢菌门Ascomycota (99%)中酵母菌亚门Saccharomycotina中的德巴利酵母科Debaryomycetaceae中的德巴利汉逊酵母Debaryomyces hansenii,以及盘菌亚门Pezizomycotina中青霉属Penicillium等为主。以上试验结果表明：东北传统发酵酸菜发酵液和大酱中微生物复杂多样,几乎涵盖了细菌和真菌的各个门目的种属,均以厚壁菌门Firmicutes和变形菌门Proteobacteria细菌和子囊孢菌门Ascomycota中的酵母菌Saccharomycotina亚门真菌为主。细菌又以乳杆菌目属种占优势,真菌以德巴利汉逊酵母Debaryomyces hansenii为主,这些属种大多为有益菌群,但大酱较酸菜发酵液中微生物构成更为复杂,具体菌群种类和数量也不尽相同。更多还原

【Abstract】 As the specialty foods, suan-cai and da-jiang made by the traditional method are still very popular because of its unique flavor in the northeast of China, which occupies a large proportion of the northeast people’s daily diet. After a long period of natural selection and training, a lot of lactic acid bacteria, yeast and other beneficial bacteria are enriched in suan-cai and da-jiang. Therefore, the traditional fermented food suan-cai and da-jiang are always considered as one of valuable natural resources to research and develop beneficial bacteria. However, due to the defects of traditional cultured method, the composition of microbe and the main microbial groups involved in the northeast suan-cai and da-jiang are still not very clear and objective. The production is difficult to industrialization completely. The current results are urgently needed to be comprehended and supplemented. Thus it can able to guide the research and production well. In the present study,97suan-cai samples and43da-jiang samples were collected from located peasant household in different areas of three provinces in Northeast China. All samples were made by traditional natural fermentation method. Based on the combination of traditional pure cultured method and16S or26S rDNA sequence analysis, and PCR-DGGE, as well as a new pyrosequencing technology, the study developed a systematic research of the community structure of lactic acid bacteria and yeast and other major fermentation microorganisms involved in suan-cai and da-jiang in three different aspects and angles. The study may be helpful to protect and utilize the lactic acid bacteria and yeasts and other major fermentation microorganisms in traditional fermented food collected in northeast of China. The concrete research content was divided into the following seven tests:Test1:A total of133isolates were obtained from97samples of naturally fermented suan-cai broth by traditional screened cultivation, that were showing to be lactic acid bacteria. According to16S rDNA sequence analysis, the isolates were grouped in three genera and8species, including Lactobacillus plantarum (34strains,25.56%), Lactobacillus coryniformis (23strains,17.29%), Lactobacillus curvatus (21strains,15.79%), Lactobacillus sakei (18strains,13.53%), Lactobacillus brevis (12strains,9.02%), Enterococcus faecium (13strains,3.01%), Enterococcus faecalis (4strains,3.01%), and Leuconostoc mesenteroides (8strains,6.02%). In general, Lactobacillus plantarum, Lactobacillus coryniformis, Lactobacillus curvatus and Lactobacillus sakei probably can be regarded as the predominate lactic acid bacteria species in suan-cai.Test2:A total of74isolates were obtained from43samples of naturally fermented da-jiang by traditional screened cultivation, that were showing to be lactic acid bacteria. According to16S rDNA sequence analysis, the isolates were grouped in8species, including Tetragenococcus halophilus (30strains,40.54%), Lactobacillus plantarum (14strains,18.92%). Lactobacillus sakei (13strains,17.57%), Lactobacillus fermentum (9strains. 12.16%), Lactobacillus casei (2strains,2.7%), Lactobacillus brevis (2strains,2.7%), Enterococcus durans (2strains,2.7%). In general, Tetragenococcus halophilus, Lactobacillus plantarum and Lactobacillus sakei probably can be regarded as the predominate lactic acid bacteria species in da-jiang.Test3:A total of93isolates were obtained from43samples of naturally fermented da-jiang by traditional screened cultivation that was showing to be yeast. According to26S rDNAD1/D2sequence analysis, the isolates were grouped in9species, including Zygosaccharomyces bisporus (28strains,30.11%), Candida parapsilosis (25strains,26.88%), Debaryomyces hansenii (18strains,19.35%), Yarrowia Lipolytica (8strains,8.60%), Rhodotorula mucilaginosa (7strains,7.53%), Candida zeylanoides (2strains,2.15%), Cryptococcus liquefaciens (2strains,2.15%), and Candida sorbosivorans (1strains,1.08%). In general, Zygosaccharomyces bisporus, Candida parapsilosis and Debaryomyces hansenii were probably can be regarded as the predominate yeast species in da-jiang.Test4:Diversity of lactic acid bacteria involved in30selected suan-cai samples was analyzed by denaturing gradient gel electrophoresis (DGGE). The results indicated that Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus sakei and Lactobacillus curvatus were the predominant microflora in samples. Cocci-shaped strains of Pediococcus sp. and Lactococcus lactis were also identified in some samples.Test5:In this study,30samples of naturally fermented da-jiang were analyzed by denaturing gradient gel electrophoresis (DGGE) to determine the diversity of the bacteria involved in fermentation. The results indicate that lactic acid bacteria, including Lactobacillus plantarum, uncultured Leuconostoc mesenteroides, Leuconostoc gasicomitatum, Enterococcus faecium, and Tetragenococcus halophilus, were the predominant species. This is the first report of Enterococcus spp. and Leuconostoc spp. in the Chinese fermented soybean paste dajiang using DGGE. The presence of Bacillus spp.(including Bacillus firmus), Oceanobacillus spp., and Paenibacillus glycanilyticus in the dajiang samples may be due to their salt tolerance. Potentially pathogenic Alphaproteobacteria and Staphylococcus epidermidis strains were also detected in this study. Moreover, three uncultured bacterium clones were found in some samples and require further study. The results reveal a high level of bacterial diversity in da-jiang.Test6:In the study, we firstly examined the microbe diversity of suan-cai broth using high-throughput barcoded pyrosequencing. The results showed that the member of Firmicutes (41%) and Proteobacteria (34%) were the main bacteria. Lactobacillales, including Enterococcaceae, Lactobacillus, Pediococcus, Lactococcus, Leuconostocaceae and Weissella, account72%of the total bacteria in Firmicutes. In Proteobacteria, Alcaligenaceae was the most genera which belonging to Alphaproteobacteria. In fungi, Ascomycota occupied87%, and Saccharomycotina was the most genera which mainly included Debaryomycetaceae, Dipodascaceae, Saccharomycetaceae, Zygosaccharomyces.Test7:Furthermore, we also examined the microbe diversity of da-jiang using high-throughput barcoded pyrosequencing. The results showed that the member of Firmicutes (46%) and Proteobacteria (30%) were the main bacteria. Lactobacillales, including Lactobacillus, Enterococcaceae, Tetragenococcus, Lactococcus, Streptococcus, Leuconostocaceae and Weissella, account70%of the total bacteria in Firmicutes. Besides, Bacillus licheniformis was found as the main genera involved in Bacillales which accounted24%of Firmicutes. Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria were the most genera in Proteobacteria. In fungi, Ascomycota occupied99%, and Saccharomycotina and Pezizomycotina were the most genera in da-jiang which mainly included Debaryomyces hansenii and Penicillium.In conclusion, the above experimental results showed that the microbial involved in traditional fermentation suan-cai and dajiang which collected in northeast of China was complex. Firmicutes and Proteobacteria, as well as Saccharomycotina were the predominant bacteria and fungi in both two kind of traditional fermentation. In bacteria, the specie of Lactobacilli was dominate, and Debaryomyces hansenii was dominate in fungi. The genera involved are mostly the beneficial bacterium group. Besides, the microbe composition involved in da-jiang is more complicated than suan-cai. The species and quantity involved in suan-cai and da-jiang are also different.更多还原