牦牛奶酪中乳酸菌的表型、基因型和益生特性的研究
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摘要
牦牛奶酪乳酸菌是我国重要而且独特的生物资源,有极其重要的收集、整理、保存、研究的价值。本研究从西藏、云南、新疆三个地区采集17份牦牛奶酪样品,分离纯化出了其中的乳酸菌,研究了乳酸菌的表型、基因型和益生特性。
表型特性的研究即用传统的菌落形态、革兰氏染色显微形态、生理生化、在不同环境(温度、pH值、盐度)中的生长特生,碳水化合物发酵方式等实验项目,将乳酸菌划分到属,进而初步判断出菌种,此外,还分析了产酸活性、利用柠檬酸盐等表型特性,以获得乳酸菌的基本数据,
基因型特性的研究包括16S rRNA序列分析和随机扩增多态性DNA技术。本研究建立了属特异性PCR→16S rRNA序列分析→种特异性PCR的快速鉴定模式,构建了系统进化树,以分析乳酸菌菌株的遗传亲缘关系。建立了适用于乳酸菌的RAPD反应体系和程序,用于分离鉴定出的乳酸菌,用NTsys2.10e软件对其图谱做聚类分析,与16S rRNA序列分析的结果相比较,分析菌株之间的遗传亲缘关系,分析菌株聚类方式与地域之间的相关性。
益生特性的研究包括乳酸菌菌株的产细菌素、产胞外多糖、产叶酸、降胆固醇的益生特性,并研究了菌株对抗生素的抗性、体内环境的耐受性、黏附活性,筛选出有突出益生功能,抗逆性较好的菌株,有希望作为益生菌加以开发应用。
主要的研究结果如下：
(1)西藏、云南、新疆三个地区的样品平均pH值分别为4.31±0.39；4.69±0.29：4.52±0.22,地区之间不存在显著性差异(P=0.176>0.05),三个地区菌落计数平均值分别为4.17±0.43；4.30±0.68；3.9±0.59log CFU/g,也不存在显著性差异(P=0.468>0.05)
(2)从17份样品中分离出39株乳酸菌,通过表型方法初步判断出菌种。35株菌为杆菌,被分为7个菌种：L.buchneri2株(Y15.Y16),L.casei16株(T7.Y19,X21,X22,X23,X26,X27,X28,X29,X30,X31,X32,X33,X34,X35,Y36)L.diolivorans3株(Y18,X24,X25),L.fermentum3株(T1,T2,T3),L.helveticus3株(T6,T8,Y9),L.kefiri3株(Y10,Y12,Y20),L.plantarum5株(T4,Y13,Y37,X38,X39)。4株菌为球菌,被分为2个菌种：Pediococcus acidilactici1株(T5),Pediococcus pentosaceus3株(Y11、Y14、Y17)。
(3)本研究测出6株乳杆菌产酸活性达到了起酵菌株的标准：X24、X38、X29、X25、X21、X22。选出发酵柠檬酸盐活性较强的菌株有：T4、T7、Y10、Y13、X25、X28、Y36、Y37,这些菌株的优良特性是在长期适应环境、以及生产实践中积累形成的。
(4)16S rRNA序列分析：将39株乳酸菌用16S rRNA序列分析法鉴定到种,并构建了系统进化树,进一步验证了菌种的划分,与表型特性是一致的。
(5)随机扩增多态性研究：做出了39株乳酸菌的RAPD指纹图谱,除X23、Y36和Y37之外,其余菌株均按照不同的菌种聚类,与16S rRNA序列分析法得出的结果基本是一致的,菌株聚类方式与地域有较强的相关性,说明RAPD技术在分类鉴定中有一定的应用价值,可解析菌株之间的遗传亲缘关系。
(6)检出31株菌产细菌素,菌株Y13能够拮抗5种致病菌：Bacillus cereus ATCC10876. Escherichia coli0157:H7、 Listeria monocytogenes、Citrobacter freundii ATCC8090、Salmonella Typhimurium ATCC13311,抑菌谱最宽X29和X30能拮抗4种致病菌：Bacillus cereus ATCC10876、Citrobacter freundii ATCC8090. Salmonella Typhimurium ATCC13311、Enterobacter cloacae ATCC23355。所有乳酸菌细菌素对嗜热链球菌Streptococcus thermophilus都没有拮抗活性,在发酵乳制品时,这些乳杆菌可与嗜热链球菌合用,相互之间没有拮抗作用。
(7)筛选出产胞外多糖的菌株有T3、T4、T5、T7、T8、Y11、Y14、Y17、X29,用苯酚-硫酸法定量检测所有筛出菌株的产量,与文献报道的产量相比处于中等水平,最高产菌株T8产量为193.72mg/L。
(8)筛选出9株菌可以产叶酸：T3、T7、T8、X23、X25、X26、X27、X29、Y37。最高产的菌株是Y37,总产量达到了85.98±5.09ng/ml,其次是X25,总产量为81.17±2.08ng/ml,与文献报道的产率17-100ng/ml相比,本实验筛选出来的菌株X25和Y37已达到了很高的产量。
(9)研究了乳酸菌在MRS-CHOL培养基中对胆固醇去除率,Y15、X25和X27对胆固醇降解率分别为45.59%,50.01%和53.18%,都达到40%以上,与文献报道过的乳酸菌菌株相比属于比较高的水平。
(10)用纸片扩散法测了39株菌对抗生素的抗性。39株乳酸菌对氨苄青霉素、氯霉素、红霉素敏感的菌株分别占74.4%；82.1%和82.1%,对萘啶酮酸、链霉素、万古霉素和新霉素有抗性的菌株分别占100%；97.4%；92.3%和82.05%。
(11)对体内环境耐受性很强菌株有3株：Y15、X25和X27,粘附性也很好。
(12)本研究鉴定并筛选出3株乳酸菌能耐受体内环境、有可能定植于肠道中,并有特定的保健功能,有可能作为益生菌开发。Y15(.L.buchneri)能产胞外多糖(50.26±1.25mg/L),降胆固醇(去除率为45.59%),对萘啶酮酸、链霉素、万古霉素有抗性。X25(L.diolivorans)能产叶酸(81.17±2.08ng/mL),能降胆固醇(去除率为50.01%),对萘啶酮酸、链霉素、四环素、万古霉素有抗性。X27(L.casei)能产胞外多糖(38.62±1.21mg/L),产叶酸(77.83±2.52ng/mL),能降胆固醇(去除率为53.8%),所产细菌素对Bacillus cereus ATCC10876, Staphylococcus aureus、Escherichia coli O157:H7有拮抗活性,对新霉素、萘啶酮酸、链霉素、万古霉素有抗性。
本论文的创新之处在于
(1)牦牛奶酪乳酸菌是我国重要而且独特的生物资源,有极其重要的收集、整理、保存、研究的价值。本研究是对牦牛奶酪中的乳酸菌进行的最完整、系统、全面的基础研究。
(2)建立了属特性PCR→16S rRNA序列分析→种特异性PCR的菌种鉴定模式,可用于对大量野生菌株快速鉴定菌种,能有效提高鉴定效率和准确性。
(3)构建了重现性好的RAPD反应体系和程序,用于乳酸菌菌株的基因分型,其聚类分析结果与16S rRNA序列分析法基本是一致的,说明它在乳酸菌的分类鉴定中有较好的区分效率,可与其它基因型鉴定方法结合使用,提高菌种的鉴定效率。菌株RAPD图谱聚类方式与地域之间有较强的相关性,可用于菌株原产地的分析,保护菌种资源。
(4)从菌株中筛选出了三株有突出益生功能、抗逆性较好的菌株,有希望作为益生菌开发应用。
There are various kinds of lactic acid bacteria existing in Chinese yak milk cheeses, which is an extremely precious natural microbiology resource. It is a research work of great values that collecting, organizing, preserving and investigating those lactic acid bacteria. This study collected17yak milk cheese samples from Tibet, Yunnan and Xinjiang province, isolated lactic acid bacteria, studied their phenotypic, genotypic and probiotic characterization.
The phenotypic characterization study included species identification of isolated lactic acid bacteria with classic taxonomy methods, such as colony morphology, Gram stain and microscopic morphology, primary biochemical and physiological properties, growth capability in different conditions (temperature, pH value, and salt concentration), carbohydrates fermentation patterns, acid producing activity, capability of utilizing citrate and so on. Isolated lactic acid bacteria were assigned to genus level, and preliminary species level. Phenotypic experiment results provided a comprehensive data of lactic acid bacteria. The capability of producing acid and utilizing citrate directly demonstrated lactic acid bacteria's role in fermentation.
The genotypic characterization study included16S rRNA sequenc analysis and randomly amplified polymorphic DNA (RAPD). This study established a rapid identification mode:genus specific PCR→16S rRNA sequence analysis→species specific PCR. Isolated lactic acid bacteria were identified to species level with this mode, phylogenetic tree was constructed based on16S rDNA sequences to confirm species assignment and demonstrate strains'genetic relationship. This study also established a stable RAPD reaction system and amplification program for lactic acid bacteria. Isolated lactic acid bacteria were subjected to this RAPD analysis, NTsys2.10e software was used to perform cluster analysis of strains'RAPD fingerprinting patterns. The cluster result were compared with that of16S rRNA sequence analysis to figure out genetic relations of isolated strains, as well as the correlativity between strains'cluster patterns and origin regions.
The probiotic characterization study included bacteriocin, extracellular polysaccharide and folic acid producing capability, cholesterol-reducing ability, antibiotic resistance, tolerance ability to in vivo conditions, adhesion activity. Strains with excellent health care functions and tolerance ability were selected out to be evaluated their potential as probiotic strains.
The main results were summarized as follows:
(1) The average pH value of yak milk cheese samples collected from Tibet, Yunnan and Xinjiang province were4.31±0.39,4.69±0.29,4.52±0.22respectively, no significant difference between three regions(P=0.176>0.05). The average colony enumeration of samples collected from Tibet, Yunnan and Xinjiang province were4.17±0.43;4.30±0.68;3.9±0.59log CFU/g respectively, no significant difference between three regions (P=0.468>0.05).
(2)39strains of lactic acid bacteria were isolated from17yak milk samples. Those39strains were identified to genus level and preliminary to species level with classic taxonomy methods. Among the39strains,35strains were rod shape, assigned to7species:2strains L.buchneri (Y15, Y16),16strains L.casei(T7, Y19, X21, X22, X23, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, Y36),3strains L.diolivorans (Y18, X24, X25),3strains L.fermentum (T1, T2, T3),3strains L.helveticus (T6, T8, Y9),3strains L.kefiri (Y10, Y12, Y20),5strains L.plantarum (T4, Y13, Y37, X38, X39)。4strains were coccus shape, assigned to2species:1strain Pediococcus acidilactici (T5),3strains Pediococcuspentosaceus (Y11、Y14、 Y17)。
(3)6Lactobacillus strains were selected out by acid producing test because they had such a high acid producing capability to meet the standard of starter cultures, and they were X24、X38.X29、X25、X21、X22. Eight strains with excellent ability to utilize citrate were also selected out:T4、T7、Y10、Y13、X25、X28、Y36、Y37. The excellent ability of strains may be developed during their evolution in the plateau climate and long-term dairy-producing practice.
(4)39strains were subjected to16S rRNA analysis to identify species with genotypic method. Phylogenetic tree was also constructed based on16S rDNA sequences. The species assignment result with phenotypic method was confirmed and strains'genetic relationship was demonstrated.
(5)39strains were subjected to RAPD fingerprinting analysis. Except for strains X23, Y36and Y37, the fingerprinting band of strains were clustered to different groups according to their species; this result corresponded well with that of16S rDNA sequences analysis. The cluster analysis also showed excellent correlative relationship between group patterns and origin regions. It is proposed that RAPD method showed excellent efficiency in taxonomy and genomic relationship analysis.
(6)31strains were tested to produce bacteriocin. Bacteriocin produced by strain Y13had the antagonism action against5pathogenic reference strains:Bacillus cereus ATCC10876、Escherichia coli O]57:H7、Listeria monocytogens、Citrobacter freundii ATCC8090、Salmonella Typhimurium ATCC13311. Strain X29and X30had the antagonism action against4pathogenic reference strains:Bacillus cereus ATCC10876, Citrobacter freundii ATCC8090, Salmonella Typhimurium ATCC13311, Enterobacter cloacae ATCC23355. All39strains had no antagonism action against Streptococcus thermophiles which was a widely used strain in dairy industry. All strains were suitable for use with Streptococcus thermophiles in dairy fermentation.
(7) Nine strains were selected out because they have capability to produce extracellular polysaccharide:T3. T4、T5、T7、T8、Y1K、Y14、Y17、X29. The yield of extracellular polysaccharide was tested with phenol-sulphuric acid method. Strain gave the highest yield result193.72mg/L, which was a relatively low level by comparison with literature report.
(8) Nine strains were selected out because they have capability to produce folic acid:T3、T7、T8、X23、X25、X26、X27、X29、Y37. Strain Y37gave the highest yield result85.98±5.09ng/ml. X25ranked in second place, amounted to81.17+2.08ng/ml, which was a very high level by comparison with literature report17-1OOng/ml.
(9) Strains Y15. X25and X27were selected out because they had excellent capability to remove cholesterol from MRS-CHOL medium, the removal percentages were amounted to45.59%,50.01%and53.18%, which was a very high level by comparison with literature report.
(10) The antibiotic resistance of39strains were tested with disc diffusion method. Among39strains,74.4%,82.1%and82.1%were sensitive to ampicillin; chloramphenicol and erythromycin respectively,100%,97.4%,92.3%and82.05%were resistant to nalidixic acid, streptomycin, vancomycin and neomycin.
(11) Strains Y15, X25and X27were selected out because they have excellent tolerance ability to in vivo conditions, as well as excellent adhesion activity.
(12) By summarizing all experiment data, Strains Y15, X25and X27were selected out as potential probiotic strains. Strain Y15(L.buchneri) had the ability to produce extracellular polysaccharide (50.26±1.25mg/L), reducing cholesterol (removing percentage45.59%), resistance to nalidixic acid, streptomycin and vancomycin. Strain X25(L.diolivorans) had the ability to produce forlic acid (81.17±2.08ng/mL), reducing cholesterol (removing percentage50.01%), resistance to nalidixic acid, streptomycin, tetracycline and vancomycin. Strain X27(L.casei)had the ability to produce extracellular polysaccharide (38.62±1.21mg/L), produce folic acid (77.83±2.52ng/mL), reducing cholesterol (removing percentage53.8%), resistance to neomycin, nalidixic acid, streptomycin and vancomycin, producing bacteriocin which showed antagonism action against pathogenic reference strains Bacillus cereus ATCC10876, Staphylococcus aureus and Escherichia coli O157:H7.
The creativeness and innovation of this study:
(1) This study investigated yak milk samples from a broad area, Tibet, Yunnan and Xinjiang province, which covers1/3-1/4of our country's area. It is the most comprehensive and systematic fundamental and application research work on lactic acid bacteria from yak milk cheeses.
(2) This study established a rapid identification mode:genus specific PCR→16S rRNA sequence analysis→species specific PCR, which is suitable for massive identification work of natural lactic acid bacteria, promoting efficiency and precision.
(3) This study established a RAPD reaction system and amplification program with excellent reproducibility and stability for lactic acid bacteria. The result of this method corresponded well with that of16S rRNA sequence analysis. It is efficient to figure out genetic relations of isolated strains, as well as the correlativity between strains'cluster patterns and origin regions.
(4) This study selected out three potential probiotic strains with excellent health care function and tolerance capability.
表型特性的研究即用传统的菌落形态、革兰氏染色显微形态、生理生化、在不同环境(温度、pH值、盐度)中的生长特生,碳水化合物发酵方式等实验项目,将乳酸菌划分到属,进而初步判断出菌种,此外,还分析了产酸活性、利用柠檬酸盐等表型特性,以获得乳酸菌的基本数据,
基因型特性的研究包括16S rRNA序列分析和随机扩增多态性DNA技术。本研究建立了属特异性PCR→16S rRNA序列分析→种特异性PCR的快速鉴定模式,构建了系统进化树,以分析乳酸菌菌株的遗传亲缘关系。建立了适用于乳酸菌的RAPD反应体系和程序,用于分离鉴定出的乳酸菌,用NTsys2.10e软件对其图谱做聚类分析,与16S rRNA序列分析的结果相比较,分析菌株之间的遗传亲缘关系,分析菌株聚类方式与地域之间的相关性。
益生特性的研究包括乳酸菌菌株的产细菌素、产胞外多糖、产叶酸、降胆固醇的益生特性,并研究了菌株对抗生素的抗性、体内环境的耐受性、黏附活性,筛选出有突出益生功能,抗逆性较好的菌株,有希望作为益生菌加以开发应用。
主要的研究结果如下：
(1)西藏、云南、新疆三个地区的样品平均pH值分别为4.31±0.39；4.69±0.29：4.52±0.22,地区之间不存在显著性差异(P=0.176>0.05),三个地区菌落计数平均值分别为4.17±0.43；4.30±0.68；3.9±0.59log CFU/g,也不存在显著性差异(P=0.468>0.05)
(2)从17份样品中分离出39株乳酸菌,通过表型方法初步判断出菌种。35株菌为杆菌,被分为7个菌种：L.buchneri2株(Y15.Y16),L.casei16株(T7.Y19,X21,X22,X23,X26,X27,X28,X29,X30,X31,X32,X33,X34,X35,Y36)L.diolivorans3株(Y18,X24,X25),L.fermentum3株(T1,T2,T3),L.helveticus3株(T6,T8,Y9),L.kefiri3株(Y10,Y12,Y20),L.plantarum5株(T4,Y13,Y37,X38,X39)。4株菌为球菌,被分为2个菌种：Pediococcus acidilactici1株(T5),Pediococcus pentosaceus3株(Y11、Y14、Y17)。
(3)本研究测出6株乳杆菌产酸活性达到了起酵菌株的标准：X24、X38、X29、X25、X21、X22。选出发酵柠檬酸盐活性较强的菌株有：T4、T7、Y10、Y13、X25、X28、Y36、Y37,这些菌株的优良特性是在长期适应环境、以及生产实践中积累形成的。
(4)16S rRNA序列分析：将39株乳酸菌用16S rRNA序列分析法鉴定到种,并构建了系统进化树,进一步验证了菌种的划分,与表型特性是一致的。
(5)随机扩增多态性研究：做出了39株乳酸菌的RAPD指纹图谱,除X23、Y36和Y37之外,其余菌株均按照不同的菌种聚类,与16S rRNA序列分析法得出的结果基本是一致的,菌株聚类方式与地域有较强的相关性,说明RAPD技术在分类鉴定中有一定的应用价值,可解析菌株之间的遗传亲缘关系。
(6)检出31株菌产细菌素,菌株Y13能够拮抗5种致病菌：Bacillus cereus ATCC10876. Escherichia coli0157:H7、 Listeria monocytogenes、Citrobacter freundii ATCC8090、Salmonella Typhimurium ATCC13311,抑菌谱最宽X29和X30能拮抗4种致病菌：Bacillus cereus ATCC10876、Citrobacter freundii ATCC8090. Salmonella Typhimurium ATCC13311、Enterobacter cloacae ATCC23355。所有乳酸菌细菌素对嗜热链球菌Streptococcus thermophilus都没有拮抗活性,在发酵乳制品时,这些乳杆菌可与嗜热链球菌合用,相互之间没有拮抗作用。
(7)筛选出产胞外多糖的菌株有T3、T4、T5、T7、T8、Y11、Y14、Y17、X29,用苯酚-硫酸法定量检测所有筛出菌株的产量,与文献报道的产量相比处于中等水平,最高产菌株T8产量为193.72mg/L。
(8)筛选出9株菌可以产叶酸：T3、T7、T8、X23、X25、X26、X27、X29、Y37。最高产的菌株是Y37,总产量达到了85.98±5.09ng/ml,其次是X25,总产量为81.17±2.08ng/ml,与文献报道的产率17-100ng/ml相比,本实验筛选出来的菌株X25和Y37已达到了很高的产量。
(9)研究了乳酸菌在MRS-CHOL培养基中对胆固醇去除率,Y15、X25和X27对胆固醇降解率分别为45.59%,50.01%和53.18%,都达到40%以上,与文献报道过的乳酸菌菌株相比属于比较高的水平。
(10)用纸片扩散法测了39株菌对抗生素的抗性。39株乳酸菌对氨苄青霉素、氯霉素、红霉素敏感的菌株分别占74.4%；82.1%和82.1%,对萘啶酮酸、链霉素、万古霉素和新霉素有抗性的菌株分别占100%；97.4%；92.3%和82.05%。
(11)对体内环境耐受性很强菌株有3株：Y15、X25和X27,粘附性也很好。
(12)本研究鉴定并筛选出3株乳酸菌能耐受体内环境、有可能定植于肠道中,并有特定的保健功能,有可能作为益生菌开发。Y15(.L.buchneri)能产胞外多糖(50.26±1.25mg/L),降胆固醇(去除率为45.59%),对萘啶酮酸、链霉素、万古霉素有抗性。X25(L.diolivorans)能产叶酸(81.17±2.08ng/mL),能降胆固醇(去除率为50.01%),对萘啶酮酸、链霉素、四环素、万古霉素有抗性。X27(L.casei)能产胞外多糖(38.62±1.21mg/L),产叶酸(77.83±2.52ng/mL),能降胆固醇(去除率为53.8%),所产细菌素对Bacillus cereus ATCC10876, Staphylococcus aureus、Escherichia coli O157:H7有拮抗活性,对新霉素、萘啶酮酸、链霉素、万古霉素有抗性。
本论文的创新之处在于
(1)牦牛奶酪乳酸菌是我国重要而且独特的生物资源,有极其重要的收集、整理、保存、研究的价值。本研究是对牦牛奶酪中的乳酸菌进行的最完整、系统、全面的基础研究。
(2)建立了属特性PCR→16S rRNA序列分析→种特异性PCR的菌种鉴定模式,可用于对大量野生菌株快速鉴定菌种,能有效提高鉴定效率和准确性。
(3)构建了重现性好的RAPD反应体系和程序,用于乳酸菌菌株的基因分型,其聚类分析结果与16S rRNA序列分析法基本是一致的,说明它在乳酸菌的分类鉴定中有较好的区分效率,可与其它基因型鉴定方法结合使用,提高菌种的鉴定效率。菌株RAPD图谱聚类方式与地域之间有较强的相关性,可用于菌株原产地的分析,保护菌种资源。
(4)从菌株中筛选出了三株有突出益生功能、抗逆性较好的菌株,有希望作为益生菌开发应用。
There are various kinds of lactic acid bacteria existing in Chinese yak milk cheeses, which is an extremely precious natural microbiology resource. It is a research work of great values that collecting, organizing, preserving and investigating those lactic acid bacteria. This study collected17yak milk cheese samples from Tibet, Yunnan and Xinjiang province, isolated lactic acid bacteria, studied their phenotypic, genotypic and probiotic characterization.
The phenotypic characterization study included species identification of isolated lactic acid bacteria with classic taxonomy methods, such as colony morphology, Gram stain and microscopic morphology, primary biochemical and physiological properties, growth capability in different conditions (temperature, pH value, and salt concentration), carbohydrates fermentation patterns, acid producing activity, capability of utilizing citrate and so on. Isolated lactic acid bacteria were assigned to genus level, and preliminary species level. Phenotypic experiment results provided a comprehensive data of lactic acid bacteria. The capability of producing acid and utilizing citrate directly demonstrated lactic acid bacteria's role in fermentation.
The genotypic characterization study included16S rRNA sequenc analysis and randomly amplified polymorphic DNA (RAPD). This study established a rapid identification mode:genus specific PCR→16S rRNA sequence analysis→species specific PCR. Isolated lactic acid bacteria were identified to species level with this mode, phylogenetic tree was constructed based on16S rDNA sequences to confirm species assignment and demonstrate strains'genetic relationship. This study also established a stable RAPD reaction system and amplification program for lactic acid bacteria. Isolated lactic acid bacteria were subjected to this RAPD analysis, NTsys2.10e software was used to perform cluster analysis of strains'RAPD fingerprinting patterns. The cluster result were compared with that of16S rRNA sequence analysis to figure out genetic relations of isolated strains, as well as the correlativity between strains'cluster patterns and origin regions.
The probiotic characterization study included bacteriocin, extracellular polysaccharide and folic acid producing capability, cholesterol-reducing ability, antibiotic resistance, tolerance ability to in vivo conditions, adhesion activity. Strains with excellent health care functions and tolerance ability were selected out to be evaluated their potential as probiotic strains.
The main results were summarized as follows:
(1) The average pH value of yak milk cheese samples collected from Tibet, Yunnan and Xinjiang province were4.31±0.39,4.69±0.29,4.52±0.22respectively, no significant difference between three regions(P=0.176>0.05). The average colony enumeration of samples collected from Tibet, Yunnan and Xinjiang province were4.17±0.43;4.30±0.68;3.9±0.59log CFU/g respectively, no significant difference between three regions (P=0.468>0.05).
(2)39strains of lactic acid bacteria were isolated from17yak milk samples. Those39strains were identified to genus level and preliminary to species level with classic taxonomy methods. Among the39strains,35strains were rod shape, assigned to7species:2strains L.buchneri (Y15, Y16),16strains L.casei(T7, Y19, X21, X22, X23, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, Y36),3strains L.diolivorans (Y18, X24, X25),3strains L.fermentum (T1, T2, T3),3strains L.helveticus (T6, T8, Y9),3strains L.kefiri (Y10, Y12, Y20),5strains L.plantarum (T4, Y13, Y37, X38, X39)。4strains were coccus shape, assigned to2species:1strain Pediococcus acidilactici (T5),3strains Pediococcuspentosaceus (Y11、Y14、 Y17)。
(3)6Lactobacillus strains were selected out by acid producing test because they had such a high acid producing capability to meet the standard of starter cultures, and they were X24、X38.X29、X25、X21、X22. Eight strains with excellent ability to utilize citrate were also selected out:T4、T7、Y10、Y13、X25、X28、Y36、Y37. The excellent ability of strains may be developed during their evolution in the plateau climate and long-term dairy-producing practice.
(4)39strains were subjected to16S rRNA analysis to identify species with genotypic method. Phylogenetic tree was also constructed based on16S rDNA sequences. The species assignment result with phenotypic method was confirmed and strains'genetic relationship was demonstrated.
(5)39strains were subjected to RAPD fingerprinting analysis. Except for strains X23, Y36and Y37, the fingerprinting band of strains were clustered to different groups according to their species; this result corresponded well with that of16S rDNA sequences analysis. The cluster analysis also showed excellent correlative relationship between group patterns and origin regions. It is proposed that RAPD method showed excellent efficiency in taxonomy and genomic relationship analysis.
(6)31strains were tested to produce bacteriocin. Bacteriocin produced by strain Y13had the antagonism action against5pathogenic reference strains:Bacillus cereus ATCC10876、Escherichia coli O]57:H7、Listeria monocytogens、Citrobacter freundii ATCC8090、Salmonella Typhimurium ATCC13311. Strain X29and X30had the antagonism action against4pathogenic reference strains:Bacillus cereus ATCC10876, Citrobacter freundii ATCC8090, Salmonella Typhimurium ATCC13311, Enterobacter cloacae ATCC23355. All39strains had no antagonism action against Streptococcus thermophiles which was a widely used strain in dairy industry. All strains were suitable for use with Streptococcus thermophiles in dairy fermentation.
(7) Nine strains were selected out because they have capability to produce extracellular polysaccharide:T3. T4、T5、T7、T8、Y1K、Y14、Y17、X29. The yield of extracellular polysaccharide was tested with phenol-sulphuric acid method. Strain gave the highest yield result193.72mg/L, which was a relatively low level by comparison with literature report.
(8) Nine strains were selected out because they have capability to produce folic acid:T3、T7、T8、X23、X25、X26、X27、X29、Y37. Strain Y37gave the highest yield result85.98±5.09ng/ml. X25ranked in second place, amounted to81.17+2.08ng/ml, which was a very high level by comparison with literature report17-1OOng/ml.
(9) Strains Y15. X25and X27were selected out because they had excellent capability to remove cholesterol from MRS-CHOL medium, the removal percentages were amounted to45.59%,50.01%and53.18%, which was a very high level by comparison with literature report.
(10) The antibiotic resistance of39strains were tested with disc diffusion method. Among39strains,74.4%,82.1%and82.1%were sensitive to ampicillin; chloramphenicol and erythromycin respectively,100%,97.4%,92.3%and82.05%were resistant to nalidixic acid, streptomycin, vancomycin and neomycin.
(11) Strains Y15, X25and X27were selected out because they have excellent tolerance ability to in vivo conditions, as well as excellent adhesion activity.
(12) By summarizing all experiment data, Strains Y15, X25and X27were selected out as potential probiotic strains. Strain Y15(L.buchneri) had the ability to produce extracellular polysaccharide (50.26±1.25mg/L), reducing cholesterol (removing percentage45.59%), resistance to nalidixic acid, streptomycin and vancomycin. Strain X25(L.diolivorans) had the ability to produce forlic acid (81.17±2.08ng/mL), reducing cholesterol (removing percentage50.01%), resistance to nalidixic acid, streptomycin, tetracycline and vancomycin. Strain X27(L.casei)had the ability to produce extracellular polysaccharide (38.62±1.21mg/L), produce folic acid (77.83±2.52ng/mL), reducing cholesterol (removing percentage53.8%), resistance to neomycin, nalidixic acid, streptomycin and vancomycin, producing bacteriocin which showed antagonism action against pathogenic reference strains Bacillus cereus ATCC10876, Staphylococcus aureus and Escherichia coli O157:H7.
The creativeness and innovation of this study:
(1) This study investigated yak milk samples from a broad area, Tibet, Yunnan and Xinjiang province, which covers1/3-1/4of our country's area. It is the most comprehensive and systematic fundamental and application research work on lactic acid bacteria from yak milk cheeses.
(2) This study established a rapid identification mode:genus specific PCR→16S rRNA sequence analysis→species specific PCR, which is suitable for massive identification work of natural lactic acid bacteria, promoting efficiency and precision.
(3) This study established a RAPD reaction system and amplification program with excellent reproducibility and stability for lactic acid bacteria. The result of this method corresponded well with that of16S rRNA sequence analysis. It is efficient to figure out genetic relations of isolated strains, as well as the correlativity between strains'cluster patterns and origin regions.
(4) This study selected out three potential probiotic strains with excellent health care function and tolerance capability.
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