新疆酸驼乳中微生物种群结构的分子解析及优势菌群的分离与鉴定
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摘要
舒巴特(Shubat)是一种新疆特有的传统发酵酸驼乳,其口感特殊,风味浓郁,营养丰富,极具民族特色。研究发现,驼乳可以帮助糖尿病患者减少对胰岛素的依赖,对婴儿也很有益。而且,酸驼乳对消化道溃疡,高血压等疾病都有辅助治疗作用。
酸驼奶的传统制作方法十分奇特,新鲜驼奶不经过消毒直接放入专用的,萨克民族称之为“撒巴”羊皮囊中,室温放置发酵24-48小时便酿制成酸驼奶。酸驼奶被饮用后再补充相同体积的新鲜驼奶,这种发酵过程可以持续数月。从“撒巴”制作酸驼奶的独特工艺来看,其中一定蕴藏着丰富的微生物资源,极有可能成为优良益生菌的来源。
本研究以新疆牧民家庭自然发酵的酸驼奶(Shubat)为材料,采用PCR-DGGE指纹技术分析酸驼奶中乳酸菌及酵母菌的分子多样性,并结合传统平板技术分离优势菌种；通过单菌株在驼奶里的生长研究其对驼奶发酵的影响,以期阐明酸驼乳发酵的机理,为民族产品的工业化生产提供了理论依据。主要研究结果如下：
1.按照传统方法制备酸驼乳,研究发酵过程中乳酸菌、酵母菌及大肠菌群等主要微生物群的动态变化；并对发酵奶pH值、理化指标(灰分、脂肪、蛋白质)粘度以及和微生物指标(大肠菌群、乳酸菌、酵母菌和霉菌)进行了分析和研究。结果显示,自然发酵过程中乳酸菌和酵母菌随时间而数量增加,发酵终止时两者数量分别为4.3×107和2.8×106CFU/mL;新鲜驼奶中大肠菌群数量为102CFU/mL以上。随后大肠菌群数量急剧下降,到发酵完成时,大肠菌群为阴性。霉菌始终没有检出。理化分析结果表明,酸驼乳中蛋白质、脂肪、灰分的含量分别为4.21%、2.81%和0.98%；调查发现不同地区采集的酸驼乳样品其酸度、pH值、粘度等有明显差异；感官评价表明,酸驼乳可分成Ⅰ、Ⅱ两种类型：类型Ⅰ奶质稀薄、酸味柔和,类型Ⅱ奶质粘稠、酸味浓烈。
2.采用PCR-DGGE指纹技术,比较了9份传统发酵酸驼乳中微生物种群结构,图谱相似性分析结果表明不同来源的酸驼乳中微生物组份存在差异,样品间细菌种群结构的相似性为78%-84%,酵母菌种群结构的相似性为80%-92%。分别对酸驼奶细菌和酵母菌DGGE图谱上主要条带的DNA进行回收、PCR扩增、测序,通过序列分析和相似性比较,结果表明酸驼乳中细菌群落结构包括巨型球菌Macrococcus caseolyticus,瑞士乳杆菌(Lactobacillus helveticus),短乳杆菌(Lactobacillus brevis),香肠魏氏菌(Weissella hellenica),清酒乳杆菌(Lactobacillus sakei),肠球菌(Enterococcus durans),屎肠球菌(Enterococcus faecium)及乳酸明串珠菌(Leuconostoc lactis)等；酵母群落结构主要包括为巨大克洛维酵母(Kluyveromyces marxianus)和单孢酿酒酵母(Kazachstania unispora),嗜酒假丝酵母(Candida ethanolica)及一种地霉(Geotrichum sp)。此结果对进一步从自然发酵酸驼乳中分离筛选优势乳酸菌和酵母菌有重要指导意义。
3.从7份采自新疆维吾尔自治区喀纳斯湖周边及布尔津县的自然发酵酸驼奶共分离到48株菌株乳酸菌和15株酵母菌菌株。乳酸菌菌株根据其细胞形态,二氧化碳产生,精氨酸产氨,乳酸构型等生理生化特性以及在不同温度和盐度条件下的生长情况等分成6组,分别称之为组Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ.采用API50CH鉴定系统进行生理生化鉴定结合16srDNA序列分析鉴定,6组乳酸菌菌株被鉴定为清酒乳杆菌(Lactobacillus sakei),瑞士乳杆菌(Lactobacillus helveticus),短乳杆菌(Lactobacillus brevis),粪肠球菌(Enterococcus faecium),屎肠球菌(Enterococcus feacalis),乳明串珠菌(Leuconostoc lactis)及香肠魏氏菌(Weissella hellenica),其中Lb. sakei分离频率最高的乳酸菌(占分离株总数26%),其次是Enterococcus faecium(占14%)和Lactobacillus helveticus。酵母菌菌株根据其26S rDNA的D1区PCR-DGGE图谱分为3组,通过对26SrDNA ITS序列进行扩增和测定并与数据库序列比对,结果表明3组酵母菌分别被鉴定为巨大克洛维酵母(Kluyveromyces marxianus),单孢酿酒酵母(Kazachstania unispora),嗜酒精假丝酵母(Candida ethanolica),其中Kluyveromyces marxianus是分离频率最高的优势酵母菌(占14%)。
4.本研究建立了一种基于特有菌落形态与katA基因相结合的清酒乳杆菌检测方法。利用此方法跟踪驼乳的发酵过程,发现所有低粘、低酸型(即类型Ⅰ)自然发酵酸驼乳中都含有清酒乳杆菌在存在。用不含清酒乳杆菌酸驼乳和含有清酒乳杆菌的酸驼乳作为自然发酵剂分别接种新鲜消毒驼乳,结果表明接种含有清酒乳杆菌的酸驼乳的新鲜驼乳其粘度随着时间延长而逐渐下降；相反接种不含清酒乳杆菌酸驼乳的新鲜消毒驼乳,其粘度随着时间延长而有增高。进一步用分离自酸驼乳的清酒乳杆菌与6株其他乳酸菌分别接种新鲜消毒驼乳,结果表明接种清酒乳杆菌的驼乳粘度均有下降；相反接种其他乳酸菌的驼乳粘度均有不同程度的增加。这一结果初步揭示了在低酸、低粘型传统驼乳发酵中清酒乳杆菌起重要作用。
Shubat is a special fermented product, prepared from unheated two-humped camel milk through indigenous fermentation process. Although it is more or less similar to yoghurt in appearance, there are important differences between these two products since shubat is liquid rather than creamy, sparking due to its CO2production and has a high degree of sourness. In the Xinjiang in China, shubat have been widely used both as beverage and folk medicine. The indigenous populations have believed that fermented camel milk (shubat) is safe and even has medicinal properties such as antidiabetic, anti-cancer and anti-tuberculosis activities.
Traditionally, shubat is homemade by using a semi-continuous or fed-batch fermentation process that was handed down throughout generations. Whenever part of the product is withdrawn for consumption, a portion of raw camel milk is added to make up volume and this process of retrieval and replacement of milk continues for months. The spontaneous fermentation of unheated milk takes advantage of natural microflora inherent in milk and environmental contaminants. However, many reports about traditional dairy products have shown that they have unique and different microflora dependent on the production technology as well as on the ecological localities where they have been produced. It is well known that specific characteristics of dairy products depend on the present microflora. Therefore, this knowledge can contribute to improving the process of the shubat manufacture, as well as to the fermentation condition for obtaining the product of better quality.
The present study aimed to isolate and identify predominant microflora in homemade shubat by using phenotypic and molecular methods, which can further developed starter cultures for camel milk fermentation. To the best of our knowledge, no similar investigation into microflora of shubat from two-humped camel milk in China has been carried out so far.
The main results are given as follows:
1. Microbiological and chemical characters of shubat prepared by natural fermention procotol were characteried in this study, including LAB, yeast and mould analysis as well as protein and fat ash analysis. The results showed that number of LAB and yeast were4.3×107and2.8×106CFU/mL, respectively. The coliform was more than102cfu/ml at fresh camel milk, but disappeared at Shubat. The moulds were not detected in Shubat. The protein, fat and ash contents of Shubat were4.21%,2.81%and0.98%, respectively. According to the pH, viscosity and acidity, Shubat from defferent regions were divided into two types:Type I have a low viscosity and mild acidity; while Type II have a high viscosity and strong acidity.
2. The microbial communities structures in traditional fermented camel milk (shubat) were analysed by using the PCR-DGGE fingerprint technique. The results showed that bacteria and yeast community similarity were78%-84%and80-92%among shubat samples from different regions, respectively. By sequencing V6-V8region of16s rDNA, bacteria community were composed of Macrococcus caseolyticus, Lactobacillus helveticus, Lactobacillus brevis, Weissella hellenica, Lactobacillus sakei, Enterococcus durans, Enterococcus faecium and Leuconostoc lactis et al. Yeast community were found to be Kluyveromyces marxianus, Kazachstania unispora, Candida ethanolica and Geotrichum sp. By compared with sequence similarity of D1region of26s rDNAgene. This results provided a theoretical foundation for developing the culture starter of shubat.
3. A total of48LAB and15yeast isolates were isolated from fermented camel milk(shubat),which collected from Kanas areas and Borjin county, Xinjiang China. According to their observed features such as microscopy appearance, CO2production from glucose, NH3production from arginine, the ability to grow under different temperature and salt conditions, these isolates were grouped into six groups,named group Ⅰ,Ⅱ,Ⅲ,Ⅳ Ⅴ and VI. By use of API50CH system and16s rDNA sequence analysis, the six groups were clearly identified as Lactobacillus sakei, Lb. helvelticus, Enterococcus feacium, Ec. feacalis, Lb. brevis and Leuconstoc lactis, respectively. Among the LAB microfora, Lb. sakei were the most frequently isolated species, then were Enterococcus faecium (14%) and Lactobacillus helveticus (13%). The yeast microfora in shubat were identified as Kluyveromyces marxianus and Kazachstania unispora as well as Candida ethanolica, with Kl. marxianus being dominant yeast.
4. Based on special colony character combined with detecting katA gene sequence of the pure culture strains, a new detection procotol specific for Lb. sakei have been developed in this study. Using this new method, we have confirmed that Lb. sakei played important role in Shubat fermentation. It is the growth of Lb. sakei that Camel milk have a mild acidity and lower viscosity, which forms the flavor of Shubat. It can be reduced if the existence LB.sakei in Shubat.
酸驼奶的传统制作方法十分奇特,新鲜驼奶不经过消毒直接放入专用的,萨克民族称之为“撒巴”羊皮囊中,室温放置发酵24-48小时便酿制成酸驼奶。酸驼奶被饮用后再补充相同体积的新鲜驼奶,这种发酵过程可以持续数月。从“撒巴”制作酸驼奶的独特工艺来看,其中一定蕴藏着丰富的微生物资源,极有可能成为优良益生菌的来源。
本研究以新疆牧民家庭自然发酵的酸驼奶(Shubat)为材料,采用PCR-DGGE指纹技术分析酸驼奶中乳酸菌及酵母菌的分子多样性,并结合传统平板技术分离优势菌种；通过单菌株在驼奶里的生长研究其对驼奶发酵的影响,以期阐明酸驼乳发酵的机理,为民族产品的工业化生产提供了理论依据。主要研究结果如下：
1.按照传统方法制备酸驼乳,研究发酵过程中乳酸菌、酵母菌及大肠菌群等主要微生物群的动态变化；并对发酵奶pH值、理化指标(灰分、脂肪、蛋白质)粘度以及和微生物指标(大肠菌群、乳酸菌、酵母菌和霉菌)进行了分析和研究。结果显示,自然发酵过程中乳酸菌和酵母菌随时间而数量增加,发酵终止时两者数量分别为4.3×107和2.8×106CFU/mL;新鲜驼奶中大肠菌群数量为102CFU/mL以上。随后大肠菌群数量急剧下降,到发酵完成时,大肠菌群为阴性。霉菌始终没有检出。理化分析结果表明,酸驼乳中蛋白质、脂肪、灰分的含量分别为4.21%、2.81%和0.98%；调查发现不同地区采集的酸驼乳样品其酸度、pH值、粘度等有明显差异；感官评价表明,酸驼乳可分成Ⅰ、Ⅱ两种类型：类型Ⅰ奶质稀薄、酸味柔和,类型Ⅱ奶质粘稠、酸味浓烈。
2.采用PCR-DGGE指纹技术,比较了9份传统发酵酸驼乳中微生物种群结构,图谱相似性分析结果表明不同来源的酸驼乳中微生物组份存在差异,样品间细菌种群结构的相似性为78%-84%,酵母菌种群结构的相似性为80%-92%。分别对酸驼奶细菌和酵母菌DGGE图谱上主要条带的DNA进行回收、PCR扩增、测序,通过序列分析和相似性比较,结果表明酸驼乳中细菌群落结构包括巨型球菌Macrococcus caseolyticus,瑞士乳杆菌(Lactobacillus helveticus),短乳杆菌(Lactobacillus brevis),香肠魏氏菌(Weissella hellenica),清酒乳杆菌(Lactobacillus sakei),肠球菌(Enterococcus durans),屎肠球菌(Enterococcus faecium)及乳酸明串珠菌(Leuconostoc lactis)等；酵母群落结构主要包括为巨大克洛维酵母(Kluyveromyces marxianus)和单孢酿酒酵母(Kazachstania unispora),嗜酒假丝酵母(Candida ethanolica)及一种地霉(Geotrichum sp)。此结果对进一步从自然发酵酸驼乳中分离筛选优势乳酸菌和酵母菌有重要指导意义。
3.从7份采自新疆维吾尔自治区喀纳斯湖周边及布尔津县的自然发酵酸驼奶共分离到48株菌株乳酸菌和15株酵母菌菌株。乳酸菌菌株根据其细胞形态,二氧化碳产生,精氨酸产氨,乳酸构型等生理生化特性以及在不同温度和盐度条件下的生长情况等分成6组,分别称之为组Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ.采用API50CH鉴定系统进行生理生化鉴定结合16srDNA序列分析鉴定,6组乳酸菌菌株被鉴定为清酒乳杆菌(Lactobacillus sakei),瑞士乳杆菌(Lactobacillus helveticus),短乳杆菌(Lactobacillus brevis),粪肠球菌(Enterococcus faecium),屎肠球菌(Enterococcus feacalis),乳明串珠菌(Leuconostoc lactis)及香肠魏氏菌(Weissella hellenica),其中Lb. sakei分离频率最高的乳酸菌(占分离株总数26%),其次是Enterococcus faecium(占14%)和Lactobacillus helveticus。酵母菌菌株根据其26S rDNA的D1区PCR-DGGE图谱分为3组,通过对26SrDNA ITS序列进行扩增和测定并与数据库序列比对,结果表明3组酵母菌分别被鉴定为巨大克洛维酵母(Kluyveromyces marxianus),单孢酿酒酵母(Kazachstania unispora),嗜酒精假丝酵母(Candida ethanolica),其中Kluyveromyces marxianus是分离频率最高的优势酵母菌(占14%)。
4.本研究建立了一种基于特有菌落形态与katA基因相结合的清酒乳杆菌检测方法。利用此方法跟踪驼乳的发酵过程,发现所有低粘、低酸型(即类型Ⅰ)自然发酵酸驼乳中都含有清酒乳杆菌在存在。用不含清酒乳杆菌酸驼乳和含有清酒乳杆菌的酸驼乳作为自然发酵剂分别接种新鲜消毒驼乳,结果表明接种含有清酒乳杆菌的酸驼乳的新鲜驼乳其粘度随着时间延长而逐渐下降；相反接种不含清酒乳杆菌酸驼乳的新鲜消毒驼乳,其粘度随着时间延长而有增高。进一步用分离自酸驼乳的清酒乳杆菌与6株其他乳酸菌分别接种新鲜消毒驼乳,结果表明接种清酒乳杆菌的驼乳粘度均有下降；相反接种其他乳酸菌的驼乳粘度均有不同程度的增加。这一结果初步揭示了在低酸、低粘型传统驼乳发酵中清酒乳杆菌起重要作用。
Shubat is a special fermented product, prepared from unheated two-humped camel milk through indigenous fermentation process. Although it is more or less similar to yoghurt in appearance, there are important differences between these two products since shubat is liquid rather than creamy, sparking due to its CO2production and has a high degree of sourness. In the Xinjiang in China, shubat have been widely used both as beverage and folk medicine. The indigenous populations have believed that fermented camel milk (shubat) is safe and even has medicinal properties such as antidiabetic, anti-cancer and anti-tuberculosis activities.
Traditionally, shubat is homemade by using a semi-continuous or fed-batch fermentation process that was handed down throughout generations. Whenever part of the product is withdrawn for consumption, a portion of raw camel milk is added to make up volume and this process of retrieval and replacement of milk continues for months. The spontaneous fermentation of unheated milk takes advantage of natural microflora inherent in milk and environmental contaminants. However, many reports about traditional dairy products have shown that they have unique and different microflora dependent on the production technology as well as on the ecological localities where they have been produced. It is well known that specific characteristics of dairy products depend on the present microflora. Therefore, this knowledge can contribute to improving the process of the shubat manufacture, as well as to the fermentation condition for obtaining the product of better quality.
The present study aimed to isolate and identify predominant microflora in homemade shubat by using phenotypic and molecular methods, which can further developed starter cultures for camel milk fermentation. To the best of our knowledge, no similar investigation into microflora of shubat from two-humped camel milk in China has been carried out so far.
The main results are given as follows:
1. Microbiological and chemical characters of shubat prepared by natural fermention procotol were characteried in this study, including LAB, yeast and mould analysis as well as protein and fat ash analysis. The results showed that number of LAB and yeast were4.3×107and2.8×106CFU/mL, respectively. The coliform was more than102cfu/ml at fresh camel milk, but disappeared at Shubat. The moulds were not detected in Shubat. The protein, fat and ash contents of Shubat were4.21%,2.81%and0.98%, respectively. According to the pH, viscosity and acidity, Shubat from defferent regions were divided into two types:Type I have a low viscosity and mild acidity; while Type II have a high viscosity and strong acidity.
2. The microbial communities structures in traditional fermented camel milk (shubat) were analysed by using the PCR-DGGE fingerprint technique. The results showed that bacteria and yeast community similarity were78%-84%and80-92%among shubat samples from different regions, respectively. By sequencing V6-V8region of16s rDNA, bacteria community were composed of Macrococcus caseolyticus, Lactobacillus helveticus, Lactobacillus brevis, Weissella hellenica, Lactobacillus sakei, Enterococcus durans, Enterococcus faecium and Leuconostoc lactis et al. Yeast community were found to be Kluyveromyces marxianus, Kazachstania unispora, Candida ethanolica and Geotrichum sp. By compared with sequence similarity of D1region of26s rDNAgene. This results provided a theoretical foundation for developing the culture starter of shubat.
3. A total of48LAB and15yeast isolates were isolated from fermented camel milk(shubat),which collected from Kanas areas and Borjin county, Xinjiang China. According to their observed features such as microscopy appearance, CO2production from glucose, NH3production from arginine, the ability to grow under different temperature and salt conditions, these isolates were grouped into six groups,named group Ⅰ,Ⅱ,Ⅲ,Ⅳ Ⅴ and VI. By use of API50CH system and16s rDNA sequence analysis, the six groups were clearly identified as Lactobacillus sakei, Lb. helvelticus, Enterococcus feacium, Ec. feacalis, Lb. brevis and Leuconstoc lactis, respectively. Among the LAB microfora, Lb. sakei were the most frequently isolated species, then were Enterococcus faecium (14%) and Lactobacillus helveticus (13%). The yeast microfora in shubat were identified as Kluyveromyces marxianus and Kazachstania unispora as well as Candida ethanolica, with Kl. marxianus being dominant yeast.
4. Based on special colony character combined with detecting katA gene sequence of the pure culture strains, a new detection procotol specific for Lb. sakei have been developed in this study. Using this new method, we have confirmed that Lb. sakei played important role in Shubat fermentation. It is the growth of Lb. sakei that Camel milk have a mild acidity and lower viscosity, which forms the flavor of Shubat. It can be reduced if the existence LB.sakei in Shubat.
引文
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5. Agrawal, R.P., et al., Zero prevalence of diabetes in camel milk consuming Raica community of north-west Rajasthan, India. Diabetes Research and Clinical Practice,2007.76(2):p.290-296.
6.赵电波,白艳红,and张文彬,自然发酵双峰驼驼乳营养价值的研究.乳业科学与技术,2007(06)：305-308.
7. Elagamy, E.I., Effect of heat treatment on camel milk proteins with respect to antimicrobial factors: a comparison with cows'and buffalo milk proteins. Food Chemistry,2000.68(2):p.227-232.
8. Abdelgadir, W., et al., A traditional Sudanese fermented camel's milk product, Gariss, as a habitat of Streptococcus infantarius subsp. infantarius. International Journal of Food Microbiology,2008.127(3):p. 215-219.
9. Elagamy, E.I., et al., Purification and characterization of lactoferrin, lactoperoxidase, lysozyme and immunoglobulins from camel's milk. International Dairy Journal,1996.6(2):p.129-145.
10.古丽孜亚·卡克巴依,新.那.,新疆哈萨克族传统发酵驼乳抗炎作用的初步研究.中国乳品工业,2007.09：8-10
11. SHUANGQUAN, Microflora in traditional fermented camel's milk from Inner Mongolia. Milchwissenschaft,2004.59::p.649-652.
12.聂小苗,巴.阿.,新疆传统发酵酸驼乳中乳酸菌的分离和鉴定.食品研究与开发,2008.04.
13.孙天松,et al.,中国新疆地区酸马奶中乳酸菌生物多样性研究.微生物学通报,2007(03).
14. ABDEL, M.E.S., A.I. ABDALLA, and I. AHMAD E Chemical and microbiological quality of Garris, Sudanese fermented camel's milk product.2006:International Journal of Food Science and Technology, p.321-328.
15. Lore, T.A., S.K. Mbugua, and J. Wangoh, Enumeration and identification of microflora in suusac, a Kenyan traditional fermented camel milk product. Lebensmittel-Wissenschaft und-Technologie,2005. 38(2):p.125-130.
16. Abdelgadir, W.S., et al., Characterisation of the dominant microbiota of Sudanese fermented milk Rob. International Dairy Journal,2001.11(1-2):p.63-70.
17. Hekmat, S., H. Soltani, and G. Reid, Growth and survival of Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 in yogurt for use as a functional food. Innovative Food Science & Emerging Technologies,2009.10(2):p.293-296.
18. Muyzer G, D.W.E.C., Uitterlinden A G., Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16SrRNA. 1993:Applied and Environmental Microbiology, p.695-700.
19. Fasoli, S., et al., Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. International Journal of Food Microbiology,2003.82(1):p.59-70.
20.杨佐毅,et al., PCR-DGGE指纹分析技术在食品微生物检测中的应用.食品工业科技,2006(02)201-203.
21. Ampe, F., A. Sirvent, and N. Zakhia, Dynamics of the microbial community responsible for traditional sour cassava starch fermentation studied by denaturing gradient gel electrophoresis and quantitative rRNA hybridization. International Journal of Food Microbiology,2001.65(1-2):p.45-54.
22. Van Beek, S. and F.G.Priest, Evolution of the lactic acid bacterial community during malt whisky fermentation:a polyphasic study.2002:Appl. Environ. Microbiol. p.297-305.
23. Ercolini, D., PCR-DGGE fingerprinting:novel strategies for detection of microbes in food. Journal of Microbiological Methods,2004.56(3):p.297-314.
24. Vila-Costa, M., et al., Number and phylogenetic affiliation of bacteria assimilating dimethylsulfoniopropionate and leucine in the ice-covered coastal Arctic Ocean. Journal of Marine Systems,2008.74(3-4):p.957-963.
25. Kolehmainen, R.E., M. Tiirola, and J.A. Puhakka, Spatial and temporal changes in Actinobacterial dominance in experimental artificial groundwater recharge. Water Research,2008.42(17):p.4525-4537.
26. Ampe, F., et al., Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations.1999:Appl Environ Microbiol. p.5464-73.
27. Abriouel, H., et al., Culture-independent analysis of the microbial composition of the African traditional fermented foods poto poto and degue by using three different DNA extraction methods. International Journal of Food Microbiology,2006.111(3):p.228-233.
28.高蕙文,吕欣.,董明盛PCR-DGGE指纹技术在食品微生物研究中的应用.食品科学,2005.08：465-468
29. Cocolin, L., L.F. Bisson, and D.A. Mills, Direct profiling of the yeast dynamics in wine fermentations. FEMS Microbiology Letters,2000.189(1):p.81-87.
30. Cocolin, L., et al., An application of PCR-DGGE analysis to profile the yeast populations in raw milk. International Dairy Journal,2002.12(5):p.407-411.
31. Franklin, R.B. and A.L. Mills, Multi-scale variation in spatial heterogeneity for microbial community structure in an eastern Virginia agricultural field. FEMS Microbiology Ecology,2003.44(3): p.335-346.
32. S. Coppola, G.B., D. Ercolini, G. Moschetti,, Molecular evaluation of microbial diversity occurring in different types of Mozzarella cheese. Journal of Applied Microbiology,2001.90(3):p.414-420.
33. Blaiotta, G., et al., Combining Denaturing Gradient Gel Electrophoresis of 16S rDNA V3 Region and 16S-23S rDNA Spacer Region Polymorphism Analyses for the Identification of Staphylococci from Italian Fermented Sausages. Systematic and Applied Microbiology,2003.26(3):p.423-433.
34. Zhou, J.-z., et al., Application of the Mixture Design to Design the Formulation of Pure Cultures in Tibetan kefir. Agricultural Sciences in China,2007.6(11):p.1383-1389.
35. Kim, M. and J. Chun, Bacterial community structure in kimchi, a Korean fermented vegetable food, as revealed by 16S rRNA gene analysis. International Journal of Food Microbiology,2005.103(1):p. 91-96.
36. Randazzo, C.L., et al., Lactobacillus casei, dominant species in naturally fermented Sicilian green olives. International Journal of Food Microbiology,2004.90(1):p.9-14.
37. Choi, S.-Y., et al., Fermentation and sensory characteristics of kimchi containing potassium chloride as a partial replacement for sodium chloride. International Journal of Food Microbiology,1994.21(4):p. 335-340.
38. Champomier, M.-C., et al., Erratum to "Lactobacillus sakei:recent developments and future prospects":[Research in Microbiology 152 (2001) 839]. Research in Microbiology,2002.153(2):p. 115-123.
39. Dolci, P., et al., Microbiological characterization of artisanal Raschera PDO cheese:Analysis of its indigenous lactic acid bacteria. Food Microbiology,2008.25(2):p.392-399.
40. AI Jassim, R.A.M., et al., The genetic diversity of lactic acid producing bacteria in the equine gastrointestinal tract. FEMS Microbiology Letters,2005.248(1):p.75-81.
41. Vishniac, H., Yeast Biodiversity in the Antarctic, in Biodiversity and Ecophysiology of Yeasts.2006. p.419-440.
42. Kurtzman, C. and J. Pikur, Taxonomy and phylogenetic diversity among the yeasts, in Comparative Genomics.2006. p.29-46.
43.游银伟,et al.,沼气发酵液中一株酵母菌菌株的鉴定与系统发育分析.中国沼气,2008(04)：7-10.
44. Hammes, W.P. and C. Hertel, New developments in meat starter cultures. Meat Science,1998. 49(Supplement 1):p. S125-S138.
45. Najjari, A., et al., Method for reliable isolation of Lactobacillus sakei strains originating from Tunisian seafood and meat products. International Journal of Food Microbiology,2008.121(3):p. 342-351.
46. R.A, F., review:probiotics in man and animal. [J].Journal of Applied Bacteriology.,1989,.66: p.365-378.
47.国立东,et al.,传统乳制品中乳酸菌的分离及性能研究.食品科学,2006(03)：60-64.
48. R., F., Probiotics:an Overview. Human Health:The Contribution of Microorgnisms. [M],1992:p.63-73.
49.李妍、张兰威,几株乳酸菌益生潜力及降胆固醇的研究.微生物学通报,2007(06)：1146-1149.
1.赵电波,白艳红,and张文彬,自然发酵双峰骆驼乳营养价值的研究.乳业科学与技术,2007(06)：305-307.
2. Zhou, J.-z., et al., Application of the Mixture Design to Design the Formulation of Pure Cultures in Tibetan kefir. Agricultural Sciences in China,2007.6(11):p.1383-1389.
3. Vishniac, H., Yeast Biodiversity in the Antarctic, in Biodiversity and Ecophysiology of Yeasts.2006. p.419-440.
4. Wouters, J.T.M., et al., Microbes from raw milk for fermented dairy products. International Dairy Journal,2002.12(2-3):p.91-109.
5.赵虹,et al.,45份活性乳酸菌饮料微生物指标检测结果的分析.中国微生态学杂志,2003(01)：p.31-33.
6.朱秋劲,et al.,酸奶加工的全程物理性质变化的研究.食品科学,2003(02)：46-50
7.肜豪峰,et al.,青海湖地区几种乳制品营养成分的初步研究.安徽农业科学,2008(08)：p.3234-3235.
8.谢爱春and朱姝,延长初发酵培养时间对大肠菌群检测结果的影响.中国卫生检验杂志,2009(01)129-130
9.黄薇,黄薇；王盛良；熊剑娟；,饮用水中霉菌、酵母菌计数滤膜测定法.环境与健康杂志,1999(03)：p.169-170.
10.梅林,王志耕,酸奶、乳酸菌饮料风味及其部分理化特性的研究.乳业科学与技术,2004(01)：p.44-47.
11. Dimitonova, S.P., et al., Antimicrobial activity and protective properties of vaginal lactobacilli from healthy Bulgarian women. Anaerobe,2007.13(5-6):p.178-184.
1.熊素玉,et al.,酸马奶中乳酸菌的分离、纯化与鉴定.新疆农业科学,2007(05)：696-701.
2. Cocolin, L., et al., An application of PCR-DGGE analysis to profile the yeast populations in raw milk. International Dairy Journal,2002.12(5):p.407-411.
3. Ercolini, D., et al., Microbial diversity in Natural Whey Cultures used for the production of Caciocavallo Silano PDO cheese. International Journal of Food Microbiology,2008.124(2):p.164-170.
4. Ercolini, D., PCR-DGGE fingerprinting:novel strategies for detection of microbes in food. Journal of Microbiological Methods,2004.56(3):p.297-314.
5. Beh, A., et al., Evaluation of Molecular Methods for the Analysis of Yeasts in Foods and Beverages, in Advances in Food Mycology.2006. p.69-106.
6. Cocolin, L., et al., The late blowing in cheese:a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process. International Journal of Food Microbiology, 2004.90(1):p.83-91.
7. Dolci, P., et al., Microbiological characterization of artisanal Raschera PDO cheese:Analysis of its indigenous lactic acid bacteria. Food Microbiology,2008.25(2):p.392-399.
8. Lee, J.-S., et al., Analysis of kimchi microflora using denaturing gradient gel electrophoresis. International Journal of Food Microbiology,2005.102(2):p.143-150.
9. Florez, A.B. and B. Mayo, Microbial diversity and succession during the manufacture and ripening of traditional, Spanish, blue-veined Cabrales cheese, as determined by PCR-DGGE. International Journal of Food Microbiology,2006.110(2):p.165-171.
10. Fasoli, S., et al., Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. International Journal of Food Microbiology,2003.82(1):p.59-70.
11. El-Baradei, G., A. Delacroix-Buchet, and J.C. Ogier, Bacterial biodiversity of traditional Zabady fermented milk. International Journal of Food Microbiology,2008.121(3):p.295-301.
12. Narvhus, J.A. and T.H. Gadaga, The role of interaction between yeasts and lactic acid bacteria in African fermented milks:a review. International Journal of Food Microbiology,2003.86(1-2):p.51-60.
1.张培业,加大驼产品开发力度促进养驼业恢复和发展.内蒙古畜牧科学,2001.05：29-31.
2.杨佐毅,et al.,PCR-DGGE指纹分析技术在食品微生物检测中的应用.食品工业科技,2006(02)：201-203.
3. Cocolin, L., et al., The late blowing in cheese:a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process. International Journal of Food Microbiology, 2004.90(1):p.83-91.
4. Fasoli, S., et al., Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. International Journal of Food Microbiology,2003.82(1):p.59-70.
5. Aquilanti, L., et al., The microbial ecology of a typical Italian salami during its natural fermentation. International Journal of Food Microbiology,2007.120(1-2):p.136-145.
6. Bhadra, B., et al., Yeasts and Yeast-Like Fungi Associated with Tree Bark:Diversity and Identification of Yeasts Producing Extracellular Endoxylanases. Current Microbiology,2008.56(5):p. 489-494.
7. Cappa, F. and P.S. Cocconcelli, Identification of fungi from dairy products by means of 18S rRNA analysis. International Journal of Food Microbiology,2001.69(1-2):p.157-160.
8. Chin-Wen, L., C. Hsiao-Ling, and L. Je-Ruei, Identification and characterisation of lactic acid bacteria and yeasts isolated from Kefir grains in Taiwan. Australian Journal of Dairy Technology,1999. 54(1):p.14.
9. Cocolin, L., L.F. Bisson, and D.A. Mills, Direct profiling of the yeast dynamics in wine fermentations. FEMS Microbiology Letters,2000.189(1):p.81-87.
10. Bergman, A., et al., Rapid identification of pathogenic yeast isolates by real-time PCR and two-dimensional melting-point analysis. European Journal of Clinical Microbiology & Infectious Diseases,2007.26(11):p.813-818.
11. Ampe, F., et al., Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations.1999:Appl Environ Microbiol. p.5464-73.
12. Zhou, J., et al., Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis. Food Microbiology,2009.26(8):p.770-775.
13.孙天松,et al.,中国新疆地区酸马奶中乳酸菌生物多样性研究.微生物学通报,2007(03)451-454.
14.聂小苗,新疆传统发酵酸驼乳中乳酸菌的分离和鉴定.食品研究与开发,2008.04：53-55.
15.孟和毕力格,et al.,不同地区酸马奶中乳杆菌的分离及其生物学特性的研究.中国乳品工业,2004(11)：6-9
16. SHUANGQUAN, Microflora in traditional fermented camel's milk from Inner Mongolia. Milchwissenschaft,2004.59::p.649-652.
17. ABDEL, M.E.S., A.I. ABDALLA, and I. AHMAD E Chemical and microbiological quality of Garris, Sudanese fermented camel's milk product.2006:International Journal of Food Science and Technology, p.321-328.
18. Lore, T.A., S.K. Mbugua, and J. Wangoh, Enumeration and identification of microflora in suusac, a Kenyan traditional fermented camel milk product. Lebensmittel-Wissenschaft und-Technologie,2005. 38(2):p.125-130.
19. Abdelgadir, W.S., et al., Characterisation of the dominant microbiota of Sudanese fermented milk Rob. International Dairy Journal,2001.11(1-2):p.63-70.
20. Abdelgadir, W., et al., A traditional Sudanese fermented camel's milk product, Gariss, as a habitat of Streptococcus infantarius subsp. infantarius. International Journal of Food Microbiology,2008.127(3):p. 215-219.
1. Hammes, W.P. and C. Hertel, New developments in meat starter cultures. Meat Science,1998. 49(Supplement 1):p. S125-S138.
2. Noonpakdee, W., et al., Expression of the catalase gene katA in starter culture Lactobacillus plantarum TISTR850 tolerates oxidative stress and reduces lipid oxidation in fermented meat product. International Journal of Food Microbiology,2004.95(2):p.127-135.
3. Dal Bello, F. and C. Hertel, Oral cavity as natural reservoir for intestinal lactobacilli. Systematic and Applied Microbiology,2006.29(1):p.69-76.
4. Ammor, S., et al., Characterization and selection of Lactobacillus sakei strains isolated from traditional dry sausage for their potential use as starter cultures. Food Microbiology,2005.22(6):p. 529-538.
5. Rantsiou, K., et al., Molecular characterization of Lactobacillus species isolated from naturally fermented sausages produced in Greece, Hungary and Italy. Food Microbiology,2005.22(1):p.19-28.
6. Zhou, J., et al., Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis. Food Microbiology,2009.26(8):p.770-775.
7. Dimitonova, S.P., et al., Antimicrobial activity and protective properties of vaginal lactobacilli from healthy Bulgarian women. Anaerobe,2007.13(5-6):p.178-184.
8. Najjari, A., et al., Method for reliable isolation of Lactobacillus sakei strains originating from Tunisian seafood and meat products. International Journal of Food Microbiology,2008.121(3):p. 342-351.
9. Champomier-Verges, M.-C., et al., Lactobacillus sakei:recent developments and future prospects. Research in Microbiology,2001.152(10):p.839-848.
2. Ganter, P., Yeast and Invertebrate Associations, in Biodiversity and Ecophysiology of Yeasts.2006. p.303-370.
3.张培业,加大驼产品开发力度促进养驼业恢复和发展.内蒙古畜牧科学,2001.05：29-31
4. Agrawal, R.P., et al., Camel milk as an adjunct to insulin therapy improves long-term glycemic control and reduction in doses of insulin in patients with type-1 diabetes:A 1 year randomized controlled trial. Diabetes Research and Clinical Practice,2005.68(2):p.176-177.
5. Agrawal, R.P., et al., Zero prevalence of diabetes in camel milk consuming Raica community of north-west Rajasthan, India. Diabetes Research and Clinical Practice,2007.76(2):p.290-296.
6.赵电波,白艳红,and张文彬,自然发酵双峰驼驼乳营养价值的研究.乳业科学与技术,2007(06)：305-308.
7. Elagamy, E.I., Effect of heat treatment on camel milk proteins with respect to antimicrobial factors: a comparison with cows'and buffalo milk proteins. Food Chemistry,2000.68(2):p.227-232.
8. Abdelgadir, W., et al., A traditional Sudanese fermented camel's milk product, Gariss, as a habitat of Streptococcus infantarius subsp. infantarius. International Journal of Food Microbiology,2008.127(3):p. 215-219.
9. Elagamy, E.I., et al., Purification and characterization of lactoferrin, lactoperoxidase, lysozyme and immunoglobulins from camel's milk. International Dairy Journal,1996.6(2):p.129-145.
10.古丽孜亚·卡克巴依,新.那.,新疆哈萨克族传统发酵驼乳抗炎作用的初步研究.中国乳品工业,2007.09：8-10
11. SHUANGQUAN, Microflora in traditional fermented camel's milk from Inner Mongolia. Milchwissenschaft,2004.59::p.649-652.
12.聂小苗,巴.阿.,新疆传统发酵酸驼乳中乳酸菌的分离和鉴定.食品研究与开发,2008.04.
13.孙天松,et al.,中国新疆地区酸马奶中乳酸菌生物多样性研究.微生物学通报,2007(03).
14. ABDEL, M.E.S., A.I. ABDALLA, and I. AHMAD E Chemical and microbiological quality of Garris, Sudanese fermented camel's milk product.2006:International Journal of Food Science and Technology, p.321-328.
15. Lore, T.A., S.K. Mbugua, and J. Wangoh, Enumeration and identification of microflora in suusac, a Kenyan traditional fermented camel milk product. Lebensmittel-Wissenschaft und-Technologie,2005. 38(2):p.125-130.
16. Abdelgadir, W.S., et al., Characterisation of the dominant microbiota of Sudanese fermented milk Rob. International Dairy Journal,2001.11(1-2):p.63-70.
17. Hekmat, S., H. Soltani, and G. Reid, Growth and survival of Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 in yogurt for use as a functional food. Innovative Food Science & Emerging Technologies,2009.10(2):p.293-296.
18. Muyzer G, D.W.E.C., Uitterlinden A G., Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16SrRNA. 1993:Applied and Environmental Microbiology, p.695-700.
19. Fasoli, S., et al., Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. International Journal of Food Microbiology,2003.82(1):p.59-70.
20.杨佐毅,et al., PCR-DGGE指纹分析技术在食品微生物检测中的应用.食品工业科技,2006(02)201-203.
21. Ampe, F., A. Sirvent, and N. Zakhia, Dynamics of the microbial community responsible for traditional sour cassava starch fermentation studied by denaturing gradient gel electrophoresis and quantitative rRNA hybridization. International Journal of Food Microbiology,2001.65(1-2):p.45-54.
22. Van Beek, S. and F.G.Priest, Evolution of the lactic acid bacterial community during malt whisky fermentation:a polyphasic study.2002:Appl. Environ. Microbiol. p.297-305.
23. Ercolini, D., PCR-DGGE fingerprinting:novel strategies for detection of microbes in food. Journal of Microbiological Methods,2004.56(3):p.297-314.
24. Vila-Costa, M., et al., Number and phylogenetic affiliation of bacteria assimilating dimethylsulfoniopropionate and leucine in the ice-covered coastal Arctic Ocean. Journal of Marine Systems,2008.74(3-4):p.957-963.
25. Kolehmainen, R.E., M. Tiirola, and J.A. Puhakka, Spatial and temporal changes in Actinobacterial dominance in experimental artificial groundwater recharge. Water Research,2008.42(17):p.4525-4537.
26. Ampe, F., et al., Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations.1999:Appl Environ Microbiol. p.5464-73.
27. Abriouel, H., et al., Culture-independent analysis of the microbial composition of the African traditional fermented foods poto poto and degue by using three different DNA extraction methods. International Journal of Food Microbiology,2006.111(3):p.228-233.
28.高蕙文,吕欣.,董明盛PCR-DGGE指纹技术在食品微生物研究中的应用.食品科学,2005.08：465-468
29. Cocolin, L., L.F. Bisson, and D.A. Mills, Direct profiling of the yeast dynamics in wine fermentations. FEMS Microbiology Letters,2000.189(1):p.81-87.
30. Cocolin, L., et al., An application of PCR-DGGE analysis to profile the yeast populations in raw milk. International Dairy Journal,2002.12(5):p.407-411.
31. Franklin, R.B. and A.L. Mills, Multi-scale variation in spatial heterogeneity for microbial community structure in an eastern Virginia agricultural field. FEMS Microbiology Ecology,2003.44(3): p.335-346.
32. S. Coppola, G.B., D. Ercolini, G. Moschetti,, Molecular evaluation of microbial diversity occurring in different types of Mozzarella cheese. Journal of Applied Microbiology,2001.90(3):p.414-420.
33. Blaiotta, G., et al., Combining Denaturing Gradient Gel Electrophoresis of 16S rDNA V3 Region and 16S-23S rDNA Spacer Region Polymorphism Analyses for the Identification of Staphylococci from Italian Fermented Sausages. Systematic and Applied Microbiology,2003.26(3):p.423-433.
34. Zhou, J.-z., et al., Application of the Mixture Design to Design the Formulation of Pure Cultures in Tibetan kefir. Agricultural Sciences in China,2007.6(11):p.1383-1389.
35. Kim, M. and J. Chun, Bacterial community structure in kimchi, a Korean fermented vegetable food, as revealed by 16S rRNA gene analysis. International Journal of Food Microbiology,2005.103(1):p. 91-96.
36. Randazzo, C.L., et al., Lactobacillus casei, dominant species in naturally fermented Sicilian green olives. International Journal of Food Microbiology,2004.90(1):p.9-14.
37. Choi, S.-Y., et al., Fermentation and sensory characteristics of kimchi containing potassium chloride as a partial replacement for sodium chloride. International Journal of Food Microbiology,1994.21(4):p. 335-340.
38. Champomier, M.-C., et al., Erratum to "Lactobacillus sakei:recent developments and future prospects":[Research in Microbiology 152 (2001) 839]. Research in Microbiology,2002.153(2):p. 115-123.
39. Dolci, P., et al., Microbiological characterization of artisanal Raschera PDO cheese:Analysis of its indigenous lactic acid bacteria. Food Microbiology,2008.25(2):p.392-399.
40. AI Jassim, R.A.M., et al., The genetic diversity of lactic acid producing bacteria in the equine gastrointestinal tract. FEMS Microbiology Letters,2005.248(1):p.75-81.
41. Vishniac, H., Yeast Biodiversity in the Antarctic, in Biodiversity and Ecophysiology of Yeasts.2006. p.419-440.
42. Kurtzman, C. and J. Pikur, Taxonomy and phylogenetic diversity among the yeasts, in Comparative Genomics.2006. p.29-46.
43.游银伟,et al.,沼气发酵液中一株酵母菌菌株的鉴定与系统发育分析.中国沼气,2008(04)：7-10.
44. Hammes, W.P. and C. Hertel, New developments in meat starter cultures. Meat Science,1998. 49(Supplement 1):p. S125-S138.
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