内蒙古传统发酵驼乳中乳酸菌和酵母菌的分离鉴定及其生物多样性分析

采用传统培养方法对4份内蒙古阿拉善盟传统发酵驼乳样品中的乳酸菌和酵母菌进行分离纯化,运用16S rRNA基因序列分析方法进行种属鉴定,同时采用Pac Bio SMRT三代测序技术对样品中的细菌和真菌多样性进行研究。发酵驼乳中共分离到8株乳酸菌和5株酵母菌,乳酸菌包括1株胚芽乳杆菌、2株瑞士乳杆菌、2株嗜热链球菌、2株假肠膜明串珠菌和1株开菲尔基质乳杆菌。酵母菌包括1株马克斯克鲁维酵母、2株酿酒酵母、1株诞沫假丝酵母和1株芽殖酵母。Pac Bio SMRT测序结果显示,硬壁菌门为4份发酵驼乳样品中的优势细菌门,乳杆菌属为优势细菌属,以及瑞士乳杆菌为优势细菌种;子囊菌门为发酵驼乳样品中的优势真菌门,克鲁维酵母菌属为优势真菌属以及马克斯克鲁维酵母为优势真菌种。     

基于高通量测序技术比较不同来源原料乳中的细菌特征

本文采用二代高通量测序技术研究北京、河北、内蒙古、天津四地区不同季节原料乳样品中的细菌多样性并对其进行功能预测。收集四地区不同季节原料乳样品23份,提取基因组DNA后使用Illumina Mi Seq测序平台对细菌16S rRNA的V3-V4"可变区"进行测序,结合数据库对原料乳中微生物群落分布、相对丰度和细菌功能基因代谢途径进行分析。原料乳中的细菌可分为33门732属,其中变形菌门(Proteobacteria),厚壁菌门(Firmicutes),拟杆菌门(Bacteroidetes)和放线菌门(Actinobacteria)为主要菌门,平均相对丰度占微生物总数的99.37%。优势菌属为假单胞菌属(Pseudomonas)和不动杆菌属(Acinetobacter),平均相对丰度之和为60.55%。不同地区、不同季节原料乳微生物群落的结构组成和相对丰度存在差异,且假单胞菌属和气单胞菌属的相对丰度在不同季节原料乳中存在显著性差异(p0.05)。氨基酸代谢、碳水化合物代谢和膜运输为原料乳细菌主要的代谢途径。     

基于三代测序技术分析新疆鲜马奶和酸马奶中乳酸菌菌群结构

传统酸马奶受制作环境和工艺的影响,其菌群结构有所不同.本文运用Pacbio SMRT测序技术,基于乳酸菌特异性引物对新疆部分地区酸马奶和鲜马奶的乳酸菌菌群进行研究,结果表明:鲜马奶中乳酸菌丰富,以瑞士乳杆菌(35.52％)、唾液链球菌(10.11％)、乳酸乳球菌(9.49％)、嗜热链球菌(5.96％)以及德氏乳杆菌(5...     

不同地区奶酪中乳酸菌多样性研究

乳酸菌作为奶酪中重要的微生物可改善奶酪风味及营养，其代谢产物有利于人体健康。为探究不同地区奶酪乳酸菌的多样性，以14份奶酪样品为研究对象，通过乳酸菌特异性引物与PacBio SMRT三代测序技术相结合的方法进行测序。结果表明：在14份奶酪样品中共发现14个乳酸菌属和65个乳酸菌种，其中乳酸乳球菌（27.10%）和嗜热链球菌（14.35%）为主要优势菌种，不同样品中乳酸菌菌群结构存在较大差异。通过主坐标分析发现：将奶酪样品分为3组，各组奶酪样品的主要优势菌属各有特点，这可能是由于采样地、气候和原料等因素所致。奶酪中鸡乳杆菌、植物乳杆菌和德氏乳杆菌等部分低丰度乳酸菌之间存在显著相关性。本研究结果表明：奶酪样品中乳酸菌菌群结构复杂多样，地理环境和气候是影响奶酪菌群结构的重要因素。本文通过研究不同地区奶酪乳酸菌的多样性，以期为乳酸菌资源开发提供参考依据。     

乳酸乳球菌乳酸亚种IMAU11823全基因组测序及胞外多糖基因簇分析

目的:乳酸乳球菌乳酸亚种IMAU11823是从内蒙古锡林郭勒盟自然发酵乳制品嚼口中分离得到的1株具有优良发酵特性且高产胞外多糖的乳酸菌。为探究其产胞外多糖的作用机制,对其进行全基因组测序并解析基因组序列信息。方法:使用PacBio SMRT三代测序技术对该菌株进行全基因组测序,采用生物信息学方法对其进行序列组装、基因预测和功能注释,并深入挖掘可能参与胞外多糖生物合成相关的基因信息。结果:乳酸乳球菌乳酸亚种IMAU11823包含1条染色体DNA(基因组大小为2 458 520 bp,GC含量为35.2%)和3个环状质粒(pIMAU11823A、pIMAU11823B、pIMAU11823C,基因组大小和GC含量分别为127 965,24 554,7 734 bp和35.7%,39.5%,32.7%),同时发现该菌株基因组内具有乳糖、纤维二糖、蔗糖、果糖、甘露糖、半乳糖转运系统和7种糖核苷酸合成相关基因以及1个胞外多糖合成基因簇epsRXCDBEF(GTF1)H(GTF2)JKM。结论:本研究揭示了乳酸乳球菌乳酸亚种IMAU11823的基因组特征,并预测了其胞外多糖的合成机制,为研究其产生的胞外多糖结构特征提供了理论依据,同时为该菌株在工业生产中的应用奠定理论基础。     

毛细管电泳法对乳及乳制品中乳源蛋白的研究

采用毛细管电泳方法对原料乳、市售鲜奶、不同厂家的巴氏灭菌乳、不同厂家和产地超高温灭菌乳(UHT)、调味乳、乳酸饮料、复原乳、酸奶、奶粉中蛋白成分进行检测。选择聚乙烯醇涂层毛细管,采用柠檬酸缓冲体系,在紫外检测214nm、分离电压20kV条件下对乳及乳制品中的α一乳白蛋白(α-La)、β一乳球蛋白(β-Lg)、α-酪蛋白(α-CN)、β-酪蛋白(β-CN)和k-酪蛋白(k-CN)进行分离测定。结果表明:五种蛋白的含量在原料乳(巴氏灭菌乳、市售鲜奶)、UHT乳、酸奶、调味乳、乳酸饮料、复原乳中依次降低,而UHT乳含量随保质期的增加而减少,奶粉中蛋白质含量因其适应人群而有差异。乳及乳制品中蛋白质的含量与其存在形式、产地及加工工艺相关。     

基于纯培养方法和PacBio三代测序技术研究蒙古国传统酸马奶中乳酸菌多样性

通过纯培养方法结合PacBio三代测序技术对采自蒙古国的12份传统酸马奶中的乳酸菌多样性进行研究。应用传统的纯培养技术从12份传统酸马奶样品中分离出62株乳酸菌,通过16S rRNA基因序列分析和系统进化关系研究,62株乳酸菌被鉴定为5个属,6个种,分别是:25株瑞士乳杆菌、20株植物乳杆菌、7株产马乳酒乳杆菌、5株屎肠球菌、3株嗜热链球菌和2株粪肠球菌。采用PacBio SMRT测序技术研究其中8份样品中乳酸菌的多样性,宏基因组测序得到的优势菌为乳酸菌,同时也检测到一些痕量的其它种属细菌。共检测到5个乳酸菌的属,分别是:乳杆菌属、乳球菌属、链球菌属、明串珠菌属和肠球菌属,20个乳酸菌的种,即:瑞士乳杆菌、乳酸乳球菌、副乳链球菌、棉子糖乳球菌、肠膜阴串珠球菌和产马乳酒乳杆菌。宏基因组测序相对含量表明:蒙古国酸马奶样品中乳酸菌优势属为乳杆菌属(95.56%),优势种为瑞士乳杆菌(94.64%),纯培养方法和宏基因组三代测序方法都表明瑞士乳杆菌为蒙古国传统酸马奶中的优势菌种。宏基因组测序技术除检测到纯培养获得的菌株外,还获得14个低丰度乳酸菌,如棉籽糖乳球菌、肠膜明串珠球菌和开菲尔乳杆菌等,这些菌在酸马奶中含量较少。通过PacBio SMRT测序技术可以全面了解酸马奶中乳酸菌的多样性,为通过纯培养方法获得低丰度乳酸菌提供指导。     

原料乳中腐败微生物对液态乳品质的影响及防控

乳因其营养丰富备受关注,被称为人体的“白色血液”,同时乳也是微生物良好的培养基,因此原料乳品质和液态乳加工过程都会受到严格把控,但目前液态乳在储藏期、流水线或货架期内发生腐败变质现象仍屡屡发生,引起行业对乳及乳制品安全的高度重视。原料乳作为液态乳生产的原料,是其品质保障的基础,原料乳中腐败微生物随着储藏时间的延长而大量繁殖,经过热处理后未被完全杀死的腐败菌及其产生的耐热酶仍会继续影响液态乳品质,此外生物膜的形成也为腐败菌在管道内残留提供了机会,加大了腐败微生物防控的难度。因此本文将从原料乳中腐败微生物多样性进行阐述并探究腐败微生物对于液态乳的不良影响,挖掘腐败微生物破坏液态乳可能存在的作用机制,总结现阶段国内外对腐败微生物的防控手段并探讨其商业应用价值,旨在为今后液态乳质量安全保障及延长货架期提供理论指导。     

生产全脂加糖奶粉时原料乳的标准化

所谓标准化,就是调整原料乳中的脂肪含量及计算出应该加入的蔗糖量,其目的是使两者符合质量标准中的规定。从国标GB5412—85规定中可以看出,脂肪与蔗糖两项指标的规定幅度较大,由于两者间又有价格上的差别,所以切实做好标准化,可以提高奶粉生产的经济效益,这是一项十分重要的工作。关于标准化的计算国内很多书籍、刊物中已作了介绍,但都是采用加入脱脂乳(原料乳脂肪含量偏高时)或加入稀奶油(原料乳脂肪含量不足时)来进行调整的。其实乳品厂(尤其是中小型厂)对原料乳进行标准化时,往往采取上列方法调整脂肪含量。当原料乳脂肪含量偏高时,直接从原料乳中分     

超高温灭菌乳的主要质量问题及控制措施

本文对影响超高温灭菌乳质量的主要问题进行了阐述，其主要影响因素是原料乳的质量和超高温灭菌过程的几个重要方面。对超高温生产过程中的杀菌温度和保温时间，脱气和均质，无菌输送，无菌包装和无菌灌装阶段工艺设备进行分析，参照应用设备结构工艺特点，找出各阶段中影响超高温灭菌乳质量的主要因素，并提出控制措施。另一方面，对影响超高温灭菌乳质量的另一重要因素原料乳质量，根据乳的特性及采用的加工方法，提出控制措施，保证产品质量的稳定。     

Single-molecule real-time sequencing reveals differences in bacterial diversity in raw milk in different regions and seasons in China

The quality of raw milk is a key factor influencing the whole dairy processing chain. The richness and diversity of bacteria in raw milk affect its quality and safety. However, traditional microbial detection methods mainly depend on the known microbe culture and are often time consuming. Thus, the development of efficient ways for supervising any possible microbiological contamination is desiderated. In the current work, single-molecule real-time (SMRT) sequencing, developed by Pacific Biosciences (PacBio), was applied to acquire long reads and applied for discrimination of bacteria at species level. Forty samples of raw milk obtained from Beijing, Hebei, Inner Mongolia, Shanghai, and Guangdong in China during summer, autumn, and winter were investigated. Among 35 bacteria species identified in these samples, Acinetobacter albensis, Pseudomonas gessardii, Pseudomonas weihenstephanensis, and Rahnella inusitata were the bacteria with the highest relative abundance in the overall sample, whereas the bacteria with the highest relative abundance in raw milk samples of different origins and seasons are different. Significant differences in bacterial richness and bacterial community diversity in raw milk grouped according to different production areas and different sampling seasons were confirmed by Welch's t-test. Interestingly, the transport distance and transport time positively correlated with the relative abundance of Pseudomonas weihenstephanensis, suggesting that the content of this bacteria was expected to be a standard for evaluating the freshness of raw milk. Pathogens Bacillus cereus and Klebsiella pneumoniae were detected in most samples, indicating that the raw milk was at risk of contamination by pathogenic bacteria. Moreover, the findings of this study provide important evidence for quality and safety monitoring and biological control of raw milk.     

我国南北不同地域自备水源水质指标及微生物群落结构特征比较

目的 比较我国南北不同地域自备水源的水质指标及微生物群落结构特征,评价其安全性。方法 本研究对我国南北5省边远散地域(北京郊区、黑龙江边境地域、内蒙古偏远地域、广西边境地域和云南边境地域)的317个自备水源进行采样,对浑浊度、总硬度和pH等水质指标进行分析,以高通量测序法对水样的微生物群落结构进行解析。结果 不同地域自备水源的水质和微生物群落结构存在较大的差异性,内蒙古偏远地域的阴离子指标最大值超标严重,在内蒙古-东和内蒙古-西两组水样中SO₄^2-最大浓度值分别为543.12和666.33 mg/L, Cl^-与NO₃^-也存在超标情况。变形菌门(Proteobacteria)是5省自备水源水样的第一优势菌门,其相对丰度为56.87%-99.38%。具有一定致病性的短波单胞菌(Brevundimonas)在各个样品中广泛分布(1.50%-27.27%)。反硝化过程的典型菌株假单胞菌(Pseudomonas)存在于北京、黑龙江和内蒙古水样中(2.96%-38.41%),广西和云南并未检出。北京、黑龙江和内蒙古水样中各种功能表达基因的数量远高于广西和云南水样。结论 我国南北5省自备水源在水质和微生物群落特征方面均存在差异;较之南方自备水源,北方自备水源普遍受到了人类活动的影响。     

阿尤恩地区自然发酵牛乳中乳酸菌分离鉴定及多样性研究

为了分离、保藏自然发酵乳中乳酸菌菌种,丰富自然发酵乳中乳酸菌多样性信息.本文采用传统的纯培养分离方法和宏基因组16S rRNA基因测序技术对阿尤恩地区自然发酵牛乳的乳酸菌多样性进行研究.纯培养结果表明:5份自然发酵牛乳中共分离出111株乳酸菌,鉴定为5个属10个种,其中Lactococcus lactis,占总分离株的...     

Both sampling seasonality and geographic origin contribute significantly to variations in raw milk microbiota,but sampling seasonality is the more determining factor

Accurately profiling and characterizing factors shaping raw milk microbiota would provide practical information for detecting microbial contamination and unusual changes in milk. The current work was an observational study aiming to profile the microbiota of raw milk collected across wide geographic regions in China in different seasons and to investigate the contribution of geographical, seasonal, and environmental factors in shaping the raw milk microbiota. A total of 355 raw cow milk samples from healthy Holsteins and 41 environmental samples (farm soil and surface of milking room floor) were collected from 5 dairy farms in 5 Chinese provinces (namely, Daqing in Heilongjiang province, Jiaozuo in Henan province, Qingyuan in Guangdong province, Suqian in Jiangsu province, and Yinchuan in Ningxia Hui Autonomous Region) in January, May, and September 2018. The microbial communities in raw milk and farm environmental samples were determined using the PacBio small-molecule real-time circular consensus sequencing, which generated high-fidelity microbiota profiles based on full-length 16S rRNA genes; such technology was advantageous in producing accurate species-level information. Our results showed that both seasonality and sampling region were significant factors influencing the milk microbiota; however, the raw milk microbiota was highly diverse according to seasonality, and sampling region was the less determining factor. The wide variation in raw milk microbial communities between samples made it difficult to define a representative species-level core milk microbiota. Nevertheless, 3 most universal milk-associated species were identified: Lactococcus lactis, Enhydrobacter aerosaccus, and Acinetobacter lwoffii, which were consistently detected in 99%, 95%, and 94% of all analyzed milk samples, respectively (n = 355). The top taxa accounting for the overall seasonal microbiota variation were Bacillus (Bacillus cereus, Bacillus flexus, Bacillus safensis), Lactococcus (Lactococcus lactis, Lactococcus piscium, Lactococcus raffinolactis), Lactobacillus (Lactobacillus helveticus, Lactobacillus delbrueckii), Lactiplantibacillus plantarum, Streptococcus agalactiae, Enhydrobacter aerosaccus, Pseudomonas fragi, and Psychrobacter cibarius. Unlike the milk microbiota, the environmental microbiota did not exhibit obvious pattern of seasonal or geographic variation. However, this study was limited by the relatively low number and types of environmental samples, making it statistically not meaningful to perform further correlation analysis between the milk and environmental microbiota. Nevertheless, this study generated novel information on raw milk microbiota across wide geographic regions of China and found that seasonality was more significant in shaping the raw milk microbiota compared with geographic origin     

Investigating the bacterial microbiota of traditional fermented dairy products using propidium monoazide with single-molecule real-time sequencing

Traditional fermented dairy foods have been the major components of the Mongolian diet for millennia. In this study, we used propidium monoazide (PMA; binds to DNA of nonviable cells so that only viable cells are enumerated) and single-molecule real-time sequencing (SMRT) technology to investigate the total and viable bacterial compositions of 19 traditional fermented dairy foods, including koumiss from Inner Mongolia (KIM), koumiss from Mongolia (KM), and fermented cow milk from Mongolia (CM); sample groups treated with PMA were designated PKIM, PKM, and PCM. Full-length 16S rRNA sequencing identified 195 bacterial species in 121 genera and 13 phyla in PMA-treated and untreated samples. The PMA-treated and untreated samples differed significantly in their bacterial community composition and α-diversity values. The predominant species in KM, KIM, and CM were Lactobacillus helveticus, Streptococcus parauberis, and Lactobacillus delbrueckii, whereas the predominant species in PKM, PKIM, and PCM were Enterobacter xiangfangensis, Lactobacillus helveticus, and E. xiangfangensis, respectively. Weighted and unweighted principal coordinate analyses showed a clear clustering pattern with good separation and only minor overlapping. In addition, a pure culture method was performed to obtain lactic acid bacteria resources in dairy samples according to the results of SMRT sequencing. A total of 102 LAB strains were identified and Lb. helveticus (68.63%) was the most abundant, in agreement with SMRT sequencing results. Our results revealed that the bacterial communities of traditional dairy foods are complex and vary by type of fermented dairy product. The PMA treatment induced significant changes in bacterial community structure.     

基于16S rDNA高通量测序分析水牛初乳与常乳中细菌多样性

目的:利用16S rDNA高通量测序研究广西水牛研究所种畜厂三品杂水牛初乳与常乳的细菌多样性。方法:提取当天采集的生水牛乳样本的细菌总DNA,PCR扩增其16S rDNA,利用纯化后的扩增片段构建其菌群的16S rDNA文库,采用Miseq PE300平台进行高通量测序以及BLAST比对。结果:初乳组样本共得到19个门,292个属和437个种。而常乳两组样本共得到7个门,203个细菌属和330个细菌种,初乳组的细菌多样性高于常乳组。初乳组中的优势菌属为金黄杆菌属（Chryseobacterium）、不动杆菌属（Acinetobacter）、假单胞菌属（Pseusomonas）,而常乳组中的优势菌则为金黄杆菌属（Chryseobacterium）、乳球菌属（Lactococcus）、肠球菌属（Enterococcus）。样本层级聚类显示:初乳组与常乳组组间群落差异不显著。PCA与PLS-DA分析表明:两组样本组间菌群结构差异显著。结论:三品杂水牛初乳与常乳均呈现较丰富的多样性,常乳组中细菌多样性显著高于初乳组。     

我国生牛乳蛋白质含量现状及影响因素调查

目的研究我国当前生牛乳的蛋白质含量和影响因素。方法分析覆盖17个省(市、自治区)18个乳品企业2014年1月—2015年2月连续12个月共30 531份生牛乳样品蛋白质含量数据,利用SPSS 19.0软件进行数据处理。结果生牛乳中蛋白质的平均含量为(3.14±0.15)g/100 g,规模化牧场、养殖小区和散户的生牛乳中蛋白质平均含量分别为(3.18±0.14)、(3.07±0.12)和(3.00±0.14)g/100 g,南方、西部、华北、东北内蒙古和大城市周边地区生牛乳蛋白质平均含量分别为(3.17±0.14)、(3.16±0.14)、(3.14±0.17)、(3.09±0.17)和(3.09±0.10)g/100 g,春、夏、秋、冬季生牛乳蛋白质平均含量分别为(3.09±0.13)、(3.07±0.13)、(3.21±0.13)、(3.20±0.14)g/100 g。结论我国生牛乳的蛋白质含量有八成以上达到3.0 g/100 g。养殖模式、养殖区域、季节和月份均会影响生牛乳中的蛋白质含量。乳脂肪、非脂乳固体、相对密度与乳蛋白含量呈正相关。     

基于16S rDNA高通量测序方法比较新疆西北部地区乳品中微生物的多样性

为更加全面地了解新疆特色乳品中微生物多样性,比较不同动物来源的原奶和酸奶的细菌群落结构,运用高通量测序技术,对乳品中细菌16S r DNA V4-V5区测序,进而对新疆克州和塔城地区牛奶、驼奶、马奶、羊奶、酸牛奶、酸驼奶和酸马奶7种乳品中细菌群落组成和多样性进行分析。研究共获得539 557条有效序列,379个OTU。多样性分析表明,原奶样品中细菌Shannon-Wiener指数明显高于酸奶样品。微生物群落组成分析发现,不同乳品之间菌群组成差异较大。7种乳品中的菌群均以厚壁菌门和变形菌门为主,但原奶样品主要以变形菌门为主,而酸奶样品主要以厚壁菌门为主。在属水平上,牛奶主要以假单胞菌属(Pseudomonas)为主,驼奶主要以埃希菌属-志贺菌属(Escherichia-Shigella),马奶主要以明串珠菌属(Leuconostoc)为主,羊奶中的优势菌属为乳球菌属(Lactococcus),而酸牛奶、酸驼奶和酸马奶都是以乳杆菌属(Lactobacillus)为优势菌属。不同动物来源的原奶和酸奶样品中的微生物多样性存在显著差异,并且原奶中检测到的环境污染菌和致病菌(或条件致病菌)的丰度也相对较高。本研究结果将为准确评估乳品中的微生物群落对新疆地区少数民族健康的影响提供一定的数据基础。     

益生乳酸菌Lactobacillus casei Zhang从基础研究到产业化开发

2001年-2009年,内蒙古农业大学乳品生物技术与工程教育部重点实验室从采集自中国西部7个省市及蒙古国14个省市不同少数民族地区951份自然发酵乳制品等样品中分离鉴定出3 388株乳酸菌,建立了中国首个原创性乳酸菌菌种资源库;从菌种库中分离自传统发酵酸马奶(koumiss)的243株乳杆菌中筛选获得1株性能优良的乳酸菌—Lactobacillus casei Zhang(L.casei Zhang)。采用5种动物模型和人体试验进行了功能评价,并利用基因组学和蛋白质组学研究技术对L.casei Zhang益生分子机制进行了深入剖析。经过多年系统的基础研究,自主解决了其产业化的关键技术问题,包括其直投式发酵剂、发酵乳制品和发酵豆乳益生菌饮料的研发并实现了产业化。