原料乳中嗜冷菌的检测

针对原料乳中嗜冷菌的一种快速检测方法进行了初步研究。由于嗜冷菌能在乳中产生大量的耐热性脂肪酶,所以我们在脂肪酶活与嗜冷菌数之间建立了一种线性关系,进而通过4-硝基苯酚游离释放法测定脂肪酶的活力来得到嗜冷菌数。      

原料乳中嗜冷菌产脂肪酶条件的优化

研究了原料乳中嗜冷菌产生脂肪酶的几组影响条件。主要从pH值、培养温度、产酶培养基组成几个方面考虑。优化产酶条件，从而可提高脂肪酶的检测活力，为原料乳中嗜冷菌分泌的脂肪酶的研究奠定理论基础。     

原料乳中嗜冷菌及其主要热稳定性酶类的研究进展

原料乳中的嗜冷菌及其热稳定性胞外酶是引起液态乳制品多种质量问题的主要原因之一。阐述了嗜冷菌及其热稳定性蛋白酶和脂肪酶的概念及特性，对嗜冷菌在原料乳中的控制提出了自己建议，并对嗜冷菌、蛋白酶、脂肪酶的检测方法及理论进行了展望。     

原料乳中嗜冷菌快速检测新技术研究进展

嗜冷菌在原料乳低温储藏过程中能大量繁殖,其生长过程中产生的脂肪酶和蛋白酶对乳制品后期储藏的风味品质有很大影响。因此,针对原料乳中嗜冷菌的检测,国内外开展了大量的研究,其中快速、自动化的现代技术蓬勃发展。本文在整理这些主要技术的基础上,重点介绍了近些年来发展较快且灵敏度高的嗜冷菌快速检测新技术及其优缺点,并对未来该领域快速检测技术的发展做出展望。     

原料乳中优势嗜冷菌株的确定及其微生物学特征研究

对原料乳样品进行嗜冷菌的分离筛选,结果得到4株优势嗜冷菌。本文研究了这4株优势嗜冷菌的生长特性及产脂肪酶特性,分别得到了4株优势嗜冷菌株的最佳生长条件及最佳产脂肪酶条件。      

原料乳中嗜冷菌的危害分析及控制研究

采用分离培养基从牧场的原料乳样品中分离得到嗜冷菌,经鉴定为荧光假单胞菌.进一步研究了不同杀菌方式下嗜冷菌对贮存过程中乳品品质的影响,结果表明,原料乳经115℃高温灭菌,然后进行55℃保温1h灭酶处理,有助于控制嗜冷菌的生长,与25℃贮存相比,在4℃贮存8d后,其蛋白质和脂肪含量相对较高.     

原料乳嗜冷菌分离株微生物学特征研究

从155份原料乳样品中分离纯化得到嗜冷菌分离物16株，经微生物分类学性状特点鉴定，确定为假单胞菌10株，微球菌4株，产碱杆菌2株。由拮抗试验表明，16株嗜冷菌分离物可分成5个Dienes型；经药敏试验表明，16株嗜冷菌分离物分别对3种药物（CFP、AKN、OXA）敏感，经方差分析，菌株问差异不显著；经系统聚类分析，16株嗜冷菌分离物可分成4个大类，均对GEN、CMP、EPY、TET等抗生素耐药。     

原料乳嗜冷菌的危害分析及控制

从相关的8个牧场的原料乳样品中分离纯化得到嗜冷菌分离物。然后重点探讨嗜冷菌对原料乳的危害及对嗜冷菌的生长控制。对嗜冷菌数动态增加与蛋白酶和脂肪酶活力变化、pH值的变化和总醛类物质浓度增加的相关性进行了分析研究。结果表明，壳聚糖和二氧化氯对嗜冷菌有较好的抑菌作用。     

原料奶中嗜冷菌的测定

１概述如果ＵＨＴ奶制品中发现有耐热蛋白酶存在（产品有凝块或发苦），或者有脂肪酶存在（产品有脂肪氧化味）时，则应对原奶中的嗜冷菌进行检验。２材料及仪器内装９９ｍｌ无菌稀释液的烧瓶；氯化钠水溶液（浓度０．７％，用于制备稀释液）；蒸馏水；无菌培养皿（预先灭...     

原料乳中嗜冷菌菌群多样性及分析方法研究进展

嗜冷菌是原料乳中主要危害性腐败菌群之一,其种类繁多,菌群结构和多样性受到多种因素影响,这为原料乳中优势嗜冷菌的鉴定带来了困难.结合国内外文献,综述了储藏温度、储藏时间、地域和季节等因素对原料乳中嗜冷菌菌群结构和多样性的影响,原料乳中嗜冷菌可能的外界来源.评述了高通量测序、随机扩增多态性DNA、变性凝胶电泳等现代分子生物...     

The main spoilage-related psychrotrophic bacteria in refrigerated raw milk

Refrigerated raw milk may contain psychrotrophic microorganisms that produce thermoresistant exoproteases and lipases, which may compromise the quality of processed fluid milk and dairy products during storage. The aim of this work was to quantify and identify the deteriorating psychrotrophic microbiota in Brazilian refrigerated raw milk using genetic diversity analysis. The mean psychrotrophic count was 1.1 × 10⁴ cfu/mL. Of the total isolates, 47.8 and 29.8% showed deteriorating activity at 35°C within 48 h and 7°C within 10 d, respectively. Among the proteolytic species, more isolated by this study were Lactococcus lactis (27.3%), Enterobacter kobei (14.8%), Serratia ureilytica (8%), Aerococcus urinaeequi (6.8%), and Bacillus licheniformis (6.8%). Observed among lipolytics were E. kobei (17.7%), L. lactis (15.6%), A. urinaeequi (12.5%), and Acinetobacter lwoffii (9.4%). The isolates S. ureilytica, E. kobei, Pseudomonas spp., and Yersinia enterocolitica potentially produced alkaline metalloprotease (aprX). Despite the low counts, a considerable portion of the psychrotrophic microbiota presented spoilage potential, which reaffirms the need for rigor in the control of contamination and the importance of rapid processing as factors that maintain the quality of milk and dairy products.     

Detection of the apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated raw milk

Bacteria of the genus Pseudomonas have been associated with the spoilage of raw milk and dairy products due to the production of thermostable proteolytic enzymes. The apr gene encodes for alkaline metalloprotease in Pseudomonas and other related bacteria. Its presence in psychrotrophic proteolytic bacteria isolated from raw milk collected from cooling tanks was verified. A polymerase chain reaction (PCR) technique was used with degenerate primers. Total DNA from 112 isolates was pooled in different groups and then used as template for the amplification reactions. Controls consisted of DNA extracted from 26 cultures. An expected DNA fragment of 194 bp was detected in groups that contained bacteria identified as Pseudomonas. The PCR product was observed only when DNA from control cultures of Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia marcescens and Aeromonas hydrophila were used. A detection limit assay indicated that the apr gene could be directly amplified from pasteurized milk inoculated with 10(8) CFU/ml of P. fluorescens. With this method it was possible to detect proteolytic bacteria at 10(5) CFU/ml in reconstituted skim milk powder if cells were recovered for DNA extraction before amplification.     

Proteolytic activity among psychrotrophic bacteria isolated from refrigerated raw milk

Psychrotrophic bacteria were isolated from refrigerated raw milk from a processing plant in Southern Brazil. Psychrotrophic counts were between 4.9 and 7.8 log cfu/mL, and 5.3 to 7.2 log cfu/mL, for samples collected at the truck and the milk storage silo, respectively. Among the bacterial isolates, 90% were Gram-negative. Most strains presented low proteolytic activity, but strains of Burkholderia cepacia, Klebsiella oxytoca and Aeromonas sp. showed higher than 20 U/mL on azocasein as substrate. Crude proteases from selected strains were resistant to conventional heat treatments and caused coagulation of UHT milk after 5 days storage at room temperature.     

Molecular identification of mesophilic and psychrotrophic bacteria from raw cow's milk

The aim of this study was to use molecular techniques to assess the microbiota of eight raw cow's milk samples at biotype and species level. Sixty-six isolates from raw milk samples were screened by Randomly amplified polymorphic DNA–PCR (RAPD–PCR) biotyping and representative strains of RAPD–PCR profiles were identified by 16S rRNA gene sequencing. Pseudomonas spp. were the most commonly occurring contaminants along with Enterobacteriaceae such as Hafnia alvei, Serratia marcescens and Citrobacter freundii. Moreover, Gram-positive isolates belonging to the genera Staphylococcus and Lactococcus were also found. Experiments of growth at different temperatures showed that more than 50% of the Gram-negative isolates could grow at chill temperatures and that 65% of the Pseudomonas spp. strains grew at 7 °C within 5 days. Only 13 Gram-negative isolates displayed proteolytic activity on milk agar, suggesting that not all the biotypes of milk contaminating species are able to perform this spoilage-associated activity. Among the Gram negative, the proteolytic strains were mainly Peudomonas spp. that displayed the activity at both 7 °C and 20 °C. A reliable molecular identification of raw milk microbiota is important for the study of the microbiological quality of raw milks and for the assessment of the ecology at species level in order to develop improved systems, preventing contamination and having the best conditions for the storage of milk.     

原料乳嗜冷菌的快速分离鉴定

利用API 20NE细菌鉴定系统快速鉴定出原料乳中主要的嗜冷菌。大大缩短常规嗜冷菌鉴定所用的时间。     

原料乳中典型嗜冷菌增菌培养条件优化

针对原料乳中典型嗜冷菌的生长条件进行了选择优化.研究了其培养的初始pH值、温度、刺激其分裂增殖的营养成分.确定了最佳生长pH值、温度、碳源、氮源、和无机盐离子,从而达到增菌的目的.     

牛乳中嗜冷菌数与胞外蛋白酶活性相关性的研究

主要研究了牛乳中嗜冷菌数与其产生的耐热性胞外蛋白酶活性的相关性.从原料乳中分离出一株优势嗜冷菌,实验室初步鉴定后用API20NE系统进行鉴定,鉴定该菌为荧光假单胞菌(Pseudomonas Fluorescens),产酶实验结果表明,该菌产耐热性胞外蛋白酶.利用该菌株研究牛乳中单一嗜冷菌菌数与蛋白酶活性的关系,结果表明菌数与蛋白酶活性呈一定正相关,回归方程为Y=0.4548ln(X) 0.4178,决定系数R2=0.8172,相关系数R=0.9039,差异极显著(P<0.01);自然状态下,原料乳中嗜冷菌数与蛋白酶活性关系实验结果表明,原料乳在不同条件下贮存,嗜冷菌数有明显的增加趋势,但原料奶中嗜冷菌数和蛋白酶活性之间没有多少相关性.     

Genetic diversity of Gram-negative, proteolytic, psychrotrophic bacteria isolated from refrigerated raw milk

The random amplified polymorphic DNA (RAPD) fingerprinting technique was used to assess the genetic diversity of 70 isolates of Gram-negative proteolytic psychrotrophic bacteria that were isolated from refrigerated raw milk. Three oligonucleotides, which generated 87 fragments of polymorphic DNA, were used in the amplification reactions. The genetic distance values calculated using Jaccard's coefficient showed there was high genetic variability among the isolates. Cluster analysis procedures suggested that the genetic variability among isolates belonging to the same species was as high as the variability among different species. Clustering by the UPGMA hierarchical method and data graph dispersion indicated a tendency of the isolates to group according to whether they did or did not ferment glucose.