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.     

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。     

原料乳中嗜冷菌计数及产脂肪酶特性的研究

本文主要对乳中的嗜冷菌数的检测方法进行了初步研究。同时,考虑到嗜冷菌能在乳中产生大量的耐热性脂肪酶,力求在脂肪酶活与菌数之间建立联系。即对不同时间测得的酶活与菌数之间的关系进行初步探讨。     

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。     

原料乳中高产蛋白酶嗜冷菌的分离及产酶培养基的研究

以原料乳为对象,使用脱脂乳平板筛出三株有较强蛋白酶水解性的嗜冷菌,通过时间-酶活曲线复筛出一株高产蛋白酶的嗜冷菌Px-1。针对其产蛋白酶培养基进行了正交优化,研究了其产酶培养基的主要成分,结果表明酵母浸出粉对产酶影响显著。通过本实验最终确定了最佳培养基组成:蔗糖4%,α-乳糖4%,葡萄糖2%;酵母浸出粉0.7%,干酪素0.5%,胰蛋白胨0.3%以及0.05%的吐温80。在此条件下,Px-1产蛋白酶活为42.5451U/mL,高于优化前的测定值30.2291U/mL。      

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.     

Development of a novel loop-mediated isothermal amplification assay for the detection of lipolytic Pseudomonas fluorescens in raw cow milk from north China

Lipases secreted by psychrotrophic bacteria are known to be heat resistant and can remain active even after the thermal processing of milk products. Such enzymes are able to destabilize the quality of milk products by causing a rancid flavor. Rapid detection of a small amount of heat-resistant lipase-producing psychrotrophic bacteria is crucial for reducing their adverse effects on milk quality. In this study, we established and optimized a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Pseudomonas fluorescens in raw cow milk, as the most frequently reported heat-resistant lipase-producing bacterial species. Pseudomonas fluorescens-specific DNA primers for LAMP were designed based on the lipase gene sequence. Reaction conditions of the LAMP assay were tested and optimized. The detection limit of the optimized LAMP assay was found to be lower than that of a conventional PCR-based method. In pure culture, the detection limit of the LAMP assay was found to be 4.8 × 10¹ cfu/reaction of the template DNA, whereas the detection limit of the PCR method was 4.8 × 10² cfu/reaction. Evaluation of the performance of the method in P. fluorescens-contaminated pasteurized cow milk revealed a detection limit of 7.4 × 10¹ cfu/reaction, which was 10² lower than that of the PCR-based method. If further developed, the LAMP assay could offer a favorable on-farm alternative to existing technologies for the detection of psychotrophic bacterial contamination of milk, enabling improved quality control of milk and milk products.     

生乳细菌总数检测仪测定生乳细菌总数的应用

以还原法为理论基础,研制生乳细菌总数检测仪进行生乳细菌总数快速检测,以克服经典还原法目测比色主观性强的缺点,而且检测结果可以定量表示。该仪器连续检测加入试剂后的生乳样品颜色(RGB),通过数理统计分析,采用斜率法以R值斜率表示样品颜色的变化速度。研究以标准平板计数法(SPC法)为标准,建立了R值斜率与菌落总数之间的标准方程,经验证生乳细菌总数检测仪检测结果与SPC法检测结果差异不显著。与流式细胞仪比较,在与SPC法的相关性、运行成本及便携性等方面具有一定优势。     

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.     

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.     

液体乳中嗜冷菌数的测定

研究了液体乳中嗜冷菌的快速菌落计数方法，通过实验得出，样品中嗜冷菌在21℃条件下培养48h，结果表明，该方法快速，准确，简便易行。     

Effect of growth phase on the subsequent growth kinetics of psychrotrophic bacteria of raw milk origin

Psychrotrophs isolated at days 1, 3 and 5 from refrigerated raw milk were monitored for growth kinetics using conductance when they had been inoculated during their lag, log and stationary growth phases. The results indicated that there was a more rapid change in the microflora during the initial stages of storage when the lag phases of the 1, 3 and 5 day isolates were analysed. There was also evidence of the 'suicide response'. In addition, the fact that the stationary phase cells showed the greatest exponential growth rate may be a contributory factor to their selective advantage.