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。     

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     

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。     

Biodiversity and technological potential of wild lactic acid bacteria from raw cows' milk

To study lactic acid bacteria (LAB) biodiversity and to evaluate their potential for use in dairy applications, eight raw cows' milk batches were sampled from five dairy factories located in different areas of the Trentino region during winter and summer milkings. A total of 370 (Gram-positive and catalase-negative) isolates were first molecularly analysed by means of randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). After strain differentiation (124 profiles), LAB were genetically identified at species level. The most frequently isolated LAB were lactococci, enterococci and streptococci. Lactobacilli, leuconostocs and pediococci were found at low levels. All strains belonging to the most numerous groups which are relevant in cheese production were characterized for their physiology, technological aptitudes and safety aspects. Although the majority of strains did not show a marked potential, the high biodiversity of wild LAB allowed the selection of a few strains with interesting properties in view of their use in traditional cheese productions as starter and non starter inocula.     

Isolation and identification of lipolytic, psychrotrophic, spore forming bacteria from raw milk

Out of 100 samples of raw milk, 48% were found to contain lipolytic, psychrotrophic, spore forming bacilli. The lipase activity of 59 selected isolates ranged from 8.5 to 42 units/ml. Forty one per cent of the total isolates exhibited enzyme activity in the range of 21–25 units/ml. On the basis of morphological and biochemical characteristics, the 59 lipolytic isolates were identified as Bacillus cereus (19), Bpolymyxa (12), B licheniformis (10), B circulans (7), B subtilis (5), B laterosporus (4) and B coagulans (2). B cereus (32.2%) was found to be the predominant organism.     

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。     

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.     

Influence of refrigeration and carbon dioxide addition to raw milk on microbial levels, free monosaccharides and myo-inositol content of raw and pasteurized milk

The influence of CO₂ treatment on free monosaccharides and myo-inositol in raw and pasteurized milk during cold storage was studied. Pasteurization did not cause significant changes in the monosaccharide fraction. No variations in the level of galactose and myo-inositol in untreated and CO₂-treated samples were observed during cold storage. The content of glucose decreased considerably during cold storage due to bacterial growth in pasteurized milk. During cold storage of pasteurized milk no changes in N-acetylgalactosamine were observed, whereas N-acetylglucosamine decreased considerably after 15 days. No differences between untreated and CO₂-treated milks were found. A substantial decrease in N-acetylglucosamine and a gradual increase in N-acetylgalactosamine were observed in raw milk during cold storage. The former was attributed to consumption of this hexosamine by microorganisms and the latter was probably due to microbial glycosidic enzymes. The addition of CO₂ to raw milk proved to be a useful treatment for milk preservation without modifying the free monosaccharide fraction. Received: 4 October 1999     

Incidence, source and some properties of psychrotrophic Bacillus spp found in raw and pasteurized milk

Spores of psychrotrophic Bacillus spp were isolated from 58% of farm bulk tank milks and about 69% of pasteurized milks. Counts of Bacillus spp in about 10% of raw milk samples reached 1 × 10⁵ cfu/ml and above within seven days at 6°C. Psychrotrophic spore counts in pasteurized milks ranged from <0.5 to 170 spores/litre with an average of about 17/1. There was little correlation between the total bacterial count of the raw milk and presence of psychrotrophic Bacillus spores. There was some evidence that the bulk tank itself may be a source of contamination. The spores in pasteurized milk probably were not the result of postpasteurization contamination. The optimum germination temperature for psychrotrophic Bacillus spores was lower than that for spores of mesophilic strains. About 50% of the psychrotrophic Bacillus strains isolated from milk were capable of growth at 2°C.     

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.     

Factors influencing the recovery of psychrotrophic, mesophilic and thermophilic Bacillus spp from bulk raw milk

Because of the wide range of physiological properties found in the genus Bacillus one of the main problems in the standard procedure for isolating these organisms from milk and dairy products is the difficulty in defining conditions which are suitable for the activation and outgrowth of all spores present. This study investigated the effects of various raw milk heat treatments, the addition of L-alanine (a nutrient encouraging germination) to the milk after heat treatment, different incubation temperature-time combinations and two confirmatory media on the recovery of psychrotrophic, mesophilic and thermophilic Bacillus spp occurring in raw bulked milk. There was no significant difference (p >0.05) between the confirmatory media used, ie, starch milk agar and milk plate count agar. However, it was found that heat treatment, addition of L-alanine and the incubation period had significant effects on the recovery of the organisms from raw milk. The highest numbers of psychrotrophic and mesophilic spores were recovered after a heat treatment of 80C for 10 min followed by the addition of L-alanine to the milk at 0.1% wlv, and incubating at 6.5°C for 15 days for the former and 30C for 15 days for the latter. The highest numbers of thermophilic spores were recovered from raw milk by a heat treatment of 80C for 30 min, addition of L-alanine and incubating at 55°C for 7 days.     

Spoilage microbiota associated to the storage of raw meat in different conditions

The spoilage of raw meat is mainly due to undesired microbial development in meat during storage. The type of bacteria and their loads depend on the initial meat contamination and on the specific storage conditions that can influence the development of different spoilage-related microbial populations thus affecting the type and rate of the spoilage process. This review focuses on the composition of raw meat spoilage microbiota and the influence of storage conditions such as temperature, packaging atmosphere and use of different preservatives on the bacterial diversity developing in raw meat. In addition, the most recent tools used for the detection and identification of meat microbiota are also reviewed.     

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.     

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。      

Rapid estimation of psychrotrophic and proteolytic bacterial counts from total bacterial counts in cooled raw milk

Cooled raw bulk milk samples were examined for total bacterial count after 3 days at 30°C, and the numbers of psychrotrophs and proteolytic psychrotrophs after 10 days at 7°C. Positive correlations were found between the total bacterial count and the numbers of psychrotrophs, enumerated on standard plate count (SPC) agar (r = 0.88, n = 65), and between the numbers of psychrotrophs counted on SPC agar and on milk agar, respectively (r = 0.89, n = 59). Only 30.4% of the bacteria were psychrotrophs but the regression shows that the percentage of psychrotrophs increases when the total bacterial count rises. On average only small differences were found between the counts of psychrotrophs on SPC agar and milk agar, suggesting that in most samples the same organisms were probably enumerated on both media. The numbers of proteolytic bacteria were less well correlated with the numbers of psychrotrophs (r = 0.65, n = 56), as wide variations occurred between the samples. It is suggested that the total bacterial count at 30°C can be a useful estimate of the number of psychrotrophs.