Genetic diversity of thermoduric spoilage microorganisms of milk from Brazilian dairy farms

When correctly pasteurized, packaged, and stored, milk with low total bacterial counts (TBC) has a longer shelf life. Therefore, microorganisms that resist heat treatments are especially important in the deterioration of pasteurized milk and in its shelf life. The aim of this work was to quantify the thermoduric microorganisms after the pasteurization of refrigerated raw milk samples with low TBC and to identify the diversity of these isolates with proteolytic or lipolytic potential by RFLP analysis. Twenty samples of raw milk were collected in bulk milk tanks shortly after milking in different Brazilian dairy farms and pasteurized. The mean thermoduric count was 3.2 (±4.7) × 10² cfu/mL (2.1% of the TBC). Of the 310 colonies obtained, 44.2% showed milk spoilage potential, 32.6% were proteolytic and lipolytic simultaneously, 31% were exclusively proteolytic, and 48 (36.4%) were only lipolytic. Regarding the diversity, 8 genera were observed (Bacillus, Brachybacterium, Enterococcus, Streptococcus, Micrococcus, Kocuria, Paenibacillus, and Macrococcus); there was a predominance of endospore-forming bacteria (50%), and Bacillus licheniformis was the most common (34.1%) species. Considering the RFLP types, it was observed that the possible clonal populations make up the microbiota of different milk samples, but the same milk samples contain microorganisms of a single species with different RFLP types. Thus, even in milk with a high microbiological quality, it is necessary to control the potential milk-deteriorating thermoduric microorganisms to avoid the risk of compromising the shelf life and technological potential of pasteurized milk.     

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.     

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。     

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。     

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.     

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。      

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.     

Aerobic spore-forming bacteria in bulk raw milk: factors influencing the numbers of psychrotrophic, mesophilic and thermophilic Bacillus spores

Psychrotrophic, mesophilic and thermophilic spore concentrations of Bacillus spp. were determined on a weekly basis in bulk raw milk samples obtained from a central processing facility, over one calendar year. These data were correlated with concentrations of metal ions, free amino acids and somatic cell counts obtained from the same samples, as well as local meteorological mean temperature and relative humidity measurements relating to the same test period. A heat treatment of 80°C for 20 min followed by the addition of L-alanine to the milk at 0.1% w/v and incubation at 55°C for 7 days gave optimal recovery conditions for thermophilic spores. Free amino acids, metal ions, somatic cell counts, temperature, and relative humidity measurements were each significantly correlated with the recovery of spores of Bacillus spp. from bulk raw milk, although no single factor was shown to demonstrate a consistent effect with the psychrotrophic, mesophilic and thermophilic spore groups studied.     

The relationship of flow cytometry results with classical measures of bacterial counts in raw refrigerated milk

Bulk milk was collected from 100 farms throughout the year and analysed after storage for either 24, 48 or 72 h, using flow cytometry. The total bacterial counts obtained by two methods – flow cytometry and standard plate count were compared and the conversion relationship between them was assessed: the results showed no effect of the age of the samples relationship between these two methods.     

Controlling psychrotrophic bacteria in raw buffalo milk preserved at 4 °C with esterified legume proteins

The activity of native and esterified legume proteins in controlling the growth of psychrotrophic bacteria contaminating raw buffalo milk kept at 4 °C as well as milk quality was followed and assessed. Supplementation of milk samples with 0.5% of methylated legume proteins has considerably reduced the levels of total bacterial count (TBC), psychrotrophic bacteria count (PBC) and Pseudomonas spp. count (PSC), during cold preservation. Raw buffalo milk samples supplemented with esterified legume proteins have reached a TBC log of 5.1-5.3 cfu ml⁻¹ surpassing the spoilage level after a relatively longer period (6 days) than in the case of non-supplemented raw milk (2 days). At the end of storage period, acidity level reached comparatively higher maximum values in the untreated raw milk (0.74 ± 0.018) compared to lower values in case of milk supplemented with esterified legume proteins (0.52 ± 0.011 to 0.55 ± 0.012). At the same time corresponding pH values of the untreated raw milk attained a lower minimum values in case of raw milk supplemented with esterified legume proteins than in non-treated raw milk. Some casein degradation started in non-treated raw milk after 2 days of preservation at 4 °C and complete casein degradation was observed after 4 days. Casein remained nearly intact in raw milk supplemented with esterified proteins for 10 days. Supplementation with esterified legume proteins has substantially delayed the coagulation response to the 8 and 10 days of preservation at 4 °C, instead of 4 days in case of the control.     

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.     

Monoclonal antibodies and an indirect ELISA for detection of psychrotrophic bacteria in refrigerated milk.

Monoclonal antibodies generated against live cells of Pseudomonas fluorescens have been used in an indirect ELISA format for the detection of Pseudomonas spp. and related psychrotrophic bacteria in refrigerated milk. The immunorecognition of monoclonal antibodies adsorbed to bacteria bound to the wells of a microtiter plate was performed with rabbit anti-mouse immunoglobulins conjugated to horseradish peroxidase. Subsequent enzymic conversion of the substrate resulted in distinct absorbance differences when assaying milk samples containing psychrotrophic bacteria in the range 10(5) to 10(9) CFU ml(-1) . The detection threshold for the ELISA assay developed in this work is 10(5) CFU ml(-1).     

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.     

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。     

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.     

原料乳中微生物的多样性

牛乳营养丰富,适合各种微生物的生长繁殖。该文介绍了原料乳中微生物的种类,综述了国内外近年来关于原料乳中微生物多样性的研究进展。并对非培养微生物学在研究原料乳中微生物多样性的应用进行了总结,高度评价了非培养方式的快速、敏感以及可定义纯培养方式无法发现的微生物的特点。