Psychrotrophic bacterial flora of raw ewes'milk, with particular reference to Gram negative rods

The microbial flora of 141 samples of raw ewes'milk was determined, before and after storage for 72 h at 4° and 7°C. Penicillin-resistant bacteria represented ca 61% of 1760 psychrotrophic isolates from refrigerated milk samples. Pseudomonas fluorescens and Pseudomonas fluorescent group-related strains predominated ( ca 86%) in the Gram negative psychrotrophic microflora. Leuconostoc dextranicum was the most frequent Gram positive psychrotrophic species isolated.     

Psychrotrophic strains of Bacillus cereus producing enterotoxin

In investigations on three outbreaks of Bacillus cereus food poisoning in Spain and The Netherlands, the causative strains grew within a temperature range of 4-37 degrees C, but not at 43 degrees C. Such psychrotrophic types were found to occur in various dairy products (including ca 25% of 35 samples of pasteurized milk) and some mousses and cook/chill meals. Growth of and enterotoxin production by psychrotrophic B. cereus could be prevented by temperatures below 4 degrees C and pH-values not exceeding 5.0.     

A numerical taxonomic study of psychrotrophic bacteria associated with lipolytic spoilage of raw milk

Raw milk samples were stored for 1-4 d and examined for bacterial growth and lipase activity. Thirty-six samples in which an increase in the heat-stable lipase activity was observed during storage were selected for further study. From these raw milk samples 205 lipolytic psychrotrophic strains were selected using butterfat agar and subsequently characterized with 86 taxonomic tests. Complete linkage cluster analysis of the taxonomic data produced two major and six minor clusters at the 83% similarity level. Pseudomonas fluorescens and Ps. fragi accounted for 63.9 and 31.2%, respectively, of the isolates.     

Prediction of the keeping quality of pasteurized milk by the detection of cytochrome c oxidase

The keeping quality (KQ) of pasteurized milk samples stored at 5 degrees C and 10 degrees C was satisfactorily predicted after 18 h pre-incubation with 0.05% benzalkonium chloride at 20 degrees C, by estimating the numbers of Gram-negative psychrotrophic bacteria using the simple, cheap and rapid (5 min) assay of cytochrome c oxidase. Correlation coefficients for the relationship between cytochrome c oxidase activity at 20 degrees C and KQ at 5 degrees C or 10 degrees C of -0.89 and -0.84 respectively were obtained. The method correctly predicted the KQ of more than 89% of the samples of pasteurized milk. The assay was not satisfactory for use on samples after pre-incubation at 30 degrees C.     

A sandwich enzyme-linked immunosorbent assay (ELISA) for detection of Pseudomcnas fluorescens and related psychrotrophic bacteria in refrigerated milk

I. GONZÁLEZ, R. MARTÍN, T. GARCÍA, P. MORALES, B. SANZ AND P.E. HERNÁNDEZ. 1993. A sandwich ELISA (enz***me-linked immunosorbent assay) was developed for detection of Pseudomonas fluorescens and related psychrotrophic bacteria in refrigerated milk. Polyclonal antibodies were raised in rabbits against protein F from the cell envelope of Pseudomonas fluorescens AH-70. The anti-protein F antibodies (anti-PF) bound to the wells of a microtitre plate were used to capture this protein from the micro-organisms on milk samples. Further immunorecognition of the captured antigen was attained with the same anti-PF antibodies conjugated to biotin. ExtrAvidin-peroxidase was used to detect the biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of substrate gave clear absorbance differences when assaying milk samples containing Ps. fluorescens strains of different origin as well as related psychrotrophic micro-organisms.     

A numerical taxonomic study of psychrotrophic bacteria associated with lipolytic spoilage of raw milk

Raw milk samples were stored for 1–4 d and examined for bacterial growth and lipase activity. Thirty-six samples in which an increase in the heat-stable lipase activity was observed during storage were selected for further study. From these raw milk samples 205 lipolytic psychrotrophic strains were selected using butterfat agar and subsequently characterized with 86 taxonomic tests. Complete linkage cluster analysis of the taxonomic data produced two major and six minor clusters at the 83% similarity level. Pseudomonas fluorescens and Ps. fragi accounted for 63.9 and 31.2%, respectively, of the isolates.     

Psychrotrophic strains of Bacillus cereus producing enterotoxin

In investigations on three outbreaks of Bacillus cereus food poisoning in Spain and The Netherlands, the causative strains grew within a temperature range of 4·37†C, but not at 43†C. Such psychrotrophic types were found to occur in various dairy products (including ca 25% of 35 samples of pasteurized milk) and some mousses and cook/chill meals. Growth of and enterotoxin production by psychrotrophic B . cereus could be prevented by temperatures below 4†C and pH-values not exceeding 5·0.     

Identification of heat stable protease of Klebsiella oxytoca isolated from raw milk

AIMS: The aim of this study was to identify and characterize heat stable proteinases of psychrotrophic proteolytic bacteria isolated from raw milk. METHODS AND RESULTS: A strain of Klebsiella oxytoca producing a high proteolytic activity when cultured on milk was isolated. Maximum proteolytic activity was observed at the stationary phase during growth on milk or casein-peptone broth. The bacterium demonstrated the capability to grow at 7 degrees C, classified as psychrotrophic. The crude enzyme showed optimum activity at 37 degrees C, and pH 5.0 and 7.0. The proteinase was very resistant to heat, maintaining 74% of initial activity after incubation at 142 degrees C. CONCLUSIONS: A heat stable protease of a psychrotrophic strain of K. oxytoca was identified and partially characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: Thermal stable proteases may constitute a serious problem to ultra-high temperature (UHT) processed milk, leading to undesirable physical and sensory alterations.     

A numerical taxonomic study of proteolytic and lipolytic psychrotrophs isolated from caprine milk

Lipolytic and proteolytic psychrotrophs were isolated from raw and pasteurized goats'milk, which had been stored at 5°C for 7d. The 241 strains isolated and 20 reference strains were examined by 149 biochemical, physiological, and morphological tests. The results yielded 195 characters suitable for taxonomic analysis. Computer-assisted complete linkage analysis, using the Jaccard coefficient, produced 22 phenons at 75% S. The results showed that Pseudomonas fluorescens was the predominant psychrotrophic bacterium, but that Pseudomonas fragi was dominant in some milk samples. Strains of Serratia liquefaciens and Flavobacterium balustinum were also identified.     

Microbiological quality of raw milk produced in Estonia

Aims: The microbial quality of farm bulk‐tank raw milk produced in Estonia during years 2004–2007 was investigated. Methods and Results: Bulk‐tank milk samples were analysed for lactic acid bacteria count (LABC), psychrotrophic bacteria count (PBC), aerobic spore‐forming bacteria count (ASFBC), total bacterial counts using BactoScan and somatic cell count (SCC) using Fossomatic. Randomly selected psychrotrophic isolates were subjected to 16S–23S PCR‐ribotyping. LABC remained below 10⁴ CFU ml^?1 in most samples, while psychrotrophic micro‐organisms dominated in 60% of farms. PBC ranged from 4·2 × 10² to 6·4 × 10⁴ CFU ml^?1, and ASFBC varied from 5 to 836 CFU ml^?1. Conclusions: In general, the microbiological quality of the farm bulk‐tank milk was good – more than 91% of samples contained <50 000 CFU ml^?1, and SCC in the majority of samples did not exceed the internationally recommended limits. Genus Pseudomonas spp. was the dominating spoilage flora with Pseudomonas fluorescens as the prevailing species. Significance and Impact of the Study: Specific bacterial groups (LABC, PBC and ASFBC), not analysed routinely by dairies, were determined in bulk‐tank raw milk of numerous dairy farms during 4‐year period. Based on the survey, dairy plants can better control their supply chains and select farms (milk) for the production of specific products, i.e. milk with low PBC and high LABC for cheesemaking.     

Use of the Direct Epifluorescent Filter Technique for predicting the keeping quality of pasteurized milk within 24 hours

The keeping quality (KQ) of pasteurized milk stored at 5 degrees C and 11 degrees C was predicted within 24 h by pre-incubating samples and counting bacteria by the Direct Epifluorescent Filter Technique (DEFT). For samples from 5 degrees C storage, 0.03% (w/v) benzalkonium chloride and 0.002% (w/v) crystal violet (final concentration) were added to inhibit the growth of Gram positive bacteria during pre-incubation. The samples from milk stored at 11 degrees C were pre-incubated without the addition of inhibitors. After pre-incubation there was a satisfactory relationship between the DEFT count and the KQ of milks at both 5 degrees C and 11 degrees C. The DEFT count following pre-incubation correctly classified greater than 80% of pasteurized milks on the basis of KQ.     

Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay

Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detection of these enzymes in milk and dairy products. In reconstituted skim milk cultures proteinase P1 was detectable when the CFU approached 10(7)/ml. Cultures in milk diluted 1:10, 1:30, or 1:100 with water showed detectable proteinase at population densities close to 10(6) CFU/ml. Aeration stimulated proteinase production; thus, a skim milk culture with shaking at 5 degrees C had a proteinase level of 36,000 ng/ml after 7 days as compared to 80 ng/ml in a stationary culture. The rate of inactivation of proteinase P1 at 150 and 55 degrees C as expressed by residual antigenic activity determined by the enzyme-linked immunosorbent assay was somewhat different from the rate determined on the basis of residual proteolytic activity. The specificity of the enzyme-linked immunosorbent assay with proteinase P1 antibodies was identical for proteinase P1 and for enzymes from six other strains of P. fluorescens, one Chromobacterium strain, and one Flavobacterium strain. Some psychrotrophic strains produced immunologically unrelated proteinase(s). These preliminary observations indicate that proteinase P1-related enzymes are common among psychrotrophs appearing as spoilage bacteria in milk.     

Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay.

Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detection of these enzymes in milk and dairy products. In reconstituted skim milk cultures proteinase P1 was detectable when the CFU approached 10(7)/ml. Cultures in milk diluted 1:10, 1:30, or 1:100 with water showed detectable proteinase at population densities close to 10(6) CFU/ml. Aeration stimulated proteinase production; thus, a skim milk culture with shaking at 5 degrees C had a proteinase level of 36,000 ng/ml after 7 days as compared to 80 ng/ml in a stationary culture. The rate of inactivation of proteinase P1 at 150 and 55 degrees C as expressed by residual antigenic activity determined by the enzyme-linked immunosorbent assay was somewhat different from the rate determined on the basis of residual proteolytic activity. The specificity of the enzyme-linked immunosorbent assay with proteinase P1 antibodies was identical for proteinase P1 and for enzymes from six other strains of P. fluorescens, one Chromobacterium strain, and one Flavobacterium strain. Some psychrotrophic strains produced immunologically unrelated proteinase(s). These preliminary observations indicate that proteinase P1-related enzymes are common among psychrotrophs appearing as spoilage bacteria in milk.     

Raw cow milk bacterial population shifts attributable to refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.     

Culturable psychrotrophic bacterial communities in raw milk and their proteolytic and lipolytic traits

During cold storage after milk collection, psychrotrophic bacterial populations dominate the microflora, and their extracellular enzymes, mainly proteases and lipases, contribute to the spoilage of dairy products. The diversity, dynamics, and enzymatic traits of culturable psychrotrophs in raw milk from four farms were investigated over a 10-month period. About 20% of the isolates were found to be novel species, indicating that there is still much to be learned about culturable psychrotrophs in raw milk. The psychrotrophic isolates were identified and classified in seven classes. Three classes were predominant, with high species richness (18 to 21 species per class) in different seasons of the year: Gammaproteobacteria in spring and winter, Bacilli in summer, and Actinobacteria in autumn. The four minor classes were Alphaproteobacteria, Betaproteobacteria, Flavobacteria, and Sphingobacteria. The dominant classes were found in all four dairies, although every dairy had its own unique "bacterial profile." Most but not all bacterial isolates had either lipolytic or both lipolytic and proteolytic activities. Only a few isolates showed proteolytic activity alone. The dominant genera, Pseudomonas and Acinetobacter (Gammaproteobacteria), showed mainly lipolytic activity, Microbacterium (Actinobacteria) was highly lipolytic and proteolytic, and the lactic acid bacteria (Lactococcus and Leuconostoc) displayed very minor enzymatic ability. Hence, the composition of psychrotrophic bacterial flora in raw milk has an important role in the determination of milk quality. Monitoring the dominant psychrotrophic species responsible for the production of heat-stable proteolytic and lipolytic enzymes offers a sensitive and efficient tool for maintaining better milk quality in the milk industry.     

Partial immunological characterization of heat-stable proteinases from Pseudomonas spp. of dairy origin

A homogeneous extracellular heat-stable proteinase from Pseudomonas fluorescens AH-70 was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relationship of the proteinases from 26 psychrotrophic Pseudomonas spp. isolated from raw milk. The inhibition of the proteinases by the antiserum, the gel precipitin reactions and the use of a double-antibody sandwich ELISA, revealed that proteinase AH-70 is immunologically related to proteinases from 8/26 other Pseudomonas strains. These results also indicate that the immunological techniques for the detection of proteolytic enzymes in raw milk require antibody preparations of broader specificity.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37 degrees and 42.5 degrees C (thermotrophs) and 30 degrees C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37 degrees and 42.5 degrees C. At 42.5 degrees C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37 degrees C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42.5 degrees C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

Formation of guaiacol in chocolate milk by the psychrotrophic bacterium Rahnella aquatilis

AIMS: The aim of this study was to identify the causative agent of a smoky/phenolic taint in refrigerated full cream chocolate milk. METHODS AND RESULTS: Microbiological examination of spoiled and unspoiled milk samples from the same processor showed high numbers of the psychrotrophic coliform Rahnella aquatilis in the spoiled samples only. Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify the taint compound as guaiacol (2-methoxyphenol) in the spoiled milk. Challenge studies in UHT chocolate and white milks inoculated with the isolate and incubated at 4-5 degrees C and 8-9 degrees C for 6 d showed the production of guaiacol in chocolate milk only, which was confirmed and quantified by GC/MS. CONCLUSIONS: The results indicate that if present in refrigerated chocolate milk, Rah. aquatilis can produce guaiacol within the expected shelf-life of the product, even without temperature abuse. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that the coliform Rah. aquatilis can produce guaiacol in refrigerated chocolate milk products.     

A numerical taxonomic study of proteolytic and lipolytic psychrotrophs isolated from caprine milk

Lipolytic and proteolytic psychrotrophs were isolated from raw and pasteurized goats' milk, which had been stored at 5 degrees C for 7 d. The 241 strains isolated and 20 reference strains were examined by 149 biochemical, physiological, and morphological tests. The results yielded 195 characters suitable for taxonomic analysis. Computer-assisted complete linkage analysis, using the Jaccard coefficient, produced 22 phenons at 75% S. The results showed that Pseudomonas fluorescens was the predominant psychrotrophic bacterium, but that Pseudomonas fragi was dominant in some milk samples. Strains of Serratia liquefaciens and Flavobacterium balustinum were also identified.     

Numerical taxonomy of proteolytic psychrotrophs from Queensland raw milks

Eighty-seven proteolytic psychrotrophic micro-organisms were isolated from 11 bulk milk supplies of two Queensland factories from different climatic regions, before and after storage at 4°C for 7 d. These isolates together with 15 reference strains formed the basis of a numerical taxonomic study involving 81 attributes. All but six isolates were pseudomonads. The strains clustered into nine groups, of which one group consisted of four yeasts. One group, containing 39 isolates, was designated as Pseudomonas fluorescens biovar 1; three groups, containing 27 isolates, as Ps. fluorescens biovar 5; and one group, containing 10 isolates, as Ps. putida biovar A. This study showed that the proteolytic psychrotrophic microflora of the 11 milks supplying the two factories was substantially different and that the proteolytic flora of 7 d refrigerated milk could not be estimated by examining the flora before storage.