Microbiological safety of UHT milk treated at 120 °C for 2 s,as estimated from the distribution of high-heat-resistant Bacillus cereus in dairy environments

Unlike common ultra-high-temperature (UHT) milk, so-called “UHT milk” in Japan is typically pasteurised at 120–130 °C for 2 s and distributed at 10 °C or less, and there is a potential risk of Bacillus cereus. To estimate the microbiological safety of UHT milk, we surveyed the distribution of high-heat-resistant B. cereus strains (defined as showing <∼3 log reduction after treatment at 120 °C for 2 s) among 200 isolates from dairy environments. Only four strains, which were isolated from the milk plant environment, showed high-heat resistance. All of them were unable to grow at 10 °C but grew at 12 °C. In contrast, heat-labile strains grew well at 10 °C. Therefore, UHT milk pasteurised at 120 °C for 2 s can be microbiologically safe, provided it is kept at 10 °C or less, within a rational shelf-life and avoiding contamination with B. cereus, especially of milk-plant-environment origin.     

The diversity and proteolytic properties of psychrotrophic bacteria in raw cows' milk from North China

Most psychrotrophic bacteria have the ability to produce thermoresistant proteases that can destroy the quality of milk and dairy products. To investigate the population dynamics of psychrotrophic bacteria during refrigeration, three raw cows' milk samples (sample A comprising milk from 10 farms in Beijing, sample B comprising milk from 5 farms in Heihe, and sample C comprising milk from 7 farms in Harbin) were refrigerated at 0–5 °C and 5–10 °C. PCR-DGGE (Polymerase chain reaction-denaturing gradient gel electrophoresis) analysis revealed that the bacterial community profiles varied from geographical site to site, and with refrigeration temperature. The dominant psychrotrophic bacteria among the samples after storage were affiliated with the order Pseudomonadales. Following isolation and identification, 8 psychrotrophic isolates were selected as stronger protease producers and their growth and proteolytic activities were assessed. The results indicate that the composition of psychrotrophic bacteria play an important role in the determination of the quality of milk and dairy products.     

Control of Yersinia enterocolitica in raw pork and pork products by γ-irradiation

γ-Radiation response of Y. enterocolitica 5692 and 152 was studied at 0°C and at −40°C in phosphate buffer (pH 7.00) as well as in 10% raw meat/salami homogenate. The strains investigated did not differ in their response and were found to be sensitive to γ-radiation but exhibited a tailing phenomenon in the survival curve. The D₁₀ in homogenate was 0.25 kGy at 0°C. This response was not affected at −40°C. Storage studies of packs, inoculated artificially with heavy inoculum of Y. enterocolitica (10⁶ cfu/g) showed that while samples of salami and cooked ham could be decontaminated at doses of 4 and 3 kGy respectively; cells could not be eliminated from raw pork meat even at the higher dose of 6 kGy. The role of different treatments given prior to irradiation for revival of Y. enterocolitica after irradiation storage was also studied. The dose of 1 kGy at −40°C was efficient in eradicating low numbers (<10³) of naturally occuring Y. enterocolitica from raw pork meat without any revival during storage at refrigeration temperature.     

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.     

Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions,seasonality and their spoilage potential

Refrigerated storage of raw milk promotes the growth of psychrotrophic bacteria, some of which produce heat-stable exoenzymes causing dairy product spoilage. The effects of storage conditions and season on the biodiversity of psychrotrophs in raw milk were examined using matrix-assisted laser desorption time of flight mass spectrometry and 16S rRNA analysis. The ability of psychrotrophs to produce protease, lipase and phospholipase C was determined. The predominant genera found were Pseudomonas (19.9%), Bacillus (13.3%), Microbacterium (5.3%), Lactococcus (8.6%), Acinetobacter (4.9%) and Hafnia (2.8%); a considerable number of isolates were hitherto unknown species and genera. Diversity varied significantly (P < 0.05), depending on the storage temperature, time, initial microbiota and season. The predominant isolates showed significantly higher heat-stable exoenzyme activities after heating at 142 °C for 4 s. Improving the quality of milk products may require differential processing of raw milk depending on the type of microbiota present, storage temperature and seasonality.     

Incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia

The purpose of this study was to evaluate the incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia. The distribution of Bacillus species in raw milk, pasteurised milk and UHT milk were 47.5%, 27.5% and 25%, respectively. Seven Bacillus species, including Bacillus pumilus (10%), Bacillus subtilis (12.5%), Brevibacillus brevis (10%), Bacillus cereus (22.5%), Bacillus sphaericus (7.5%), Bacillus licheniformis (12.5%) and Bacillus sporothermodurans (25%) were identified in different milk samples. Bacillus cereus was predominant in raw and pasteurised milk. Although B. sporothermodurans was the predominant sporogenous micro‐organism in UHT milk, B. cereus, B. sphaericus and B. licheniformis were also present. This study showed that there is a high degree of diversity, both phenotypic and genotypic, among Bacillus isolates from Tunisian milk and the persistence of spoilage risk in UHT milk.     

A survey of foodborne pathogens in bulk tank milk and raw milk consumption among farm families in pennsylvania

A 2-part study was conducted to determine the risk of exposure to human pathogens from raw milk. The first part of the study focused on determining raw milk consumption habits of dairy producers. A total of 248 dairy producers from 16 counties in Pennsylvania were surveyed. Overall, 105 (42.3%) of the 248 dairy producers consumed raw milk and 170 (68.5%) of the 248 dairy producers were aware of foodborne pathogens in raw milk. Dairy producers who were not aware of foodborne pathogens in raw milk were 2-fold more likely to consume raw milk compared with dairy producers who were aware of foodborne pathogens. The majority of dairy producers who consumed raw milk indicated that taste (72%) and convenience (60%) were the primary factors for consuming raw milk. Dairy producers who resided on the dairy farm were nearly 3-fold more likely to consume raw milk compared with those who lived elsewhere. In the second part of the study, bulk tank milk from the 248 participating dairy herds was examined for foodborne pathogens. Campylobacter jejuni (2%), Shiga toxin-producing Escherichia coli (2.4%), Listeria monocytogenes (2.8%), Salmonella (6%), and Yersinia enterocolitica (1.2%) were detected in the milk samples. Salmonella isolates were identified as S. enterica serotype Typhimurium (n = 10) and S. enterica serotype Newport (n = 5). Of the 248 bulk tank milk samples, 32 (13%) contained ≥1 species of bacterial pathogens. The findings of the study could assist in developing farm community-based educational programs on the risks of consuming raw milk.     

Characterization of Yersinia enterocolitica and related species isolated from foods and porcine tonsils in the Netherlands

Yersinia enterocolitica and related species were isolated from a great variety of foods. High isolation rates were found in raw minced pork and beef, porcine tonsils and tongues, egg products, raw vegetables and surface water. Of 310 strains isolated 221 (71%) were identified as Y. enterocolitica and 89 as either Y. frederiksenii (12%), Y. intermedia (8%), Y. kristensenii (5%) or other Yersinia species (4%). The great majority (92%) of these strains were of biotype 1. Only 4% of the Yersinia isolates belonged to serotype 0 : 3 or 0 : 9, which are the main serotypes isolated from patients with yersiniosis in The Netherlands. Based on the results of an autoagglutination virulence assay and a test for calcium dependency at 37°C only the 0 : 3 and 0 : 9 strains isolated from porcine tonsils were potentially virulent. Most of the strains were isolated using cold enrichment in phosphate-buffered saline containing sorbitol and bile salts. However, the potentially virulent strains were all isolated using modified Rappaport broth.     

Development of an improved extraction and HPLC method for the measurement of ascorbic acid in cows' milk from processing plants and retail outlets

An improved extraction (2.5% HPO₃, 5 mm dithiothreitol) and HPLC quantification methodology using a C–18 column at 35 °C and 0.1 m acetic acid (98%) and acetonitrile (2%) mobile phase was developed to quantify total ascorbic acid (AA) in commercial whole/semi‐skim/skim raw/pasteurised/UHT milk packaged in opaque bags, transparent plastic, cardboard and Tetra Brik^?. AA content ranged from 0.21 to 10 and from 3.4 to 16 mg L^?1 in milk from retail outlets and processing plants, respectively, and was higher in organic milk. For same processor/lot samples, pasteurised milk showed higher AA content than UHT milk. This was not true for retail outlets samples. AA content was similar for whole/semi‐skim and semi‐skim/skim milk, but not for whole/skim comparisons. Among UHT samples, the AA content trend was whole<semi‐skim<skim and lower for UHT milk in opaque plastic and Tetra Brik^? container. After 14 days at 4 °C in the dark, AA losses ranged 35–83% depending on milk type and preservation method with a higher AA retention in unopened containers.     

Detection and prevalence of pathogenic Yersinia enterocolitica in refrigerated and frozen dairy products by duplex PCR and dot hybridization targeting the virF and ail genes

Pathogenic Yersinia enterocolitica is involved in yersiniosis through expression of chromosome-borne or plasmid-borne virulence factors. Yersinia enterocolitica is a cold-tolerant pathogen frequently isolated from refrigerated or frozen foods. However, little attention has been focused on the prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy samples in China. In this study, we developed a new duplex PCR targeting the plasmid-borne virF gene and chromosome-borne ail gene for detection of pathogenic Y. enterocolitica isolates. We established a detection limit for the duplex PCR of 6.5 × 10² cfu/mL in artificially contaminated dairy samples. In addition, the duplex PCR could detect directly 4.5 to 5.7 cfu of Y. enterocolitica in 5 mL of brain heart infusion broth after 6 h of enrichment at 28°C. A newly developed dot hybridization assay further confirmed specificity of the duplex PCR for detection of virulent Y. enterocolitica. Furthermore, 13 Y. enterocolitica and 5 pathogenic strains, from 88 commercial frozen or refrigerated dairy products, were detected successfully by the China National Standard method (GB/T4789.8-2008) and the duplex PCR, respectively. Finally, biotypes and serotypes of pathogenic Y. enterocolitica strains were further characterized. The duplex PCR developed here is reliable for large-scale screening, routine monitoring, and risk assessment of pathogenic Y. enterocolitica in refrigerated or frozen dairy products.     

Molecular characterisation,genotyping and survival of Aeromonas hydrophila isolated from milk,dairy products and humans in Egypt

Two Aeromonas species, Aeromonas hydrophila and Aeromonas sobria, were isolated from raw milk (8% and 5.3% of samples tested, respectively), yoghurt (12% and 8% of samples tested, respectively) and cheese (4% and 2% of samples tested, respectively). Only A. hydrophila was isolated from human stool samples (18.8% of samples tested). Aerolysin and haemolysin associated genes were characterised in 12 and 3 isolates, respectively, while both genes were identified simultaneously in 9 isolates. Genotyping of the isolates by random amplified polymorphic DNA (RAPD) PCR revealed a high discriminatory index (D = 0.966). The storage of yoghurt samples inoculated with A. hydrophila showed the ability of the bacteria to survive for 14 days, resembling the shelf-life of yoghurt at 4 °C and 12 °C. To the best of our knowledge, this is the first study to assess the survival of A. hydrophila in yoghurt at refrigeration temperature.     

Effect of packaging atmosphere and pH on the virulence and growth ofYersinia enterocoliticaon pork stored at 4°C

Growth and virulence of pathogenicYersinia enterocoliticawere investigated on high (pH>6.0) and normal (pH<5.8) pH pork packaged in modified atmospheres and stored at 4°C. Modified atmospheres used in the study were vacuum packaging and saturated CO₂. Pork was packaged in a high gas barrier packaging film and examined over a 30-day period. Phenotypic characteristics were used to detect the presence of the virulence plasmid ofY. enterocoliticaafter exposure to the pork packaging and storage regimen. Phenotypic characteristics ofY. enterocoliticaisolates from pork loin stored at 4°C for 30 days that were studied included Congo red uptake, calcium dependence and autoagglutination in methyl red Voges–Proskauer broth and tissue culture medium. Numbers ofY. enterocoliticaon the lean surface of high pH pork slices increased approximately 2.7logcfucm⁻²when vacuum packaged and stored at 4°C for 30 days. Storage of inoculated normal pH pork in 100% CO₂resulted inY. enterocoliticaremaining in the lag phase over the storage period. Virulence ofY. enterocoliticawas maintained in 25 to 35% of isolates following storage for 30 days at 4°C in vacuum- and CO₂-packaged meats and was not affected by pH of the pork loin.     

Toxin production potential and the detection of toxin genes among strains of the Bacillus cereus group isolated along the dairy production chain

Isolates of the Bacillus cereus group (396 in total) from farms, silo tanks and production lines for pasteurised milk were tested for toxin production potential, and by polymerase chain reaction (PCR) for the presence of toxin genes. Comparison between the tests indicated the presence of gene polymorphisms. Highly toxigenic strains, based on production of subunit A of the nonhemolytic enterotoxin, NHE (NheA) and subunit C of the haemolytic enterotoxin, HBL (HblC), were less common among dairy isolates compared with farm and silo isolates. No producer of high levels of both toxins was found among 156 psychrotrophic dairy isolates (B. weihenstephanensis) and only 3% of all psychrotrophs were high producers of NheA. Psychrotrophic B. cereus from pasteurised milk appeared to have a low enterotoxin production potential, and they were not producers of emetic toxin or cytotoxin K and therefore may be less likely to cause illness than mesophilic strains.     

The immunological consequences of pasteurisation: Comparison of the response of human intestinally-derived cells to raw versus pasteurised milk

Microarray technology was used to identify differentially expressed biological signatures in human intestinal cell line (HT-29) exposed to raw versus pasteurised milk; 1041 differentially expressed genes (≥1.3 fold change) were identified (P < 0.001) between exposure groups. These were further identified to be contained within 179 gene ontologies. Genes more highly expressed (599 or 57.5%) in cells exposed to raw milk were predominately contained within immune-based gene ontologies. In contrast, genes showing lower expression in raw milk treated cells (442 or 42.5%) in comparison with pasteurised milk were involved in a broader range of cellular functions. The lowered immune function identified in cells exposed to pasteurised milk was intriguing and suggests that raw milk may be capable of inducing certain aspects of the immune system, including processes involved in T and B cell function/development. The results may indicate an alteration in the immunomodulatory potential of milk following pasteurisation.     

The prevalence,genetic diversity and antibiotic resistance of Staphylococcus aureus in milk,whey, and cheese from artisan farm dairies

In this study, coagulase positive staphylococci were detected in 45% of the 69 bovine milk, whey and cheese samples taken from five farm dairies, and all raw milk samples were contaminated. Genetic diversity, staphylococcal enterotoxin genes and antimicrobial susceptibility in putative Staphylococcus aureus isolates were investigated. Sixty-one percent of the 72 isolates analysed belonged to the same pulsed field gel electrophoresis group. The spa-typing revealed seven different spa types, t2678 being the most prevalent, but t127 and t197 were also detected. Sixteen different toxin gene profiles were identified in 87.5% of the isolates with sec and tst being the most frequent (52.5%), followed by seg and seh. All isolates were methicillin-sensitive S. aureus, and sensitive to the 12 antibiotics tested. The prevalence of S. aureus and the high diversity of isolates carrying enterotoxin genes constitute grounds for food safety concern in artisanal cheese making, whether pasteurised or not.     

Cathepsin D activity in quarg

Quarg cheese was produced from raw skim milk, pasteurised skim milk, raw skim milk with rennet added and ultrafiltrated raw skim milk. Quarg was also produced from raw skim milk with pepstatin added at curd cutting and from ultrafiltration retentate of raw milk with added pepstatin. No starter bacteria were used in this model system, with the reduction of pH being achieved by addition of glucono- δ-lactone. Yields ranged between 20.25 and 23.5%, with protein levels of 13.6–15.7%. Proteolysis occurred during storage of all experimental cheese samples for 3 m at 8°C. By immunoblotting using antibodies against bovine cathepsin D, immunoreactive procathepsin D was identified in all cheese samples. Presence of cathepsin D or procathepsin D-derived activity was confirmed by a specific enzyme assay in all samples, except those which contained pepstatin. Inhibition of cathepsin D-catalysed proteolysis by pepstatin was observed in chromatograms of water-soluble extracts analysed by reverse-phase HPLC. Peptides thought to be produced as a result of cathepsin D activity were observed in cheese made from both raw and pasteurised milk, suggesting that the activity at least partially survived pasteurisation.     

Development of a multiplex and semi-nested PCR assay for detection of Yersinia enterocolitica and Aeromonas hydrophila in raw milk

Single-locus (sl), multiplex (m), and semi-nested (sn) polymerase chain reaction (PCR) procedures were developed for the detection of Yersinia enterocolitica and Aeromonas hydrophila in raw milk samples. Two oligonucleotide primers for each pathogen were used in PCR, to detect the yst gene of Y. enterocolitica and aer gene of A. hydrophila. The amplified fragment size was 145 base-pair (bp) for yst gene, and 209 bp for the aer gene. The performance of the systems were evaluated with seeded milk samples, and naturally contaminated raw milk samples. PCR results were compared with conventional cultural procedures. The limits of detection of slPCR and mPCR assays were approximately 10² cfu ml⁻¹ (0·5 cfu/PCR reaction mixture) for both pathogens in seeded raw milk. When studied with naturally contaminated raw milk samples, detection rates obtained by PCR and cultural methods were 53% and 36% for Y. enterocolitica and were 23% and 14% for A. hydrophila, respectively. These results indicate that the direct PCR analysis of raw milk can be used as a rapid and specific diagnostic method for both pathogens.     

Microbiological and biochemical characteristics of Kashkaval cheese produced using pasteurised or raw milk

Microbiological quality and biochemical changes of Kashkaval cheese manufactured using sheep's raw milk without starter addition or pasteurised milk with an added commercial starter were studied. Mature cheeses had pH values 5.0–5.3, salt content 2.1–2.7%, protein content 23.3–25.1%, moisture content 36.8–39.5%, fat content 28.0–32.2%, and ash content around 5.0%. In raw milk cheeses, mesophilic non-starter lactobacilli prevailed followed by enterococci. In pasteurised milk cheeses Lactococcus lactis starter prevailed. All cheeses were safe according to the criteria in Regulation (EC) 1441/2007. The proteolysis index was around 20%. Butyric, myristic, palmitic, stearic and oleic were the principal free fatty acids in both cheeses. Ketones were abundant in pasteurised milk cheeses and esters in mature raw milk cheeses. Pasteurisation did not affect (P > 0.05) the physicochemical composition and the proteolysis of cheeses. Raw milk cheeses showed higher levels (P < 0.05) of lipolysis than pasteurised milk cheeses.     

Are we closer to understanding why viable cells of Mycobacterium avium subsp. paratuberculosis are still being reported in pasteurised milk?

Mycobacterium avium subsp. paratuberculosis (MAP) continues to be associated with Crohn’s disease. Following work in the 1990s that suggested that statutory pasteurisation of milk (72 °C, 15 s) was insufficient to destroy MAP, the UK Dairy Industry increased the holding time to 25 s. Since then, some plants have increased the lethality of pasteurisation further with a number using 78 °C for 27 s. Despite the increase in lethality, a recent survey of pasteurised milk in England found that 10.3% of pasteurised milk samples tested positive for viable MAP. This article discusses the significance of MAP and why viable MAP might be found in pasteurised milk.     

Occurrence of Listeria species in raw milk in farms on the outskirts of Mexico city

Listerosis may be transmitted by direct contact with infected animals or by consumption of contaminated vegetables or meat and milk products. In Mexico, raw milk is widely consumed and the incidence of milkborne disease is unknown. A total of 1300 raw milk samples were obtained from 20 l bulk tanks at four different dairy farms in southeast of Mexico City from June 1998 to June 1999. The samples were enriched for 48 h at 30°C and plated onto McBride's Modified Agar (MMA). Suspect colonies were biochemically tested to confirm identity. Overall, 23% of all raw milk samples examined tested positive forListeria species; 13% were positive for L. monocytogenes (45·6% were serotype-4b and 54·4% were serotype 1); 6% for L. ivanovii; 4% for L. seeligeri and 1% forL. innocua. L. monocytogenes contamination was more frequent during the spring and summer months as isolation rates were 12·2% from June to October 1998 and 17% from March to June 1999. Serotype-4b isolates were not pathogenic for the mouse, while for serotype-1, strains DL₅₀ranged from 1·8×10⁶to 4×10⁷CFU ml⁻¹. Additional studies are needed to assess the public health impact of contaminated milk in Mexico.