Harnessing Nature's Defence: The Antimicrobial Efficacy of Pasteurised Cattle Milk-Derived Extracellular Vesicles on Staphylococcus aureus ATCC 25923.

Increasing antimicrobial resistance (AMR) challenges conventional antibiotics, prompting the search for alternatives. Extracellular vesicles (EVs) from pasteurised cattle milk offer promise, due to their unique properties. This study investigates their efficacy against five pathogenic bacteria, including Staphylococcus aureus ATCC 25923, aiming to combat AMR and to develop new therapies. EVs were characterised and tested using various methods. Co-culture experiments with S. aureus showed significant growth inhibition, with colony-forming units decreasing from 2.4 × 105 CFU/mL (single dose) to 7.4 × 104 CFU/mL (triple doses) after 12 h. Milk EVs extended lag time (6 to 9 h) and increased generation time (2.8 to 4.8 h) dose-dependently, compared to controls. In conclusion, milk EVs exhibit dose-dependent inhibition against S. aureus, prolonging lag and generation times. Despite limitations, this suggests their potential in addressing AMR.

Survival of Mycobacterium avium subspecies paratuberculosis in retail pasteurised milk.

A survey of retail purchased semi-skimmed pasteurised milk (n = 368) for Mycobacterium avium subspecies paratuberculosis (MAP) was conducted between May 2014 and June 2015 across the midlands of England using the Phage-PCR assay. Overall, 10.3% of the total samples collected contained viable MAP cells, confirming that pasteurisation is not capable of fully eliminating human exposure to viable MAP through milk. Comparison of the results gained using the Phage-PCR assay with the results of surveys using either culture or direct PCR suggest that the phage-PCR assay is able to detect lower numbers of cells, resulting in an increase in the number of MAP-positive samples detected. Comparison of viable count and levels of MAP detected in bulk milk samples suggest that MAP is not primarily introduced into the milk by faecal contamination but rather are shed directly into the milk within the udder. In addition results detected an asymmetric distribution of MAP exists in the milk matrix prior to somatic cell lysis, indicating that the bacterial cells in naturally contaminated milk are clustered together and may primarily be located within somatic cells. These latter two results lead to the hypothesis that intracellular MAP within the somatic cells may be protected against heat inactivation during pasteurisation, accounting for the presence of low levels of MAP detected in retail milk.

Fatty acid profiling of Western Australian pasteurised milk using gas chromatography-mass spectrometry.

The fatty acid composition of Western Australian commercial pasteurised milk was profiled using gas chromatography-mass spectrometry (GC-MS). A total of 31 fatty acids (FA) were identified in the milk samples. The majority of FA were medium-chain fatty acids (MCFA) with 6-13 carbon atoms and long-chain fatty acids (LCFA) with 14-20 carbon atoms. The results of principal component analysis (PCA) showed significant differences in the levels of MCFA and LCFA in the different milk samples. The levels of MCFA and LCFA ranged from 10.09 % to 12.12% and 87.88% to 89.91% of total FA, respectively. C10:0 and C12:0 were the major components of MCFA comprising 3.46% and 4.22% of total FA, while C16:0 and C18:1 (cis 9-octadecenoic acid) represented the majority of LCFA with the levels of 26.18% and 23.34% of total FA, respectively. This study provides new insight into the FA composition of Western Australian pasteurised milk and differences in FA profiles which influence human health.

Preservation of high pressure pasteurised milk by hyperbaric storage at room temperature versus refrigeration on inoculated microorganisms, fatty acids, volatile compounds and lipid oxidation.

High pressure pasteurised (HPP) milk was stored by hyperbaric storage at room temperature (HS/RT) (50-100 MPa at 20 °C) and compared with refrigeration (RF), to assess the effect on two pathogens surrogates and a pathogenic, up to 120 days, and on fatty acids, volatile organic compounds (VOCs) and secondary lipid oxidation over 60 days. HS/RT (75-100 MPa) was able to inactivate at least 6.68/6.31/6.03 log CFU/mL of Escherichia coli/Listeria innocua/Salmonella Senftenberg (to below the detection limit), while RF resulted only in minor changes. Overall, fatty acids profile remained stable under HS/RT, although secondary lipid oxidation showed slightly higher values. In addition, both HS/RT and RF showed stable and similar VOCs profiles and off-flavour indicative compounds were not detected, except for the lowest pressure (50 MPa) after 40 days. HS/RT preserved HPP milk with enhanced microbial safety, shelf-life and quality compared to RF, being in addition quasi-energetically costless and more sustainable than RF.

Aflatoxin M1 in milk in Hisar city, Haryana, India and risk assessment.

Aflatoxin M1 (AFM1) contamination in 150 samples of milk, sold in market of Hisar city of Haryana, India, was investigated by using High Pressure Liquid Chromatography (HPLC). Out of these, 40 samples contained AFM1 at a concentration below the limit of detection (LOD) of 0.052 µg/kg. Among the AFM1 contaminated samples, 46 raw milk samples contained a concentration above the LOD but less than the limit of quantitation (LOQ), whereas 64 samples were above the LOQ. Of these samples, 31 exceeded the maximum limit of 0.5 µg/kg prescribed by FSSAI, India. Based on this study, the dietary intake of AFM1 for adults through consumption of milk was estimated. The results indicated that AFM1 contamination can be a food safety issue for raw and pasteurised milk consumed in India. Therefore, there is a need for a national monitoring programme to control the level of mycotoxins in milk.

Aflatoxin M1 Levels in Raw Milk, Pasteurised Milk and Infant Formula.

The incidence of contamination of aflatoxin M1 (AFM1) in milk samples collected from the Jordanian market was investigated by using the competitive enzyme linked immunosorbent assay (ELISA) technique. A total of 175 samples were collected during 2014-2015. All tested samples were contaminated with various levels of AFM1 ranging from 9.71 to 288.68 ng/kg. The concentration of AFM1 in 66% of fresh milk samples was higher than the maximum tolerance limit accepted by the European Union (50 ng/kg) and 23% higher than the maximum tolerance limit accepted by the US (500 ng/kg). Percentages of contaminated raw cow, sheep, goat and camel milk exceeding the European tolerance limit were 60, 85, 75 and 0%, respectively. Of AFM1 contaminated pasteurised cow milk samples, 12% exceeded the European tolerance limit with a range of contamination between 14.60 and 216.78 ng/kg. For infant formula samples, the average concentration of AFM1 was 120.26 ng/kg (range from 16.55 to 288.68 ng/kg), the concentration of AFM1 in 85% of infant formula samples was higher than the maximum tolerance limit accepted by the European Union and the US (25 ng/kg).

The changes of proteins fractions shares in milk and fermented milk drinks.

BACKGROUND: The aim of this research was to observe the changes which take place in the electrophoretic picture of milk proteins after pasteurisation and inoculation with different starter cultures (both traditional and probiotic). After incubation, the yoghurt, kefir, acidified milk, fermented Bifidobacterium bifidum drink and Lactobacillus acidophillus drink were chilled for 14 days to observe the changes which occurred. METHODS: The research materials were raw and pasteurised milk, as well as fermented milk- based drinks. The raw milk used for research came from Polish Holstein-Fresian black and white cows. The milk was sampled 3 times and divided into 5 parts, each of which was pasteurised at 95°C for 10 min and then cooled for inoculation: yoghurt to 45°C, kefir and acidified milk to 22°C and drinks with Bifidobacterium bifidum and Lactobacillus acidophillus to 38°C. Milk was inoculated with lyophilised, direct vat starter cultures, in an amount equal to 2% of the working starter. For the production of fermented drinks, the subsequent starters were applied: "YC-180" Christian Hansen for yoghurt, "D" Biolacta-Texel-Rhodia for kefir, CH-N--11 Christian Hansen for acidified milk, starter by Christian Hansen for the probiotic Bifidobacterium bifidum milk, starter by Biolacta-Texel-Rhodia for the probiotic Lactobacillus acidophillus milk. The analyses were conducted in raw, pasteurised and freshly fermented milk as well as in milk drinks stored for 14 days. The total solid content was estimated by the drying method; the fat content by the Gerber method; the lactose content by the Bertrand method; the protein content by the Kjeldahl method with Buchi apparatus; the density of milk was measured with lactodensimeter; acidity with a pH-meter; and potential acidity by Soxhlet-Henkl method (AOAC, 1990). The electrophoretic separation of proteins in raw and pasteurised milk, as well as in freshly produced milk drinks and those stored for 14 days, was performed with SDS-PAGE (on polyacrylamid gel) basing on procedure described by Laemmli (1970). RESULTS: It was shown that, in comparison with raw milk, the pasteurised milk had smaller amounts of αs-, ß- and κ-casein, whereas the shares of γ-casein and peptides were greater, and there were no changes in immunoglobulin, α-lactalbumin or ß-lactoglobulin levels, which indicated that hydrolysis of caseins had occurred. In all freshly fermented milk drinks, a drop in αs- and ß-casein was observed relative to raw milk. An increase in peptides and γ-casein was also noticed (with the exception of acidified milk). There were differences in α-lactalbumin and ß-lactoglobulin levels between the different drinks: raw, pasteurised or freshly fermented milk. It was shown that kefir, compared to the other drinks, had the lowest levels of αs- and ß-casein, α-lactalbumin and of peptides, as well as the highest level of γ-casein, which is evidence of an increased rate of hydrolysis in that drink. It was stated that, during the storage of fermented milk drinks, the levels of lactoferrin, serum albumin and peptides significantly increased whereas the content of κ-casein diminished. The proportions of serum albumin and lactoferrin in fermented milk drinks increased relative to raw milk and/or after storage, which is evidence of aggregation of proteins of low molecular mass into bigger conglomerates. CONCLUSIONS: The observed differences between fermented milks, including during chilled storage, in the amounts of individual proteins proves the different proteolytic abilities of starter cultures used in fermented milk production. α-lactoalbumin and ß-lactoglobulin are, besides caseins, the most allergenic milk proteins. So, kefir, because of its low α-lactoalbumin content, and Bifidobacterium bifidum milk, with the lowest content of ß-lactoglobulin, were the most advantageous and least allergenic drinks examined.

Detection of viable Mycobacterium avium subspecies paratuberculosis in powdered infant formula by phage-PCR and confirmed by culture.

Surveys from different parts of the world have reported that viable Mycobacterium avium subsp. paratuberculosis (MAP) can be cultured from approximately 2% of samples of retail pasteurised milk samples. Pasteurised milk is used for the production of powdered infant formula (PIF) and therefore there is a concern that MAP may also be present in these products. Several studies have previously reported the detection of MAP in PIF using PCR-based assays. However, culture-based surveys of PIF have not detected viable MAP. Here we describe a phage amplification assay coupled with PCR (page-PCR) that can rapidly detect viable MAP in PIF. The results of a small survey showed that the phage-PCR assay detected viable MAP in 13% (4/32) of PIF samples. Culture detected viable MAP in 9% (3/32) PIF samples, all of which were also phage-PCR positive. Direct IS900 PCR detected MAP DNA in 22% (7/32) of PIF samples. The presence of viable MAP in PIF indicates that MAP either survived PIF manufacturing or that post-production contamination occurred. Irrespective of the route of MAP contamination, the presence of viable MAP in PIF is a potential public health concern.

Seasonal patterns of aflatoxin M1 contamination in commercial pasteurised milk from different areas in Thailand.

Aflatoxin M1 (AFM1) levels were determined in pasteurised milk from five commercial trademarks produced in different areas in Thailand. One hundred and twenty milk samples were collected from local markets in Chiang Mai province, Thailand, to evaluate AFM1 concentrations using immunoaffinity columns and high-performance liquid chromatography with fluorescence detection. The overall median AFM1 level was 0.023 µg L(-1) ranging from 0.004 to 0.293 µg L(-1). All trademarks had average AFM1 concentrations lower than 0.05 µg L(-1), with those in Trademarks 3 to 5 being higher than Trademarks 1 and 2 (P < 0.01). All trademarks had different seasonal patterns of AFM1, even though operating in the same area. However, only Trademark 3 showed significant differences of AFM1 levels between seasons. The results suggested that farm management factors, rather than environment factors, were likely to be the main cause of AFM1 contamination in dairy products.