Unprocessed Cow's Milk Research Articles

Isolation and Characterization of Staphylococcus aureus from Raw Cow's Milk and Investigating the Effect of Bifidobacterium bifidum Probiotic Cell Free Supernatant on Their Enterotoxins Genes Expression.

The present reserach aimed to detect and isolate the genes involved in the staphylococcal enterotoxins (SEs) production in strains isolated from unprocessed cow's milk and to examine the impact of Bifidobacterium bifidum probiotic cell-free supernatant (CFS) on their expression. Standard techniques were used for isolation and identification of Staphylococci strains in unprocessed milk. The PCR was used to identify strains carrying enterotoxin genes. The B. bifidum CFS was applied to strains containing the target genes, and the genes expression levels were quantified using Real-time PCR. Using 16SrDNA sequencing, the phylogenic relationship of the isolated strains was determined. Analysis revealed that bacteria such as Staphylococcus species were found in the 72% of the samples. The PCR test showed the presence of various SE superantigens, including SEA (16.7%), SEC (11.7%), SED (8.3%), SEE (6.7%), and SEB (1.7%) in isolated strains. The B. bifidum CFS had obvious antimicrobial activity against strains 24, 51, 54, and 35 of Staphylococcus species, and the minimum inhibitory concentration and minimum bactericidal concentration values for these strains treated with B. bifidum CFS were in the range of 31.25-125 μg/ml. Strains 51 and 24 were clustered with S.aureus ATCC 25923, and strains 54 and 35 were clustered with S.aureus ATCC 12600, respectively. The RT-PCR exhibited that probiotics CFS suppressed the expression of SEA, SEB, SEC, and SEE genes (P<0.05). The average fold change for SEA, SEB, SEC, and SED genes was -1.681, -1.28, -1.52, and -0.84, respectively. The research demonstrated that probiotic bacteria can lower enterotoxin production by downregulating the expression of SEs genes.

Omics Analysis of Extracellular Vesicles Recovered from Infant Formula Products and Milk: Towards Personalized Infant Nutrition.

Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritional needs of the developing infant. Extracellular vesicles (EVs) in human (HM) and cow milk (CM) contain molecular cargo such as proteins and micro(mi)RNAs that serve as functional messengers between cells and may be of importance to infant health. Most IF is derived from a CM protein base, however differences between HM and CM EV molecular cargo have not been extensively studied. This study develops a pipeline using advanced proteomics and transcriptomics to enable cross-species comparison of milk and IF EVs. The number of nanoparticles per mL of IF is significantly reduced compared to unprocessed CM. 130 proteins and 514 miRNAs are differentially abundant between HM and CM EVs. While 90% of CM EV miRNAs are also identified in IF EVs, only 20% of CM EV proteins are identified in IF EVs. This workflow identifies key species-specific differences that can be used to optimize IF recipes and enhance infant nutrition. Improved preservation of EV functional molecular cargo in IF products is of critical importance to retaining molecular drivers of good health and should be the focus of future investigations.

Immune Responsiveness to LPS Determines Risk of Childhood Wheeze and Asthma in 17q21 Risk Allele Carriers.

Rationale: In murine models, microbial exposures induce protection from experimental allergic asthma through innate immunity. Objectives: Our aim was to assess the association of early life innate immunity with the development of asthma in children at risk. Methods: In the PASTURE farm birth cohort, innate T-helper cell type 2 (Th2), Th1, and Th17 cytokine expression at age 1 year was measured after stimulation of peripheral blood mononuclear cells with LPS in n = 445 children. Children at risk of asthma were defined based on single-nucleotide polymorphisms at the 17q21 asthma gene locus. Specifically, we used the SNP rs7216389 in the GSDMB gene. Wheeze in the first year of life was assessed by weekly diaries and asthma by questionnaire at age 6 years. Measurements and Main Results: Not all cytokines were detectable in all children after LPS stimulation. When classifying detectability of cytokines by latent class analysis, carrying the 17q21 risk allele rs7216389 was associated with risk of wheeze only in the class with the lowest level of LPS-induced activation: odds ratio (OR), 1.89; 95% confidence interval [CI], 1.13-3.16; P = 0.015. In contrast, in children with high cytokine activation after LPS stimulation, no association of the 17q21 risk allele with wheeze (OR, 0.63; 95% CI, 0.29-1.40; P = 0.258, P = 0.034 for interaction) or school-age asthma was observed. In these children, consumption of unprocessed cow's milk was associated with higher cytokine activation (OR, 3.37; 95% CI, 1.56-7.30; P = 0.002), which was in part mediated by the gut microbiome. Conclusions: These findings suggest that within the 17q21 genotype, asthma risk can be mitigated by activated immune responses after innate stimulation, which is partly mediated by a gut-immune axis.

Consumption of differently processed milk products and the risk of asthma in children.

Consumption of unprocessed cow's milk has been associated with a lower risk of childhood asthma and/or atopy. Not much is known about differently processed milk products. We aimed to study the association between the consumption of differently processed milk products and asthma risk in a Finnish birth cohort. We included 3053 children from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Nutrition Study. Asthma and its subtypes were assessed at the age of 5years, and food consumption by food records, at the age of 3 and 6months and 1, 2, 3, 4, and 5years. We used conventional and processing (heat treatment and homogenization)-based classifications for milk products. The data were analyzed using a joint model for longitudinal and time-to-event data. At the age of 5years, 184 (6.0%) children had asthma, of whom 101 (54.9%) were atopic, 75 (40.8%) were nonatopic, and eight (4.3%) could not be categorized. Consumption of infant formulas [adjusted hazard ratio (95% confidence intervals) 1.15 (1.07, 1.23), p<.001] and strongly heat-treated milk products [1.06 (1.01, 1.10), p=.01] was associated with the risk of all asthma. Consumption of all cow's milk products [1.09 (1.03, 1.15), p=.003], nonfermented milk products [1.08 (1.02, 1.14), p=.008], infant formulas [1.23 (1.13, 1.34), p<.001], and strongly heat-treated milk products [1.08 (1.02, 1.15), p=.006] was associated with nonatopic asthma risk. All these associations remained statistically significant after multiple testing correction. High consumption of infant formula and other strongly heat-treated milk products may be associated with the development of asthma.

Denaturation of selected bioactive whey proteins during pasteurization and their ability to modulate milk immunogenicity.

This research communication relates to the hypothesis that the consumption of raw or unprocessed cow's milk contributes to lowered prevalence of allergies. Thermal pasteurization of bovine milk can result in denaturation of minor whey proteins and loss of their bioactivity. Denaturation of bovine serum albumin (BSA), immunoglobulin G (IgG) and lactoferrin (LF) in skim milk was studied under different temperature (72, 75 or 78°C) and time (0-300 s) combinations. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that denaturation of all 3 proteins occurred at 72°C and progressed with increase in temperature and holding time. About 59% of LF and 12% of IgG denatured under high-temperature short-time (72°C/ 15 s) pasteurization, while BSA was least impacted. To assess modulation of milk immunogenicity, secretion of selected T helper (Th)-type cytokines by human peripheral blood mononuclear cells (PBMCs) was studied in vitro in response to different concentrations of BSA (0.4-1.0 mg/ml) and IgG (0.8-1.6 mg/ml) in unheated skim milk. Addition of IgG at 1.6 mg/ml induced a prominent Th1-skewed cytokine profile that may not trigger a Th2-skewed allergic reaction. BSA did not appear to modulate milk immunogenicity to any significant extent.

ω-3 fatty acids contribute to the asthma-protective effect of unprocessed cow's milk

Living on a farm has repeatedly been shown to protect children from asthma and allergies. Amajor factor involved in this effect is consumption of unprocessed cow's milk obtained directly from a farm. However, this phenomenon has never been shown in a longitudinal design, and the responsible milk components are still unknown. We sought to assess the asthma-protective effect of unprocessed cow's milk consumption in a birth cohort and to determine whether the differences in the fatty acid (FA) composition of unprocessed farm milk and industrially processed milk contributed to this effect. The Protection Against Allergy-Study in Rural Environments (PASTURE) study followed 1133 children living in rural areas in 5 European countries from birth to age 6years. In 934 children milk consumption was assessed by using yearly questionnaires, and samples of the "usually" consumed milk and serum samples of the children were collected at age 4years. Doctor-diagnosed asthma was parent reported at age 6years. In a nested case-control study of 35 asthmatic and 49 nonasthmatic children, 42 FAs were quantified in milk samples. The risk of asthma at 6years of age was reduced by previous consumption of unprocessed farm milk compared with shop milk (adjusted odds ratio for consumption at 4years, 0.26; 95% CI, 0.10-0.67). Part of the effect was explained by the higher fat content of farm milk, particularly the higher levels of ω-3 polyunsaturated FAs (adjusted odds ratio, 0.29; 95% CI, 0.11-0.81). Continuous farm milk consumption in childhood protects against asthma at school age partially by means of higher intake of ω-3 polyunsaturated FAs, which are precursors of anti-inflammatory mediators.

A Comparison of Different Methodologies for the Measurement of Extracellular Vesicles and Milk-derived Particles in Raw Milk from Cows.

Cow’s milk is economically important to the agricultural industry with the nutritive value of milk being routinely measured. This does not give full insight into normal mammary tissue turnover during the course of lactation, which could be important for both an understanding of milk production and animal welfare. We have previously demonstrated that submicron particles, including extracellular vesicles (EVs), can be measured in unprocessed cow’s milk by flow cytometry and that they correlate with stage of lactation. A number of different techniques are available to measure EVs and other milk-derived particles. The purpose of this study was to compare two different methodologies and the value of fluorescent staining for the phospholipid phosphatidylserine (PS), which is exposed on the surface of EVs (but not other milk-derived particles). We used two different flow cytometers and nanotracker analysis to detect milk-derived particles in whole and skimmed milk samples. Our findings indicate significant correlation, after staining for PS, suggesting potential for larger multicenter studies in the future.

Consumption of unprocessed cow's milk protects infants from common respiratory infections

Breast-feeding is protective against respiratory infections in early life. Given the co-evolutionary adaptations of humans and cattle, bovine milk might exert similar anti-infective effects in human infants. To study effects of consumption of raw and processed cow's milk on common infections in infants. The PASTURE birth cohort followed 983 infants from rural areas in Austria, Finland, France, Germany, and Switzerland, for the first year of life, covering 37,306 person-weeks. Consumption of different types of cow's milk and occurrence of rhinitis, respiratory tract infections, otitis, and fever were assessed by weekly health diaries. C-reactive protein levels were assessed using blood samples taken at 12 months. When contrasted with ultra-heat treated milk, raw milk consumption was inversely associated with occurrence of rhinitis (adjusted odds ratio from longitudinal models [95% CI]: 0.71 [0.54-0.94]), respiratory tract infections (0.77 [0.59-0.99]), otitis (0.14 [0.05-0.42]), and fever (0.69 [0.47-1.01]). Boiled farm milk showed similar but weaker associations. Industrially processed pasteurized milk was inversely associated with fever. Raw farm milk consumption was inversely associated with C-reactive protein levels at 12 months (geometric means ratio [95% CI]: 0.66 [0.45-0.98]). Early life consumption of raw cow's milk reduced the risk of manifest respiratory infections and fever by about 30%. If the health hazards of raw milk could be overcome, the public health impact of minimally processed but pathogen-free milk might be enormous, given the high prevalence of respiratory infections in the first year of life and the associated direct and indirect costs.

The potential mechanistic link between allergy and obesity development and infant formula feeding.

This article provides a new view of the cellular mechanisms that have been proposed to explain the links between infant formula feeding and the development of atopy and obesity. Epidemiological evidence points to an allergy- and obesity-preventive effect of breastfeeding. Both allergy and obesity development have been traced back to accelerated growth early in life. The nutrient-sensitive kinase mTORC1 is the master regulator of cell growth, which is predominantly activated by amino acids. In contrast to breastfeeding, artificial infant formula feeding bears the risk of uncontrolled excessive protein intake overactivating the infant’s mTORC1 signalling pathways. Overactivated mTORC1 enhances S6K1-mediated adipocyte differentiation, but negatively regulates growth and differentiation of FoxP3+ regulatory T-cells (Tregs), which are deficient in atopic individuals. Thus, the “early protein hypothesis” not only explains increased mTORC1-mediated infant growth but also the development of mTORC1-driven diseases such as allergy and obesity due to a postnatal deviation from the appropriate axis of mTORC1-driven metabolic and immunologic programming. Remarkably, intake of fresh unpasteurized cow’s milk exhibits an allergy-preventive effect in farm children associated with increased FoxP3+ Treg numbers. In contrast to unprocessed cow’s milk, formula lacks bioactive immune-regulatory microRNAs, such as microRNA-155, which plays a major role in FoxP3 expression. Uncontrolled excessive protein supply by formula feeding associated with the absence of bioactive microRNAs and bifidobacteria in formula apparently in a synergistic way result in insufficient Treg maturation. Treg deficiency allows Th2-cell differentiation promoting the development of allergic diseases. Formula-induced mTORC1 overactivation is thus the critical mechanism that explains accelerated postnatal growth, allergy and obesity development on one aberrant pathway.

Assessment of thiamine content in some dairy products and rice milk

The levels of thiamine were compared between different types of milk, fermented milk products and rice milk. The analysed products were mainly from the markets, so vitamin analysis may be a good parameter for the quality of commercial food. Thiamine concentrations were determined by high performance liquid chromatography (HPLC) using a reversed-phase C-18 column with fluorescence detector. The average thiamine contents were 0.46 µg/mL for ultra high temperature (UHT), 0.39 µg/mL for pasteurized and 0.38 µg/mL for chocolate milk. Unprocessed cow milk had average thiamine concentration of 0.47 µg/mL. The highest thiamine content (11.95 µg/mL) was found in rice milk. Sour cream samples had higher concentration of thiamine (0.54 µg/mL) than yogurt (0.45 µg/mL) and sour milk (0.47 µg/mL). In the opened UHT milk packages, which were stored in refrigerator for 10 days, significant thiamine loss was not observed. Investigated products contained appropriate amounts of thiamine and had satisfactory nutritional value with respect to vitamin B1.

Maternal farm exposure/ingestion of unpasteurized cowʼs milk and allergic disease

The prevalence of allergic diseases has reached a high prevalence in affluent countries. Yet, there are areas and living conditions associated with very low rates which are attributable to environmental exposures. One example is the low prevalence of childhood asthma and allergies amongst children raised on farms, particularly those exposed to farm animals and unprocessed cow's milk very early in life which are being reviewed in this article. Several studies have shown that maternal exposure to various farm animals in pregnancy is related to a reduced risk of atopic dermatitis in the child and immune responses which have generally been regarded as protecting from the development of asthma and allergies. In addition, maternal and infant consumption of unprocessed cow's milk has repeatedly been associated with protection from childhood asthma and allergies. The precise nature of the protective factors remains to be elucidated. Maternal exposures to farm animals result in increased microbial exposures, the diversity of which has been associated with protection from childhood asthma. The beneficial milk constituents may, however, not be contained in the microbial contamination of milk samples but rather in the whey fraction of unprocessed cow's milk. Better understanding of these protective exposures might in the future enable the development of new prevention strategies.

The farm effect, or

Multiple studies have shown that the prevalence of asthma and atopy is reduced in children raised on traditional dairy farms. This article discusses the temporal constraints for the protective farm effect, the components of a farming environment that are associated with protection, and novel mechanisms that may underlie protection from asthma and atopy in farming populations. Protection from asthma and allergy is strongest when exposure occurs in utero or early in life, but the protective effects can persist into adulthood. Just three exposures (contact with cows and straw and consumption of unprocessed cow's milk) account for virtually all the protective farm effect for asthma but not atopy. Whey proteins appear to be critical for the protective effects of farm milk, whereas the high microbial diversity existing in a farm environment is strongly and inversely associated with asthma, but only weakly associated with atopy. Therefore, distinct mechanisms are likely to mediate protection from asthma and atopy. The biological significance of microbial diversity is still unclear, but multiple lines of evidence link the asthma-protective and allergy-protective effects of farming to immune responses and the microbiome. Work in mouse models is revealing novel cellular and molecular mechanisms through which the microbiota may modulate immune responses and allergic inflammation, and thus contribute to the farm effect. The role of the host's genetic makeup, on the contrary, remains poorly understood. The discovery of the central role played by microbial diversity in the asthma-protective and allergy-protective effects of farming warrants metagenomic studies that concertedly and longitudinally investigate the microbiome, the genome, and the immune system of farmers and the farms they live on.

Can farm milk consumption prevent allergic diseases?

Cow's milk is an important part of human diet and a source of food allergy for some individuals. Medical guidance strongly discourages consumption of raw milk because of the known health risk associated with pathogenic bacteria present in unpasteurized milk. Despite these risks there is a growing body of epidemiological evidence suggesting that consumption of unprocessed cow's milk does not increase but rather decreases the risk of asthma, hay fever and atopic sensitisation. The article reviews the epidemiological literature and discusses components of unprocessed milk potentially responsible for this protection. It focuses on the role of bacteria in raw milk, the fatty acid profile, whey proteins and finally the role of allergens in milk. Although the epidemiological evidence consistently suggest a protective role of unprocessed cow's milk consumption on the development of asthma, hay fever and atopic sensitization the underlying mechanisms are not yet understood and the consumption of raw milk cannot be recommended as a preventive measure for allergic diseases.

Farm living: effects on childhood asthma and allergy

Numerous epidemiological studies have shown that children who grow up on traditional farms are protected from asthma, hay fever and allergic sensitization. Early-life contact with livestock and their fodder, and consumption of unprocessed cow's milk have been identified as the most effective protective exposures. Studies of the immunobiology of farm living point to activation and modulation of innate and adaptive immune responses by intense microbial exposures and possibly xenogeneic signals delivered before or soon after birth.

99th Dahlem Conference on Infection, Inflammation and Chronic Inflammatory Disorders: Farm lifestyles and the hygiene hypothesis

About 15 years have gone by since Strachan first proposed the idea that infections and unhygienic contact may confer protection from the development of allergic illnesses. The so-called 'hygiene hypothesis' has since undergone numerous modifications in the field of epidemiology, clinical science and immunology. Three main areas of research have been brought forward: to explore the role of overt viral and bacterial infections for the inception of allergic diseases; to investigate the significance of environmental exposure to microbial compounds on the development of allergies; and to study the effect of both exposures on underlying innate and adaptive immune responses. A concept unifying these various aspects has not been found, but various pieces of a complex interplay between immune responses of the host, characteristics of the invading microorganism, the level and variety of the environmental exposure and the interactions between an exposed subject's genetic background and the environmental exposures becomes apparent. A natural experiment relating to the hygiene hypothesis is the recurrent observation of a protective effect of growing up on a farm for asthma and allergies. This has been shown in a large number of epidemiological studies across the world among children and adults. The timing and duration of exposure are likely to play a critical role. The largest reduction in risk has been demonstrated for those exposed prenatally and continuously thereafter until adulthood. The protective factors in these farming environments have not been unravelled completely. Findings from various studies suggest that the contact with farm animals, at least in childhood, confers protection. Also the consumption of unprocessed cow's milk directly from the farm has been shown to protect from childhood asthma and allergies. Increased levels of microbial substances may, at least in part, contribute to the 'farm effect'. However, only few studies have measured microbial exposures in these environments and the results obtained so far suggest that the underlying protective microbial exposure(s) have not been identified, but a number of studies using metagenomic approaches are currently under way. The mechanisms by which such environmental exposures confer protection from respiratory allergies are also not well understood. There is good evidence for the involvement of innate immune responses, but translation into protective mechanisms for asthma and allergies is lacking. Furthermore, a number of gene x environment interactions have been observed.

Glucose and galactose absorption after ingestion of milk containing hydrolysed lactose in calves with diarrhoea.

SummaryTen calves which had contracted acute diarrhoea caused by rotavirus, coronavirus and Cryptosporidium were used to test the hypothesis that feeding lactose‐hydrolysed cow's milk instead of unprocessed cow's milk improves sugar absorption in diarrhoeic calves. The animals were rehydrated with an orally administered solution containing electrolytes and glucose. Thereafter the calves received one test meal of whole fresh cow's milk whose lactose had been hydrolysed by added lactase and one test meal of unprocessed cow's milk at an interval of 24 h in a cross‐over design trial. In comparison with unprocessed milk, the intake of milk containing hydrolysed lactose resulted in a slight decrease of mean breath hydrogen concentration (P = 0.18), but also a slight decrease of mean blood galactose concentration (P = 0.14). There was no treatment effect on mean plasma glucose concentration. Peak plasma glucose and blood galactose concentration tended to be delayed after the intake of lactose‐hydrolysed milk, which implies that gastric emptying was probably delayed. The results show that feeding milk which contains hydrolysed lactose does not significantly improve lactose utilization in calves that are suffering from benign infectious diarrhoea.

Relative zinc availability in human breast milk, infant formulas, and cow’s milk

The concentration and bioavailability of zinc was analyzed in 10 varieties of milk. The concentration of zinc averaged 4.1 µg/ml in unprocessed (raw) cow's milk; fortified infant formulas contained about 3 to 5 µg Zn/ml; human breast milk and processed cow's milk contained approximately 2 µg Zn/ml; sweetened condensed milk contained 1.3 µg Zn/ml, and reconstituted nonfat dry milk contained 0.4 µg Zn/ml. The mean values for zinc bioavailability to rats were as follows: sweetened condensed milk = 66%; human breast milk = 59.2%; processed cow's milk = 43.7 to 50.9%; unprocessed (raw) cow's milk = 42%; nonfat dry milk = 41.2%; and infant formulas = 26.8 to 39.5%. Assuming similar absorption of zinc in rats and humans, our experimental results provide some guidelines for estimating the quantity of zinc that would actually be absorbed from various types of milk.