An update on non-aureus staphylococci and mammaliicocci in cow milk: unveiling the presence of Staphylococcus borealis and Staphylococcus rostri by MALDI-TOF MS.

Non-aureus staphylococci and mammaliicocci (NASM) are the microorganisms most frequently isolated from milk. Given their numerosity and complexity, MALDI-TOF MS is one of the preferred species identification approaches. Nevertheless, reference mass spectra for the novel species Staphylococcus borealis were included only recently in the Bruker Biotyper System (MBT) library, and other species of veterinary interest such as S. rostri are still absent. This work provides an updated picture of the NASM species found in milk, gained by retrospectively analyzing the data relating to 21,864 milk samples, of which 6,278 from clinical mastitis (CM), 4,039 from subclinical mastitis (SCM), and 11,547 from herd survey (HS), with a spectrum library including both species. As a result, S. borealis was the second most frequently isolated NASM (17.07%) after S. chromogenes (39.38%) in all sample types, with a slightly higher percentage in CM (21.84%), followed by SCM (17.65%), and HS (14.38%). S. rostri was also present in all sample types (3.34%), reaching 8.43% of all NASM in SCM and showing a significant association (p < 0.01) with this condition. Based on our findings, the presence of S. borealis and S. rostri in milk and their potential association with mastitis might have been overlooked, possibly due to the difficulties in differentiating these species from other closely related NASM. Our results indicate that S. borealis could be a more frequent contributor to bovine udder infections than previously thought and that S. rostri should also not be underestimated considering its significant association with SCM.

A metabolomics analysis of interspecies and seasonal trends in ruminant milk: The molecular difference between bovine, caprine, and ovine milk.

Ruminant milk composition can be affected by many factors, primarily interspecies differences, but also environmental factors (e.g., season, feeding system, and feed composition). Pasture-based feeding systems are known to be influenced by seasonal effects on grass composition. Spring pasture is rich in protein and low in fiber compared with late-season pasture, potentially inducing variability in the composition of some milk metabolites across the season. This study aimed to investigate interspecies and seasonal differences in the milk metabolome across the 3 major commercial ruminant milk species from factories in New Zealand: bovine, caprine, and ovine milk. Samples of bovine (n = 41) and caprine (n = 44) raw milk were collected monthly for a period of 9 mo (August 2016-April 2017), and ovine milk samples (n = 20) were collected for a period of 5 mo (August 2016-January 2017). Milk samples were subjected to biphasic extraction, and untargeted metabolite profiling was performed using 2 separate liquid chromatography high-resolution mass spectrometry analytical methods (polar metabolites and lipids). Major differences in the milk metabolome were observed between the 3 ruminant species, with 414 of 587 (71%) polar metabolite features and 210 of 233 (87%) lipid features being significantly different between species. Significant seasonal trends were observed in the polar metabolite fraction for bovine, caprine, and ovine milk (17, 24, and 32 metabolites, respectively), suggesting that the polar metabolite relative intensities of ovine and caprine milk were more susceptible to changes within seasons than bovine milk. We found no significant seasonal difference for the triglycerides (TG) species measured in bovine milk, whereas 3 and 52 TG species changed in caprine and ovine milk, respectively, across the seasons. In addition, 4 phosphatidylcholines and 2 phosphatidylethanolamines varied in caprine milk within the season, and 8 diglycerides varied in ovine milk. The interspecies and seasonal metabolite differences reported here provide a knowledge base of components potentially linked to milk physiochemical properties, and potential health benefits of New Zealand pasture-fed dairy ingredients.

Non-aureus staphylococci and mammaliicocci isolated from bovine milk in Italian dairy farms: a retrospective investigation.

Non-aureus staphylococci and mammaliicocci (NASM) are associated with bovine mastitis and increased milk somatic cell count (SCC) but their relationships with mammary gland health at the species level are not clearly defined. Regional differences have also been reported in their specific prevalence. The implementation of MALDI-TOF MS in milk microbiology is generating large and dependable datasets with the potential of providing useful epidemiological information. We present the retrospective analysis of 17,213 milk samples sent to our laboratory in 2021-2022, including 13,146 quarter samples from cows with subclinical (SCM) or clinical mastitis (CM) from 104 farms, and 4,067 composite herd survey (HS) samples from 21 farms. NASM were isolated from 21.12% of SCM, 11.49% of CM, and 15.59% of HS milk samples. The three most frequently identified NASM in SCM milk were Staphylococcus chromogenes (33.33%), S. haemolyticus (26.07%), and S. epidermidis (10.65%); together with S. microti and S. hyicus, these species were significantly more prevalent in quarters with SCM (p < 0.05). The three most frequently identified NASM in CM milk were S. chromogenes (31.69%), S. haemolyticus (21.42%), and Mammaliicoccus sciuri (18.38%), although no significant associations were found between these NASM species and CM. The three most frequently identified NASM in HS milk were S. chromogenes (44.49%), S. epidermidis (17.84%), and S. haemolyticus (17.23%), with S. chromogenes being isolated in all the farms sending HS milk (100%). In conclusion, this retrospective study provides the first information on the NASM species isolated from cow milk in Italy, expanding our knowledge on the epidemiology of NASM at the species level and providing further insights into their relationships with mammary gland health in modern dairy farms.

Occurrence of perfluoroalkyl substances in cow's, goat's and sheep's milk - dietary intake and risk assessment.

Introduction: Milk from cows, goats and sheep was analysed in terms of content of fourteen perfluoroalkyl substances (PFASs). Material and Methods: Altogether, 73 milk samples from cows (n = 38), goats (n = 20) and sheep (n = 15) were collected from various regions of Poland. Concentrations of analytes were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: The lower-bound sum of four PFAS (∑4 PFASs) concentrations (perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid, perfluorononanoic acid and perfluorohexanesulfonic acid) were highest in sheep's (0.0055 µg/kg), lower in goat's (0.0046 µg/kg), and lowest in cow's milk (0.0008 µg/kg). Goat's and sheep's milk was statistically significantly more contaminated than cow's milk. None of the samples exceeded the indicative values set by Commission Recommendation (EU) 2022/1431, and even the maximum detected concentrations were an order of magnitude lower. The most frequently detected was linear PFOS, which was found in 33%, 76% and 93% of cow's, goat's and sheep's milk samples, respectively. Based on mean upper-bound ∑4 PFAS concentrations and average milk consumption, the estimated intake of ∑4 PFASs ranged from 0.153 to 0.266 ng/kg body weight (b.w.) for children and from 0.050 to 0.88 ng/kg b.w. for adults, which indicates that exposure is very low and is merely <7% of the tolerable weekly intake (TWI) for children and <2% of the TWI for adults. Conclusion: Regardless of the milk type, the intake of PFASs via consumption of Polish milk does not contribute significantly to the overall PFAS intake of either adults or children.

Progress of Conjugated Linoleic Acid on Milk Fat Metabolism in Ruminants and Humans.

As a valuable nutrient in milk, fat accounts for a significant proportion of the energy requirements of ruminants and is largely responsible for determining milk quality. Fatty acids (FAs) are a pivotal component of milk fat. Conjugated linoleic acid (CLA) is one of the naturally occurring FAs prevalent in ruminant dairy products and meat. Increasing attention has been given to CLA because of its anti-cancer, anti-inflammatory, immune regulation, and lipid metabolism regulation properties, and these benefits potentially contribute to the growth and health of infants. In breast milk, CLA is present in trace amounts, mainly in the form of cis-9, trans-11 CLA. Notably, cis-9, trans-11 CLA improves the milk fat rate while trans-10, cis-12 CLA inhibits it. Apart from having multiple physiological functions, CLA is also a pivotal factor in determining the milk quality of ruminants, especially milk fat rate. In response to growing interest in green and healthy functional foods, more and more researchers are exploring the potential of CLA to improve the production performance of animals and the nutritional value of livestock products. Taken together, it is novel and worthwhile to investigate how CLA regulates milk fat synthesis. It is the purpose of this review to clarify the necessity for studying CLA in ruminant milk fat and breast milk fat.

Metabolite profiling of peripheral blood plasma in pigs in early postnatal life fed whole bovine, caprine or ovine milk.

Ruminants' milk is commonly used for supplying nutrients to infants when breast milk is unavailable or limited. Previous studies have highlighted the differences between ruminants' milk composition, digestion, absorption, and fermentation. However, whether consuming different ruminants' milk impact the appearance of the circulatory blood metabolites in the early postnatal life is not well understood. The analysis conducted here aimed to determine the effect of feeding exclusively whole milk from bovine, caprine or ovine species to pigs, approximately 7 days-old for 15 days, on circulatory blood plasma metabolites. Relative intensities of plasma metabolites were detected using a liquid chromatography-mass spectrometry based metabolomic approach. Seven polar and 83 non-polar (lipids) metabolites in plasma were significantly different (false discovery rate < 0.05) between milk treatments. These included polar metabolites involved in amino acid metabolism and lipids belonging to phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, and triglycerides. Compared to the caprine or bovine milk group, the relative intensities of polar metabolites and unsaturated triglycerides were higher in the peripheral circulation of the ovine milk group. In contrast, relative intensities of saturated triglycerides and phosphatidylcholine were higher in the bovine milk group compared to the ovine or caprine milk group. In addition, correlations were identified between amino acid and lipid intake and their appearance in peripheral blood circulation. The results highlighted that consuming different ruminants' milk influences the plasma appearance of metabolites, especially lipids, that may contribute to early postnatal life development in pigs.

Effect of Heat Treatment on Protein Self-Digestion in Ruminants' Milk.

This study investigated whether heat treatments (raw, 63 °C for 30 min, and 85 °C for 5 min) affect protein hydrolysis by endogenous enzymes in the milk of ruminants (bovine, ovine, and caprine) using a self-digestion model. Self-digestion consisted of the incubation for six hours at 37 °C of the ruminants' milk. Free amino group concentration was measured by the o-phthaldialdehyde method, and peptide sequences were identified by chromatography-mass spectrometry. Results showed that heat treatments prior to self-digestion decreased the free NH2 by 59% in bovine milk heated at 85 °C/5 min, and by 44 and 53% in caprine milk heated at 63 °C/30 min and 85 °C/5 min, respectively. However, after self-digestion, only new free amino groups were observed for the raw and heated at 63 °C/30 min milk. ß-Casein was the most cleaved protein in the raw and heated at 63 °C/30 min bovine milk. A similar trend was observed in raw ovine and caprine milk. Self-digestion increased 6.8-fold the potential antithrombin peptides in the bovine milk heated at 63 °C/30 min. Enhancing bioactive peptide abundance through self-digestion has potential applications in the industry for functional products. Overall, heat treatments affected the free amino groups according to the species and heat treatment applied, which was reflected in the varying degrees of cleaved peptide bonds and peptides released during self-digestion.

The impact of heating and drying on protease activities of ruminant milk before and after in vitro infant digestion.

This study investigated the effect of heating (63°C/30 min or 75°C/15 s) and drying (spray-drying or freeze-drying) on plasmin, cathepsin D, and elastase activities in bovine, ovine, and caprine milk, compared to non-dried raw milk counterparts. Protease activities and protein hydrolysis were assessed before and after in vitro infant digestion with or without gastric and pancreatic enzymes. At 75°C/15 s, plasmin activity in caprine and ovine milk decreased (69-75%, p<0.05), while cathepsin D activity in spray-dried bovine milk heated increased (2.8-fold, p<0.05). Plasmin and cathepsin D activities increased (<1.2-fold, p<0.05) after in vitro digestion with pancreatin, regardless of milk species. Endogenous milk enzymes hydrolyzed more proteins than gastric enzymes during gastric digestion and contributed to small intestinal digestion. In summary, milk proteases remained active after processing with effects dependent on the species of milk, and they contributed to in vitro protein hydrolysis in the stomach and small intestine.

Comparison of Milk Odd- and Branched-Chain Fatty Acids among Human, Dairy Species and Artificial Substitutes.

The aim of the study was to compare odd and branched-chain fatty acids (OBCFA) of milk from sheep, goat, cow, buffalo, donkey, human, and formula milk. Ruminant, monogastric, and human milks have different concentrations of these fatty acids (FA). To highlight the differences on OBCFA, a total of 282 individual milk samples were analyzed by gas chromatography. The OBCFA were found higher in ruminant than non-ruminant milks (p < 0.05). Among ruminants, sheep milk had the highest OBCFA (4.5 g/100 g of total FAME), whereases the lowest values were found in formula milk (0.18 g/100 g of total FAME). Regarding individual linear odd-chain FA (linear-OCFA), C11:0 was found higher in donkey milk than others, while sheep and buffalo milks had the greatest concentration of C15:0. Among BCFA, the iso-BCFA were higher than anteiso-BCFA in all considered milks. The isoC17:0 showed the highest concentration in all milks except for donkey and buffalo, which showed higher concentration of isoC16:0 than others. In conclusion, ruminant milks are different in terms of these FA compared to human milk and its substitutes. However, the greatest differences were found with formula milk, suggesting that this product needs the implementation of these FA to be more similar to human milk composition.

Improvements in extraction yield by solid phase lipid extraction from liquid infant formula and human milk, and the fatty acid distribution in milk TAG analyzed by joint JOCS/AOCS official method Ch 3a-19.

The Röse-Gottlieb method is one of the most widely used methods for extracting lipids from milk samples. However, we found that lipid recovery from liquid infant formula and human breast milk was lower than expected. Better lipid recovery from these liquid matrices was obtained by solid phase extraction using silica gel; ~10% more could be recovered from liquid infant formula and ruminant milk, and 25% more from human breast milk. However, the method is not recommended for lipid extraction from dried whole milk powders.

The effects of ruminant milk treatments on hippocampal, striatal, and prefrontal cortex gene expression in pigs as a model for the human infant.

While infant formula is usually bovine milk-based, interest in other ruminant milk-based formulas is growing. However, whether different ruminant milk treatments with varying nutrient compositions influence the infant's brain development remains unknown. The aim was to determine the effects of consuming bovine, caprine, or ovine milk on brain gene expression in the early postnatal period using a pig model of the human infant. Starting at postnatal day 7 or 8, pigs were exclusively fed bovine, ovine, or caprine milk for 15 days. The mRNA abundance of 77 genes in the prefrontal cortex, hippocampus, and striatum regions was measured at postnatal day 21 or 22 using NanoString. The expression level of two hippocampal and nine striatal genes was most affected by milk treatments, particularly ovine milk. These modulatory genes are involved in glutamate, gamma-aminobutyric acid, serotonin, adrenaline and neurotrophin signaling and the synaptic vesicle cycle. The expression level of genes involved in gamma-aminobutyric acid signaling was associated with pigs' lactose intake. In contrast, milk treatments did not affect the mRNA abundance of the genes in the prefrontal cortex. This study provides the first evidence of the association of different ruminant milk treatments with brain gene expression related to cognitive function in the first 3 months of postnatal life.

Total conjugated linoleic acid content of ruminant milk: The world status insights.

Conjugated linoleic acid (CLA) content of ruminant milk reported in published research papers (n = 65) from January 1995 to March 2020 around the world were analyzed to estimate the overall mean CLA value. The CLA content of ruminant milk samples was grouped according to geographical regions (Europe, South America, North America, Oceania, Asia, and Africa). The total CLA content of milk samples from cows, sheep, goats, yaks, and llama retrieved from the collected data ranged between 0.06 and 2.96% of total fatty acids. There is a wide variation of pooled estimated mean content of CLA in milk among the study regions and were highest in Oceania with 1.33% (95% confidence interval (CI): 1.16 - 1.49%) of total fatty acids. Though several factors have been reported to influence the CLA content of milk, the effect of the "geographical origin" was only considered in the present manuscript as one of the main factors in this respect.

Structural characteristics of triacylglycerols contribute to the distinct in vitro gastric digestibility of sheep and cow milk fat prior to and after homogenisation.

The regiodistribution of fatty acids (FAs) in triacylglycerols (TAGs) is highly correlated to the digestion and absorption of dietary fats. During in vitro gastric digestion, medium-long-medium/short chain triacylglycerols (MLM) showed the fastest digestibility among 105 TAG molecular species, for both raw and homogenised milk, resulting from the higher activity of gastric lipase towards medium-chain fatty acids (MCFAs) than towards long-chain fatty acids (LCFAs). After 60 min of in vitro gastric digestion, the percentage of digestion of each MLM was 16.6-31.2% and 12.3-18.6% respectively in raw sheep and cow milk, and it reached 33.4-45.5% and 23.0-38.9% respectively, after homogenisation. Sheep milk TAGs were digested faster than cow milk TAGs in raw milk (16.1% and 12.4%, respectively) as well as after homogenisation (24.1% and 20.3%, respectively). This is due to the higher percentages of MCFAs esterified on the sn-1/3 positions of sheep milk TAGs (48.3-67.3% and 11.9-51.5% respectively for sheep and cow milk prior to and after homogenisation). Homogenisation favoured TAG lipolysis due to the increased fat globule surface area; however, it did not affect lipase regiospecificity towards TAG molecular species.

Current intakes of trans-palmitoleic (trans-C16:1 n-7) and trans-vaccenic (trans-C18:1 n-7) acids in France are exclusively ensured by ruminant milk and ruminant meat: A market basket investigation.

High circulating levels of trans-palmitoleic acid (TPA) are associated with a lower risk of type 2 diabetes in humans. Thus, the origin of circulating TPA matters. Direct intakes of TPA are ensured by dairy products, and perhaps by partially hydrogenated oils (PHOs). Indirect intakes of TPA rely on dietary trans-vaccenic acid (TVA), which occurs in ruminant-derived foods and PHOs. As it is usually assumed that PHOs are not used any longer, we analyzed here a wide range of foods currently available at retail in France. We report that TPA and TVA (1) do occur in ruminant milk and meat, dairy products and in foreign PHOs, (2) do occur in dairy fat-containing foods and (3) do not occur in dairy fat-free foods. Together, our findings demonstrate that ruminant fats are the only contributors to circulating levels of TPA in humans.

Factors affecting levels of volatile 4-alkyl branched-chain fatty acids in sheep milk from 2 contrasting farming systems in New Zealand.

Knowledge of factors influencing the levels of 4-alkyl branched-chain fatty acid (vBCFA), and consequently the "sheepy flavor" intensity of New Zealand sheep milk, is currently limited. In this study, we investigated the effects of 2 contrasting farming systems (fully housed/mid-lactation or pasture-grazed/late lactation) on the levels of vBCFA in sheep milk on a commercial farm in the North Island of New Zealand. Fully housed/mid-lactation ewes were housed 24 h/d and fed a total mixed ration. Pasture-grazed/late-lactation ewes were grazed 24 h/d and offered approximately 40% supplements because of poor pasture growth resulting from dry and hot climatic conditions. Any effects of genetics, age, lactation stage, feed composition, lambing date, or the environment in the housing barn or outdoors were confounded. The results obtained in this study were descriptive rather than definitive, because of the limitations of the experimental design. Levels of 4-methyloctanoic acid and 4-methylnonanoic acid in milk from fully housed/mid-lactation ewes increased during the trial period, but remained low in milk from pasture-grazed/late-lactation ewes. Levels of 4-ethyloctanoic acid in milk from the 2 groups of ewes were comparable throughout the trial. Increases in levels of 4-methyloctanoic acid and 4-methylnonanoic acid in sheep milk were associated with lactation stage and the proportion of lucerne silage fed to ewes. The level of free-form 4-ethyloctanoic acid was positively correlated with the proportion of soy meal in the diet and negatively correlated with the proportion of barley. Milk from fully housed/mid-lactation ewes had a higher flavor values than milk from pasture-grazed/late-lactation ewes because of its higher total amounts of vBCFA.

Trans-palmitoleic acid (trans-9-C16:1, or trans-C16:1 n-7): Nutritional impacts, metabolism, origin, compositional data, analytical methods and chemical synthesis. A review.

Since the early 2010s, dietary trans-palmitoleic acid (trans-9-hexadecenoic acid, trans-9-C16:1 in the Δ-nomenclature, trans-C16:1 n-7 in the Ω-nomenclature, TPA) has been epidemiologically associated with a lower risk of type 2 diabetes in humans. Thanks to these findings, TPA has become a nutrient of interest. However, there is a lot of unresolved crucial questions about this dietary fatty acid. Is TPA a natural trans fatty acid? What kind of foods ensures intakes in TPA? What about its metabolism? How does dietary TPA act to prevent type 2 diabetes? What are the biological mechanisms involved in this physiological effect? Clearly, it is high time to answer all these questions with the very first review specifically dedicated to this intriguing fatty acid. Aiming at getting an overview, we shall try to give an answer to all these questions, relying on appropriate and accurate scientific results. Briefly, this review underlines that TPA is indeed a natural trans fatty acid which is metabolically linked to other well-known natural trans fatty acids. Knowledge on physiological impacts of dietary TPA is limited so far to epidemiological data, awaiting for supplementation studies. In this multidisciplinary review, we also emphasize on methodological topics related to TPA, particularly when it comes to the quantification of TPA in foods and human plasma. As a conclusion, we highlight promising health benefits of dietary TPA; however, there is a strong lack in well-designed studies in both the nutritional and the analytical area.

In-depth lipidomic analysis of tri-, di-, and mono-acylglycerols released from milk fat after in vitro digestion.

Milk fat is arguably one of the most complex fats found in nature and varies widely between animal species. Analysis of its digestion products is tremendously challenging, due to the complexity, diversity, and large range of concentrations of triacylglycerols (TAGs) and their digestion products (i.e. diacylglycerols (DAGs), monoacylglycerols (MAGs), and free fatty acids (FFAs)). Therefore, a method combined the solid phase extraction (SPE), high-performance liquid chromatography (HPLC) and multi-dimension mass spectrometry (MDMS) was developed to identify and semi-quantify the TAGs, DAGs and MAGs in milk fat after in vitro digestion. Up to 105, 64, 14 and 30 species of TAGs, DAGs, MAGs, and FFAs were determined with their concentrations of 0.01-22.3, 0.01-39.2, 0.01-47.8, and 0.04-191.0 mg/g fat, respectively, during the in vitro digestion of cow and sheep milk. The validation of the method shows that this method was precise and reliable.

Effects of season and industrial processes on volatile 4-alkyl-branched chain fatty acids in sheep milk.

4-alkyl-branched-chain fatty acids (vBCFAs) are responsible for the goaty-sheepy flavour of sheep and goat milk. The levels of vBCFAs were investigated in sheep, goat and cow milk powders. The effect of season and industrial processes on the levels of vBCFAs in sheep milk was also evaluated. Sheep milk powders contained lower amounts of 4-Me-8:0 and 4-Et-8:0 (143-176 and 1.2-10.7 µg/g milk fat) than goat milk powders (335-481 and 73.1-105 µg/g milk fat), while 4-Me-9:0 was low in both sheep and goat milk powders (<8 µg/g milk fat). Odour activity values (OAVs) of sheep milk powders (17-393) were 6-fold lower than the values of goat milk powders (882-1323). Higher levels of vBCFAs were found in sheep milk produced in spring than in summer. Thermisation had little impact on vBCFAs; however, spray drying led to dramatic increases in free vBCFAs, particularly towards late lactation, which resulted in higher OAVs in sheep milk powder compared to raw milk.