Meta-Genomic Analysis of Different Bacteria and Their Genomes Found in Raw Buffalo Milk Obtained in Various Farms Using Different Milking Methods.

Milking methods have significant impacts on the microbiological composition, which could affect the quality of raw buffalo milk. Hence, the current study was conducted on the impact of milking methods on microorganisms in buffalo tank raw milk from 15 farms in Guangxi, China. The farms were divided into two groups based on the milking method: mechanical milking (MM, n = 6) and hand milking (HM, n = 9). Somatic cell counts, bacterial cell counts and nutrients of the raw buffalo milk samples were analyzed. The comparison of raw buffalo milk samples was analyzed using metagenomic sequencing to detect any differences between the two groups. There was no significant difference in the basic nutritional compositions and somatic cell count of raw buffalo milk between the two milking methods. However, the HM samples had significantly higher bacterial counts and diversity compared to the MM samples. The results showed that Staphylococcus spp., Klebsiella spp., Streptococcus spp., and Pseudomonas spp. were the major microbes present in canned raw buffalo milk. However, the differences between the two milking methods were the relative abundance of core microorganisms and their potential mastitis-causing genera, including the content of antibiotic-resistance genes and virulence genes. Our study revealed that Staphylococcus spp. and Streptococcus spp. were significantly more abundant in the MM group, while Klebsiella spp. was more abundant in the HM group. Regardless of the milking method used, Pseudomonas spp. was identified as the primary genus contributing to antibiotic resistance and virulence genes in canned raw buffalo milk. These findings affirm that there are differences in the microbial and genomic levels in canned raw milk. To prove the functional roles of the discovered genes and how these genes affect milk quality, further research and experimental validation are necessary.

Low Effectiveness of Mid-Infrared Spectroscopy Prediction Models of Mediterranean Italian Buffalo Bulk Milk Coagulation Traits.

This study evaluated the potential use of mid-infrared spectroscopy to predict milk coagulation traits in bulk milk from Mediterranean Italian buffaloes. A total of 1736 bulk milk samples from 55 farms in central Italy were collected during the official milk quality testing system. The prediction models were developed based on modified partial least-squares regression with 75% of the samples and validated with the remaining samples. All bulk milk samples coagulated between 7.37 and 29.45 min. Average values for milk coagulation traits in the calibration set were 17.71 min, 3.29 min, and 38.83 mm for rennet coagulation time, curd firming time, and curd firmness, respectively. The validation set included samples with similar mean and standard deviation for each trait. The prediction models showed the greatest coefficient of determination of external validation (0.57) and the ratio of prediction to deviation (1.52) for curd firmness. Similar fitting statistics of the prediction models were obtained for rennet coagulation time and curd firming time. In conclusion, the prediction models for all three coagulation traits were below the threshold to consider the prediction models adequate even for rough screening of the samples.

The Physicochemical, Textural, Microbiological and Sensory Properties of Skimmed Buffalo Milk Yoghurt with Tragacanth Gum During Storage.

Research background: In the food industry, research interest in the functional effects of natural polysaccharides from plants has increased in recent years. Tragacanth gum is used in dairy products because of its stabilising, thickening, fat-replacing and prebiotic properties. However, skimmed milk is considered a significant commercial loss in the production of buffalo clotted cream. Therefore, the aim of the present study is to investigate the potential of tragacanth gum in the production of yoghurt from buffalo milk residues with different concentrations of tragacanth gum (0.5, 1 and 1.5 g/L). Experimental approach: Skimmed buffalo milk with different concentrations of tragacanth gum was pasteurised and, after cooling at 45 °C, a starter culture was added to each sample. All samples were fermented to a pH=4.80±0.2. The gross composition, acidity, water activity, water-holding capacity, whey separation, mass fractions of organic acids and volatile aroma compounds, counts of total aerobic mesophilic bacteria, yeasts and moulds, Lactococcus spp. and Lactobacillus spp. as well as sensory and textural properties were analysed during 15 days storage. Results and conclusions: The results showed that the use of tragacanth gum increased the dry matter mass fraction, water-holding capacity and mass fraction of proteins in the samples, while whey separation decreased as the concentration of gum increased. The addition of gum improved textural properties and hardness of the yoghurt. In terms of consistency, the sample with 1 g/L tragacanth gum was the most reliable. In the control group, the total aerobic mesophilic bacteria count was highest on the first and last day of storage. According to the results of the sensory evaluation, the sample with 0.5 g/L tragacanth gum was the most favourable. Novelty and scientific contribution: Research has shown that the use of stabilisers in varying ratios improves the quality of yoghurt made from fat-free buffalo milk, which is a by-product of industrial production. So instead of ending up as industrial waste, it is recycled and its value is increased.

Multi-omics analysis of five species of milk and specific composition links within each species.

The gold standard of milk is human milk, not cow milk. The present study expects to explored the comprehensive nutritional value of different kinds of milk and the differences between them through multi-omics analysis and found functional components that are more similar to human milk. This study employed untargeted LC-MS/MS metabolomics, untargeted LC-MS/MS lipidomics, and 4D label-free proteomics analysis techniques. The findings revealed substantial disparities in metabolites, lipids, and proteins among the five types of milk. Notably, pig milk exhibited a remarkable abundance of N-acetylneuraminic acid (Neu5Ac) and specific polar lipids. Yak milk stood out with significantly elevated levels of creatine and lipoprotein lipase (LPL) compared to other species. Buffalo milk boasted the highest concentrations of L-isoleucine, echinocystic acid, and alkaline phosphatase, tissue-nonspecific isozyme (ALPL). The concentrations of iminostilbene and osteopontin (OPN) were higher in cow milk.

Complete genome sequence of Indonesian probiotic strain Lactiplantibacillus plantarum subsp. plantarum Dad-13.

Previous studies have investigated the probiotic properties of L. plantarum subsp. plantarum Dad-13. Nevertheless, genomic sequence data from previous studies were not yet available to support each probiotic characteristic. This study focused on the complete genome sequence of the strain to validate its role in specific probiotic properties.

Impact of cow, buffalo, goat or camel milk consumption on oxidative stress, inflammation and immune response post weaning time.

Milk is a whitish liquid that is secreted from mammary glands; and considered as the primary source of nutrition for newborns since they are not able to digest solid food. However, it contains primary nutrients, as well as growth and immune factors. Early weaning is a critical issue that face women and their babies in developing countries. To avoid infant malnutrition, they tend to use other milk types instead of baby formula. Therefore, the present study aimed to evaluate the impact of cow, buffalo, goat or camel milk consumption on oxidative stress, inflammation and immune response in male and female Sprague Dawley rats post weaning time. The amino acids, fatty acids, minerals and vitamins in the tested milk types were evaluated. Animals were divided into 5 groups (control, cow, buffalo, goat and camel milk administrated groups) (10 rats/group); each animal was administrated by 3.4 ml/day. Rats were administered with milk for 6 weeks; at the end of the 5th week, five animals of each group were isolated and the remaining five animals were immunized with sheep red blood cells (SRBCs) and kept for another week to mount immune response. The effect of different milk types on rats' immune response towards SRBCs was evaluated through pro-inflammatory cytokines, antioxidants, ESR and CRP measurement; together, with the histopathological examination of spleen samples and hemagglutination assay. Camel milk consumption reduced oxidative stress and inflammation in spleen that resulted from SRBCs immunization; in addition to, B cell stimulation that was apparent from the high level of anti-SRBCs antibodies. Camel milk is recommended for newborn consumption, due to its high-water content, unsaturated fatty acids, and vitamin C, as well as low lactose and fat content.

Draft genome sequence of Leuconostoc falkenbergense isolated from naturally fermented buffalo milk curd.

This study reports the draft genome of Leuconostoc falkenbergense strain BSMRAU-M1L5, isolated from artisanal buffalo milk curd in Bangladesh. The draft genome spans 1,776,471 bp, with 50× coverage and 96 contigs.

Milk quality of Italian Mediterranean buffalo as affected by temperature-humidity index during late spring and summer.

A total of 18 Italian Mediterranean buffalo cows were subjected to fortnightly milk sampling from May to July 2022. Air temperature and relative humidity were monitored throughout the trial. Temperature-humidity index (THI) was calculated and ranged from 69 to 79, so that 3 classes were set to study the effect of different THI on milk quality: THI less than 72 (THI<72), THI ranging from 72 to 76 (THI72-76), and THI over 76 (THI>76). Individual milk samples from buffalo cows were analyzed for milk composition and coagulation traits, fatty acid and amino acid composition, and enzymatic and mineral profiles. The THI>76 class registered higher lactose content and poorer milk clot characteristics in comparison with THI<72. Exposure to a THI over 76 resulted in increased SFA and short-chain fatty acid contents in milk, as well as in the highest atherogenic and thrombogenic index values and the lowest essential amino acid content. Moreover, the lowest values of calcium, copper, and selenium contents were found in the milk of buffalo cows exposed to a THI over 72. Results confirm that Italian Mediterranean buffalo express a good level of thermo-tolerance, even though exposure to a daily THI over 76 has a deleterious effect on some nutritional and technological properties of milk.

Genomic insights into antibiotic resistance genes in Leuconostoc citreum strains isolated from artisanal buffalo milk curd in Bangladesh through whole-genome sequencing.

We sequenced the genome of Leuconostoc citreum strains BSMRAU-M1L6 and BSMRAU-M1L13 isolated from artisanal buffalo milk curd in Bangladesh. The draft genomes of BSMRAU-M1L6 and BSMRAU-M1L13 are 1,869,891 and 1,890,611 bp, respectively, with 50.0× coverage (both) and 65 and 75 contigs, respectively.

Buffalo yogurt fermented with commercial starter and Lactobacillus plantarum originating from breast milk lowered blood pressure in pregnant hypertensive rats.

Nutritional therapy, which may have advantages over medication, is being investigated as a novel treatment for pregnancy-induced hypertension. Several studies have shown that probiotic yogurt supplementation during pregnancy has beneficial effects on maternal and fetal health. In this study, fermented buffalo milk was produced with yogurt culture and Lactobacillus plantarum B, a probiotic isolated from healthy breast milk with high angiotensin-converting enzyme inhibitory activity. The fermentation conditions under which the angiotensin-converting enzyme (ACE) inhibitory activity reached 84.51% were optimized by the response surface method as follows: 2 × 106 cfu/mL of L. plantarum B, yogurt culture 2.5 × 105 cfu/mL, and 8 h at 37°C. The distribution of ACE inhibitory peptides from fermented buffalo milk and fermented cow milk were further analyzed by liquid chromatography-mass spectrometry. By searching according to the structural features of ACE inhibitory peptides, 29 and 11 peptides containing ACE inhibitory peptide features were found in fermented buffalo milk and fermented cow milk, respectively. To investigate the in vivo antihypertensive activity of fermented buffalo milk, 18 pregnant rats were divided into 3 groups (n = 6 in each group) and administered 10 mL of normal saline, yogurt (20 mg/kg), or labetalol hydrochloride (4 mg/kg) daily from the beginning of pregnancy to parturition. To induce hypertension, methyl nitrosoarginine (125 mg/kg) was injected subcutaneously every day from d 15 of pregnancy to the day of delivery. Blood pressure was not significantly changed in the yogurt and labetalol groups after induction of hypertension and was lower compared with the normal saline group, but there was no difference between the yogurt and labetalol groups. This implied that the buffalo yogurt had a preventive and antihypertensive effect in the pregnancy-induced hypertensive rat model. Further studies to determine the mechanism of action, as well as a randomized control trial, are warranted.

Clonal spread of non-O157 Shiga toxigenic Escherichia coli O21:H25 in raw water buffalo milks.

AIMS: This study was conducted to investigate the presence of Shiga toxin-producing O157 and non-O157 E. coli in raw water buffalo milk, as well as to determine the virulence gene profiles, phylogroups, sequence types, and serotypes of the isolated strains. METHODS AND RESULTS: A total of 200 hand-milked raw water buffalo milk samples were collected from 200 different water buffaloes over a period of three months from 20 different farms. Isolation of STEC was performed using CHROMagar STEC. Presence of stx1, stx2, and eaeA genes were investigated by mPCR. Phylogroups and sequence types of E. coli strains were determined by Clermont phylotyping and MLST. Serotyping was performed using PCR or WGS. According to the results, two milk samples obtained from two different farms were found as STEC-positive. All Stx-positive E. coli isolates belonged to phylogenetic group A and were assigned to ST10. WGS results indicated that serotype of two isolates was O21:H25 and average nucleotide identity was detected at 99.99%. Thirteen additional registered E. coli O21:H25 assembled WGS data were obtained from EnteroBase and a phylogenetic tree was constructed. CONCLUSIONS: With this study, the presence of stx2 harboring E. coli O21:H25 in milk was identified for the first time. Although the identified serotype is considered a non-pathogen seropathotype, we conclude it could play an important role in the environmental circulation of Stx-phages and consequently contribute to the emergence of new STEC-related outbreaks.

Isolation of a novel pentasaccharide Ebaliose from immunostimulant fraction of buffalo milk oligosaccharide.

Buffalo milk processed by method of Kobata and Ginsburg for isolation of its oligosaccharide contents showed significant stimulation of anti-body, delayed type hypersensitivity response to sheep red blood cells in BALB/c mice. This oligosaccharide mixture also stimulated non-specific immune response in terms of MMI. In continuation to our previous studies of oligosaccharide contents of buffalo milk another novel pentasaccharide Ebaliose was isolated from immunostimulant oligosaccharide fraction of buffalo milk. Ebaliose was procured in its purest form, by combining the different chromatographic techniques like Gel filtration, Silica Gel column chromatography, and HPLC. The structure of novel oligosaccharide was determined by 1H,13C, HSQC, TOCSY, COSY, HMBC, ESI-MS, chemical transformation, and chemical degradation. The stereoscopic structure of this novel pentasaccharide was established as under.

A Genotyping Method for Detecting Foreign Buffalo Material in Mozzarella di Bufala Campana Cheese Using Allele-Specific- and Single-Tube Heminested-Polymerase Chain Reaction.

Mozzarella di Bufala Campana (MdBC) cheese is a Protected Designation of Origin (PDO) product that is important for the economy and cultural heritage of the Campania region. Food fraud can undermine consumers' trust in this dairy product and harm the livelihood of local producers. The current methods for detecting adulteration in MdBC cheese due to the use of buffalo material from foreign countries could exhibit limitations associated with the required use of expensive equipment, time-consuming procedures, and specialized personnel. To address these limits here, we propose a rapid, reliable, and cost-effective genotyping method that can detect foreign buffalo milk in a counterpart from the PDO area and in MdBC cheese, ensuring the quality and authenticity of the latter dairy product. This method is based on dedicated allele-specific and single-tube heminested polymerase chain reaction procedures. By using allele-specific primers that are designed to detect the nucleotide g.472G>C mutation of the CSN1S1Bbt allele, we distinguished an amplicon of 330 bp in the amplification product of DNA when extracted from milk and cheese, which is specific to the material originating from foreign countries. By spiking foreign milk samples with known amounts of the counterpart from the PDO area, the sensitivity of this assay was determined to be 0.01% v/v foreign to PDO milk. Based on a rough estimate of its simplicity, reliability, and cost, this method could be a valuable tool for identifying adulterated buffalo PDO dairy products.

Influence of Calcium-Sequestering Salts on Heat-Induced Changes in Blends of Skimmed Buffalo and Bovine Milk.

Heat-induced interactions of calcium and protein in milk lead to undesirable changes in the milk, such as protein coagulation, which can be minimized through the addition of calcium-sequestering salts prior to heat treatment. Thus, the present study investigated the influence of 5 mM added trisodium citrate (TSC) or disodium hydrogen phosphate (DSHP) on the heat-induced (85 °C and 95 °C for 5 min) changes in physical, chemical, and structural properties of buffalo and bovine skim milk mixtures (0:100, 25:75, 50:50, 75:25, and 100:0). Significant changes in pH and calcium activity as a result of TSC or DSHP addition subsequently resulted in higher particle size and viscosity as well as non-sedimentable protein level. These changes are mostly observed during heat treatment at 95 °C and increased proportionally to the concentration of buffalo skim milk in the milk mixture. Significant changes were affected by TSC addition in the 75:25 buffalo:bovine milk blend and buffalo skim milk, but for other milk samples, TSC addition effected comparable changes with DSHP addition. Overall, the addition of TSC or DSHP before heat treatment of buffalo:bovine milk blends caused changes in milk properties that could reduce susceptibility of milk to coagulation.

Effect of Buffalo Milk Consumption on the Growth of 10 to 12 years Old Schoolgirls in Magdalena, Laguna, Philippines.

Background: Milk contains essential nutrients that help can improve the growth and development of adolescent girls. Objectives: The study determined the effect of milk consumption on the nutritional status of schoolgirls ages 10 to 12 years old in Magdalena, Laguna, Philippines. Methodology: A quasi-experimental study design was used to determine the impact of daily consumption of 200 ml buffalo milk on the prevalence of undernutrition among the 57 schoolgirl participants before and after 160 days. One sample t-test and paired t-test were used to compare the actual and expected total increment and monthly changes in the height and body mass index (BMI) of the participants, while a one-way analysis of variance was used to compare the actual total changes in height and BMI by age. Factors that are correlated with these measurements were identified based on Spearman's correlation coefficients. Results: The percentage of stunting (31.6%-22.8%) and thinness (21.1%-15.8%) decreased after the milk feeding. Significant differences were observed in the means of total actual and expected changes in height (P < .00) and BMI (P < .00). While there were significant differences in the means of actual and expected monthly changes in height every month, this was only observed in the first 2 months for BMI. Only the mean actual changes in height had significant differences when compared by age (P = .04). Lastly, the age and education of the father were found to be correlated with the height of the schoolgirls. Conclusion: Buffalo milk consumption can improve the growth outcomes of schoolgirls.

Effect of Buffalo Breed on the Detailed Milk Composition in Guangxi, China.

Buffalo is the second source of milk in the world, and its milk is rich in nutritive components. It is well-known that breed influences milk composition. This work aimed to compare the detailed milk composition of three buffalo breeds (Murrah, Nili-Ravi, and Mediterranean) housed under the same environmental conditions. Mediterranean buffalo milk showed a significantly higher content of fat, protein, and some fatty acids. Moreover, the milk from the Mediterranean breed was characterized by the highest content of sphingomyelin (SM), cholesterol, and lanosterol. However, the Murrah buffalo milk contained the highest amount of total unsaturated fatty acids, phosphatidylinositol, and whey proteins. Furthermore, the Nili-Ravi buffalo milk was characterized by the highest content of total saturated fatty acids, phosphatidylglycerol, squalene, lathosterol, stigmasterol, beta-sitosterol, and casein fractions. Nevertheless, the lactose and amino acid profiles of the milk remained almost similar across the three buffalo breeds. The generated results in this study enable a comprehensive understanding of the milk constituent variability that is linked to buffalo breeds, which may support the acquirement of essential scientific knowledge on milk ingredient-processing interactions that will offer a foundation of knowledge for Chinese dairy processors in terms of milk processability and innovation.

The Effects of Modified Atmosphere Packaging on the Quality Properties of Water Buffalo Milk's Concentrated Cream.

Concentrated cream (CC) is a dairy product containing more than 60% milk fat. CC has a very short shelf life because it is made from unripe cream. The present study aims to determine how packaging with reducing gas (H2) and nitrogen (N2) affects the quality properties and shelf life of CC. For this purpose, lipolysis, oxidation, color, microbiological, and free fatty acid development and the fatty acid composition of modified atmosphere packaged (MAP) CC samples were studied for 28 days. For MAP1, 96% N2 + 4% H2 was used, and for MAP2, 100% N2 and air was used for the control group. During storage, MAP1 samples remained at lower lipolysis (ADV and FFA) and oxidation levels than MAP2 and the control group. The MAP1 and MAP2 methods preserved the color of the samples and reduced the microbial growth rate. A lower formation of free fatty acids was observed in the samples packed with MAP1 and MAP2 than in the control group. The results showed that hydrogen gas positively affected the quality and storage time of samples.

Staphylococcus microti Strains Isolated from an Italian Mediterranean Buffalo Herd.

S. microti is a new species among non-aureus staphylococci (NAS) frequently found in bovine milk samples and associated with subclinical mastitis (SCM). The aim of this study was to analyze the presence of S. microti in 200 composite milk samples and 104 milking parlor surface swabs collected at a buffalo farm in Southern Italy to define its presence in milk and a milking parlor environment. The samples were inoculated onto different agar plates, and the isolates were identified by MALDI-TOF MS. The strains identified as S. microti (54/304 samples, 17.8%) were collected, and their purified genomic DNA was subjected to PCR amplification and whole 16S rRNA gene sequencing. Furthermore, their phenotypic resistance profiles were evaluated by a disk diffusion method, and the genotypic characterization of the tetracycline resistance was performed for the tetM and tetK genes by multiplex PCR. Four and forty-seven S. microti isolates from milk samples of lactating animals with subclinical mastitis (SCM) and intramammary infection (IMI), respectively, and three isolates from milking parlor surfaces were recovered. The genomic DNA was purified from the bacterial isolates, and the amplification and sequencing of the 16S gene further supported the proteomic identification as S. microti. No clinical mastitis was detected in the herd during the study period. The antimicrobial susceptibility testing revealed a worrisome 100% resistance to tetracyclines, genotypically mediated by the tetM gene for all strains. This study highlights that S. microti may be commonly isolated from dairy buffalo milk and milking parlor equipment. Its association with SCM or IMI remains to be established.

In silico identification of novel ACE and DPP-IV inhibitory peptides derived from buffalo milk proteins and evaluation of their inhibitory mechanisms.

The capacity of buffalo milk proteins to release bioactive peptides was evaluated and novel bioactive peptides were identified. The sequential similarity between buffalo milk proteins and their cow counterparts was analysed. Buffalo milk proteins were simulated to yield theoretical peptides via in silico proteolysis. The potential of selected proteins to release specific bioactive peptides was evaluated by the A value obtained from the BIOPEP-UWM database (Minkiewicz et al. in Int J Mol Sci 20(23):5978, 2019). Buffalo milk protein is a suitable precursor to produce bioactive peptides, particularly dipeptidyl peptidase IV (DPP-IV) and angiotensin I-converting enzyme (ACE) inhibitory peptides. Two novel ACE inhibitory peptides (KPW and RGP) and four potential DPP-IV inhibitory peptides (RGP, KPW, FPK and KFTW) derived from in silico proteolysis of buffalo milk proteins were screened using different integrated bioinformatic approaches (PeptideRanker, Innovagen, peptide-cutter and molecular docking). The Lineweaver-Burk plots showed that KPW (IC50 = 136.28 ± 10.77 µM) and RGP (104.72 ± 8.37 µM) acted as a competitive inhibitor against ACE. Similarly, KFTW (IC50 = 873.92 ± 32.89 µM) was also a competitive inhibitor of DPP-IV, while KPW and FPK (82.52 ± 10.37 and 126.57 ± 8.45 µM, respectively) were mixed-type inhibitors. It should be emphasized that this study does not involve any clinical trial.

Effects of bovine milk and buffalo milk on lipid metabolism in mice.

Buffalo milk contains more polyunsaturated fatty acids than bovine milk. However, it is not clear about the effects of buffalo milk and bovine milk on lipid metabolism. In this study, a mouse model was used to explore the effects of buffalo milk and bovine milk on lipid metabolism in mice. The experiment was divided into three groups: a control group on a normal diet; a bovine milk group infused with bovine milk; a buffalo milk group infused with buffalo milk. We fed three groups of mice (n = 6) for 6 weeks. These results showed that bovine milk and buffalo milk had no effect on body weight gain. Bovine milk increased the content of ApoA1, ApoB and glucose in serum, compared with the control group, but buffalo milk has no profound change in serum ApoB. Remarkably, buffalo milk decreased the content of total cholesterol (TC) and triglyceride (TG) in the liver lipid profile, and also downregulated the expression of the carnitine palmitoyltransferase 2 (Cpt2) gene involved in the fatty acid oxidation in the liver. This study also found that bovine milk and buffalo milk did not cause the expression of pro-inflammatory factors in serum and colon tissues. This experiment proved that buffalo milk has beneficial effects on the regulation of lipid metabolism, and also does not affect the normal growth and pro-inflammatory response of the colon in mice. It provides a theoretical basis for future in-depth research on the special functions of buffalo milk and the development of buffalo milk functional foods.