Subclinical mastitis (SCM) is a major health problem of dairy animals in India and across the globe. An identification of potential risk factors of SCM can help for efficient udder health management in dairy animals. In this study, apparently healthy cows (HF crossbred: n = 45; Deoni: n = 43) were screened for SCM during different seasons through milk somatic cell count (SCC: reference test using 200 × 103 cells/ml as cut off value), California mastitis test (CMT) and differential electrical conductivity (DEC) test at an organized research farm. SCM positive milk samples (n = 34) were inoculated in selective media for Coliform sp., Streptococcus sp. and Staphylococcus sp. and DNA was isolated (n = 10) for species confirmation by 16s rRNA method. Both bivariate and multivariate models were used for risk assessment. We found the cumulative prevalence of 31 and 65% SCM in Deoni and crossbred cows, respectively. Screening of 328 crossbred cows under field conditions revealed point prevalence of 55% SCM. Multivariate analysis revealed stage of lactation (SOL), milk yield in previous lactation and test day milk yield in Deoni cows, as well as parity and mastitis treatment history in current lactation in HF crossbred cows as risk factors. SOL was a significant factor under field conditions. Receiver operated characteristic curve analysis revealed better accuracy of CMT than DEC. We found more mixed infections due to Staphylococcus sp. and Streptococcus sp. in culture, while 16s rRNA based molecular method revealed lesser-known pathogens associated with SCM. It is concluded that SCM prevalence rate is higher in crossbred than indigenous cows and these breeds have different risk factors for SCM. HF crossbred cows had similar SCM prevalence rate under different farming conditions, where CMT can be used for SCM diagnosis with excellent accuracy. The 16s rRNA method is useful for specific identification of lesser known and emerging mastitis pathogens.

This research communication paper addresses the hypothesis that the use of therapeutic alternatives for mastitis, such as intramammary ozone, can cure the disease with lower costs and without harmful residues for human consumption and without formation of microbial resistance like the ones caused by indiscriminate use of antibiotics in dairy farms. The study was performed in 36 mammary quarters from 12 dairy cows with subclinical mastitis grade three. The experimental units were randomly assigned into four groups and each group received a treatment. Treatments comprised (a) 20 μg/ml ozone gas; (b) 40 μg/ml ozone gas; (c) negative control treatment of 12.5 μg/ml ozonated saline and (d) positive control treatment of 100 mg of cephalexin + 100 mg of neomycin + 10 mg of prednisolone, all by intramammary injection. In all quarters, milk was collected before and after the application of treatments for California mastitis test and evaluation of milk composition, somatic cell count, and bacterial cultures. The results indicated that the use of intramammary ozone did have a therapeutic effect, and whilst this was less than that of antibiotics, ozone does confer some advantages. Treated milk had a good composition, the treatment cost was low, milk withdrawal may not be necessary and there is no risk of antibiotic resistance.

The objectives of this research were (1) to study different factors affecting milk total bacterial count (TBC) and (2) to estimate the economic value associated with TBC in Holstein dairy herds in Iran. The relationships between bulk tank TBC and farm management and economic factors were examined on 56 randomly selected intensive dairy farms. Herd management factors associated with bulk tank TBC were determined using mixed linear models. The median bulk tank TBC for the sample herds was 299 (range 81–1185) × 103 cfu/ml. The average economic premium opportunity from bulk tank TBC was US$ 1.32 per ton of milk ranging from US$ 0.02 per ton of milk for herds applying wet tissue procedures as teat cleaning material and washing the water troughs three times per day to US$ 5.20 per ton of milk for herds with dirty barns. Results showed that the following management factors were associated with low TBC and high economic value: frequency of cleaning water troughs, teat cleaning material, the frequency of milk delivery to the processor, bedding material, herd size, education level of workers, udder washing material, material of milking parlor wall, frequency of disinfection of the calving area, presence of veterinarian, water quality control, having a hospital pen and barn hygiene. In conclusion, our findings highlight the need to pay more attention to farm management issues, particularly farm hygiene practices to reduce milk TBC and so reduce the economic burden of TBC in dairy herds in Iran.

The objective of this experiment was to investigate the effect of reducing early lactation milking frequency on milk yield and persistency through lactation and early lactation fat mobilization, measured by body condition score (BCS) and BHB in milk. We hypothesized that milking cows twice per day in early lactation before milking them 3 times per day for the remaining lactation would cause less fat mobilization in early lactation, a lower peak milk yield but improved persistency throughout lactation compared with milking cows 3 times per day for the entire lactation. The experiment took place on 2 commercial dairy farms in Denmark. All cows calving in a period of nine months (n = 239) in their current first and later parities were randomly allocated at dry-off to 1 of 3 treatments based on expected calving date. The treatments were (1) cows milked 2 times per day for 1 week after calving, (2) cows milked 2 times per day for 4 weeks after calving and (3) cows milked 2 times per day for 7 weeks after calving. All cows were then milked 3 times per day for the remaining lactation. Milk yield peaked 3.3 and 3.6 d later and milk yield persistency improved with 18 and 19 g per day when cows were milked 2 times per day for 4 and 7 weeks, respectively, compared with milking 2 times per day for 1 week after calving. We found a significant highest milk BHB in treatment 2, but the underlying effect of milking cows 2 times per day for 4 weeks compared with 1 or 7 weeks was unclear. In conclusion, we did not confirm our hypothesis that milking cows 2 times per day compared to 3 times in early lactation would reduce fat mobilization and reduce peak yield. We did, however, find an improved milk yield persistency, which partially offset a numerical reduction in peak yield, and hence there was no significant effect of reducing early lactation milking frequency on total lactation (305 DIM) milk yield.

We evaluated the presence of bacterial pathogens in the milk of goats and their relationship with somatic cell count (SCC) and milk composition. The study was performed on a dairy farm in northern Slovakia. Half udder milk samples were collected from goats in June and July. The samples were divided on the basis of SCC into 4 bands (SCC1 lowest to SCC4 highest). Bacterial pathogens were only detected in 13% of samples. SCC3 and SCC4 had 15 and 25% positive samples respectively compared with SCC1 (2%) and SCC2 (14%). Coagulase-negative staphylococci (CNS) were the most common isolates (73%), of which Staphylococcus caprae was the most frequently isolated (65%). In samples with ≥ 1000 × 103 cells ml− 1 (SCC3, SCC4) there was higher somatic cell score (SCS) in the presence of a pathogen (7.48 ± 0.11) than without a pathogen (7.16 ± 0.05, P < 0.01). Statistically significant but weak negative correlations were observed between SCS and lactose, dry matter and non-fat dry matter. In conclusion, a higher percentage of bacteriologically positive milk samples was observed in both SCC3 and SCC4 groups but this does not explain the aetiology of high SCC in the milk of goats that are apparently free of bacteria. As a diagnostic tool, SCC is probably less useful in goats than in cows.

This research paper addresses the hypothesis that mast cells (MCs) contribute to the formation of mammary fibrosis. MCs are important immune regulatory and immune modulatory cells that play major roles in the inflammatory process. Since there is no detailed knowledge, this research study aimed to comparatively investigate the presence, localization, and immunophenotypes of MCs in healthy and mastitic mammary tissues. A total of 264 mammary samples were evaluated for the examination of mast cells and fibrosis. The mean mast cell number in both acute and chronic mastitis samples were very significantly higher than the control group P < 0.001). A 7.9-fold increase in the number of mast cells was found when the chronic mastitis group was compared with the control (healthy) group. Immunohistochemistry revealed presence of all three immune phenotypes in control and mastitic mammary samples (tryptase + (MCT), chymase + (MCC) and both chymase and tryptase + (MCTC). The mean MCT, MCC, and MCTC numbers in the chronic mastitis group were found to be significantly higher than the control (P < 0.001 for all three phenotypes) but did not differ significantly between control and acute mastitis samples. When the mean numbers of MCT, MCC, and MCTC in the control group and chronic mastitis group were compared, a 10.5, 7.8, and a 4.1-fold increase was observed, respectively. The amount of connective tissue was strongly increased in tissues with chronic mastitis and a 3.01-fold increase was detected compared to the control group. A statistically significant relation was also found between the amount of fibrosis and the increased number of total MCs (P < 0.001).

We studied the genetic polymorphism of beta-lactoglobulin (β-Lg) whey protein in Gangatiri zebu cows for this Research Communication. The polymorphic nature of milk protein fractions and their association with milk production traits, composition and quality has attracted several efforts in evaluating the allelic distribution of protein locus as a potential dairy trait marker. Genetic variants of β-Lg have highly significant effects on casein number (B > A) and protein recovery (B > A) and also determine the yield of cheese dry matter (B > A). Molecular techniques of polyacrylamide gel electrophoresis and high-resolution accurate mass-spectroscopy were applied to characterize the β-Lg protein obtained from the Gangatiri breed milk. Sequence analysis of β-Lg showed the presence of variant B having UniProt database accession number P02754, coded on the PAEP gene. Our study can provide reference and guidance for the selection of superior milk (having β-LgB) from this indigenous breed that could potentially give a good yield of β-Lg for industrial applications.

This research communication aimed to probe the genetic polymorphisms of alpha, beta, and kappa caseins in Gangatiri cows (an indigenous Indian cattle). Detection of variants has received considerable research interest in the dairy industry and animal breeding in recent years as a source of good quality protein, but also of bioactive peptides that may be linked to health implications. The polymorphic nature of casein fractions and their association with milk production traits, composition, and quality also attracted several efforts in evaluating the allelic distribution of different casein locus as a potential dairy trait marker. Molecular techniques of polyacrylamide gel electrophoresis and high-resolution accurate mass-spectrometry have been applied to this probe. Sequence analysis of different casein types in the cows showed the presence of four specific variants.

The performance of the starter culture is a critical factor that decides the quality of fermented milk. Dahi is a fermented milk product popular in India made using a mixed starter culture of lactic acid bacteria comprising acid and flavour producers. The prevalence of bacteriophages in the dairy environment can critically affect the activity of these starter cultures resulting in starter failure. As there is little information available on the occurrence of bacteriophages in the dairy environment of Kerala, this research communication examines the presence of lytic bacteriophages acting against three potential flavour-producing strains of Lacticaseibacillus paracasei (Lc. paracasei). Dairy effluent samples were screened for the presence of phages against the strains of Lc. paracasei by the multiple host enrichment method. Plates showing clearance zone in spot assay were confirmed for the presence of phages by double-layer agar assay. The plaques obtained in the double-layer agar assay were purified for further identification by next-generation sequencing. A bacteriophage infecting one of the three strains of Lc. paracasei was detected by the plaque assay and the blast annotation of the bacteriophage sequence found 86.05% similarity of the phage to Siphoviridae family. The study endorses the need for monitoring phages in the dairy environment to control phage-related starter failure in the state of Kerala.

This research paper aimed to locate protein modifications caused by treatment of milk and determine if the modification locations were consistent. The majority of milk for consumption is homogenised using pressure and heat, and this causes changes in the location of proteins in the milk as well as protein modifications. To investigate these proteomic changes, raw milk was pasteurised (72°C, 15 s), then, to separate the treatment for homogenisation, heated at these different pressures and temperatures: 45°C without no pressure applied, 45°C with 35 MPa, 80°C without pressure applied and 80°C, with 35 MPa. Proteomic analysis was done after separating the milk into three fractions: whey, casein and cream. Protein modifications in each fraction were examined and we found Maillard products as well as oxidation to be of interest. The proteins were also further identified and characterised to compare protein modification sites and differences in proteins present in the cream resulting from homogenisation and/or pasteurisation. This experiment showed that both heat and pressure during homogenisation can cause increases in protein modifications as a result of oxidation or the Maillard reaction. Total cysteine oxidation and total proline oxidation differed between treatments although this was only significantly different for cysteine. It was observed that protein modifications occurred in the same location in the protein sequence rather than in random locations which we highlighted by examining α-S1-casein, lactadherin and β-lactoglobulin.

The effects of high hydrostatic pressure (HHP) and heat treatments on antimicrobial protein stability and on the physico-chemical, microbiological, rheological and shelf-life properties of donkey milk were investigated. Although heat treatment at 75°C for 2 min resulted in 1.50 log CFU ml−1 microbial inactivation, losses in activities of lysozyme (58%) and lactoferrin (82%) were observed due to whey protein denaturation. By contrast, HHP application at 400 MPa caused lower enzyme activity losses (22 and 37% respectively) whilst maintaining a significant reduction of microbial load (1.80 log CFU ml−1). Color analyses showed that the lightness values of all samples decreased during storage. Higher flow consistency (viscosity) and lower flow behavior indexes were observed in heat-treated samples compared to untreated and HHP-treated ones, which can be explained by advanced protein denaturation during heat-treatment. The results suggest that HHP is a more suitable process than heat treatment for preservation of donkey milk within the conditions studied.

When implementing the transition from regular milk production to the dry period, drying off is mostly conducted simply by abrupt cessation of milking. Efforts to reduce milk synthesis before cessation of milking aim to reduce stress in cows as well as to lower the risk of mastitis. A previous study demonstrated that incomplete milking during the last ten days of lactation gradually reduced the milk yield of healthy, high-yielding cows. However, a reduction period of ten days might be too long for cows with lower yields. Therefore, a follow-up study was conducted on an organic dairy research farm with a lower average milk yield. We investigated whether automated incomplete milking can reduce milk synthesis within one week without impairing the health status of udder quarters infected with minor pathogens. Before drying off, 15 German-Holstein cows with 58 lactating quarters, 21 of which were infected with minor pathogens (coagulase-negative Staphylococci (CNS) and Corynebacterium bovis), were milked twice daily using the software module AutoDry (GEA Farm Technologies) with the 5%-step-down-per-day-program. The level of udder emptying was gradually reduced over 8–10 milkings beginning at a mean milk yield of 17.2 ± 4.4 kg d−1. During the last three milkings before drying off, milking clusters were automatically removed when a milk flow rate of 0.3 kg min−1 was reached. Quarter fore-milk samples were collected at two time points (14 d before and at the date of the last milking, ie drying off) and were analysed for mastitis pathogens and somatic cell count. The gradual reduced emptying of the udder induced a clear decline in milk yield by 0.8 ± 0.3 kg d−1. Within the reduction period, the somatic cell count of quarter foremilk did not change regardless of the infection status. Furthermore, no cow suffered from clinical mastitis. Thus, a reduction in milk synthesis could be achieved within less than one week before drying off without any impairment in udder health of cows, even when infected with minor mastitis pathogens.

This research communication delineates the quality of pasteurized cow milk sold in Brazil from 2015 to 2020. A cross-sectional study was performed gathering 1749 samples, which were evaluated for microbiological and physicochemical parameters, including Salmonella spp., total and thermotolerant coliforms, freezing point, alkaline phosphatase and lactoperoxidase. The proportion of compliant and non-compliant samples was compared through the years and jurisdiction of the inspection services. Interactions between the design and response variables were assessed by log-linear analysis. Overall, a considerable non-conformity rate (12%) was found for at least one microbiological or physicochemical parameter. Post-pasteurization contamination by coliforms was the major challenge for dairy industries. Notably, the non-compliance rate for freezing point increased during the SARS-CoV-2 pandemic. In addition, the ability to comply was linked to the type of inspection service. Thus, it is suggested that the SARS-CoV-2 pandemic is affecting the dairy industries in Brazil, and we strengthen the need for more studies monitoring the quality of milk over the years, which could assist industries and regulatory agencies to ensure the compliance of pasteurized milk.

Homogenisation is a widely used technique in manufacturing powdered milk with a direct impact on product solubility, and the homogenisation pressure is a central attribute of this process. We aimed to understand the effect of increasing homogenisation pressures (0/0, 15/5, and 75/5 MPa, 1st/2nd stages) on particle-size distribution during homogenised whole milk powder manufacture and rehydration of the final product. The fluid milk was thermally treated, homogenised, concentrated by rotary evaporation, and then dried using a spray dryer. Particle size (Dv90) was monitored at all stages of the manufacturing process. The final product (milk powder) was analysed using particle-size distribution, electronic scanning microscopy, water activity, and isotherms. The results demonstrated that increasing the homogenisation pressure leads to milk powder with smaller particle size when rehydrated (Dv90 values: 6.08, 1.48 and 0.64 μm for 0, 20 and 80 MPa, respectively). Furthermore, the volume (%) of the particles in the ‘sub-micro’ region (smaller than 1.0 μm) presented an inversely proportional profile to the homogenisation pressure (homogenised fluid milk: 86.1, 29.3 and 2.4%; concentrated milk: 86.1, 26.5 and 5.7%, and reconstituted milk powder: 84.2, 31.8 and 10.9%). Surprisingly, this pattern was not observed in the SPAN value (which corresponds to the width or range of the size distribution based on the volume). Additionally, the increase in the homogenisation pressure did not affect the sorption isotherm pattern. These results demonstrate that increasing the homogenisation pressure decreases the particle size of the reconstituted powdered milk, indicating the potential for future studies on how this phenomenon affects its physicochemical and final product properties.

This research communication was designed to evaluate the effects of different levels of diet restriction on the composition and ethanol stability (MES) of raw bovine milk. This research was carried out using three electronic databases: Scopus, Pubmed and Web of Science. The main inclusion criteria were: (i) original research, (ii) use of alcohol (ethanol) test as a method to assess milk stability, (iii) measure different levels of feed restriction and (iv) allow access to the raw data of articles. Of the nine publications that addressed the subject filtered by the systematic review, seven fitted the selection criteria and were selected to perform the meta-analysis. Feed restriction (reduction of 20, 30, 40 and 50% of the dietary dry matter offered) decreased (P < 0.01) milk yield (−18%), ethanol stability (−5%), acidity (−4%), protein (−3%) and lactose (−2%) concentrations, but did not affect the values of pH, density, fat and total solids concentrations, nor somatic cell count. The correlation between milk yield and MES was low but positive and numerically higher in the control group compared with the restriction group. The milk of cows fed the control diet presented greater ethanol stability (76.5%) compared with milk of cows fed the restrictive diet (72.8%). This decrease by up to 4 percentage units due to restriction levels ranging from 20 to 50% of diet intake may cause limitations in milk processing at the dairy industry, increasing milk rejection.

We investigated sonication as a pre-processing step to reduce acid whey generation during Greek yoghurt manufacture. The generation of a large amount of acid whey during the production of Greek yoghurt is an ongoing problem in the dairy industry and many studies are currently focusing on reducing it. We focused on the use of ultrasonication as a novel approach to minimize the casein fraction in the acid whey stream and simultaneously improve the gel properties. Ultrasound applied before the fermentation modified the structural properties and bonding behaviours of milk proteins, and enhanced the retention of casein in the yoghurt gel after the fermentation and straining steps. Therefore, the use of low-frequency ultrasonication as a pre-processing step may have the potential to provide significant economic benefits to the Greek yoghurt manufacturing process. Moreover, it improved the nutritional and physicochemical properties compared to regular Greek yoghurts.

This research paper addresses the hypothesis that powdered milk may contain amyloid fibrils. Amyloids are fibrillar aggregates of proteins. Up to this time, research on the presence of amyloids in food products are scarce. To check the hypothesis we performed thioflavin T fluorescence assay, X-ray powder diffraction, atomic force microscopy and fluorescence microscopy imaging. Our preliminary results show that commercially available milks contain fibrils that have features characteristic to amyloids. The obtained results can be interpreted in two opposite ways. The presence of amyloids could be considered as a hazard due to the fact that food products may induce amyloid related diseases. On the other hand, the presence of amyloids in traditionally consumed foodstuffs could serve as proof that fibrils of food proteins do not pose a threat for consumers.

Set yogurt's physical characteristics are greatly affected by the homogenization and heat treatment processes. In our previous study, set yogurt treated at 130°C and with the fat particle size reduced to ≤0.6 μm had equivalent curd strength, less syneresis and smoother texture than yogurt treated at 95°C. When investigating the mechanisms underlying yogurt's physical properties, it is important to evaluate the yogurt's microstructure. We conducted electron microscopy evaluations to investigate the mechanisms of changes in yogurt's physical properties caused by 130°C heat treatment and by a reduction in the fat globule size. We prepared yogurt mixtures by combining heat treatment at 95 and 130°C and homogenization pressure at 10 + 5 and 35 + 5 MPa and then fermented the mixtures in a common yogurt starter. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used for the structural observations. Fine particles were observed on the surface of the casein micelles of the yogurt treated at 95°C, and the coalescence density between micelles was high. The surface of the yogurt treated at 130°C had few fine particles, and the coalescence density between micelles was low. The yogurt treated at 130°C with 35 + 5 MPa homogenization had low coalescence density between casein micelles, but smaller-particle-size fat globules increased the network density. Approximately 30% of the fat globules were estimated to be incorporated into the yogurt networks compared to the volume of casein micelles. We speculate that 130°C heat treatment alters the structure of whey protein on the surface of casein micelles and interferes with network formation, but reducing the size of fat globules reinforces the network as a pseudoprotein.

The aim in this research paper was to investigate the effect of using calcium monophosphate (MCP) and MCP mixed with commercial phosphates salts, in total or partial replacement of calcium chloride (CaCl2) in the manufacture of Minas Frescal cheese. Initially, model cheeses were made to perform the rheological analysis during the coagulation process. Of these, the five best treatments were chosen to carry out the production of Minas Frescal cheese, used only CaCl2 and MCP, and partial replacements of MCP + polyphosphate, MCP + potassium monophosphate (MKP) and MCP. The cheeses showed no significant difference in physicochemical composition, yield and syneresis, however, the cheese with partial replacement of CaCl2 by MCP + polyphosphate and MCP + MKP showed the highest hardness values, like the control. This demonstrates that it is possible to replace calcium chloride without significant changes in the physicochemical characteristics and yield of Minas Frescal cheese, and it is still possible to modulate the hardness of the cheese produced according to the type of calcium/phosphate source used. This allows the industry to replace the source of calcium in the manufacture of Minas Frescal cheese according to the desired hardness.

This research paper aimed to examine the antibacterial activity of lactoferrin (LF) as a potential natural alternative in the dairy sector, by measuring its minimum inhibitory concentration (MIC) against a number of common food-borne pathogens as well as Pseudomonas aeruginosa, one of the major dairy product spoiling microorganisms. Additionally, a viability experiment was applied to laboratory-manufactured set yoghurt to assess its impact on the activity of starter culture, sensory properties and STEC survivability. The findings demonstrated that LF exhibited significant antimicrobial activity, particularly against E. coli and S. typhimurium with MIC values of 0.0001 and 0.01 mg/ml, respectively. However, P. aeruginosa and B. cereus were quite resistant to LF requiring higher concentrations for MIC (2.5 mg/ml). By the third day of storage, LF at 0.0001 and 0.001 mg/ml significantly reduced the survivability of Shiga toxin-producing E. coli STEC by 70 and 91.6%, respectively, in the lab-manufactured yoghurt. Furthermore, LF enhanced the sensory properties of fortified yoghurt with a statistically significant difference in comparison to the control yoghurt group. There was no interference with the activity of the starter culture throughout the manufacturing process and the storage period. In conclusion, the potent antimicrobial effect of LF opens a new avenue for the dairy industry's potential applications of LF as a natural preservative without negatively influencing the sensory properties and starter culture activity of fermented products.