Bacteria In Milk Research Articles

Phylogenetic variation in raw cow milk microbiota and the impact of forage combinations and use of silage inoculants.

The microbiota of bulk tank raw milk is known to be closely related to that of microbial niches of the on-farm environment. Preserved forage types are partof this ecosystem and previous studies have shown variations in their microbial ecology. However, little is known of the microbiota of forage ration combinations and the transfer rates of associated species to milk. We identified raw milk bacteria that may originate from forage rations encompassing either hay (H) or grass/legume silage uninoculated (GL) as the only forage type, or a combination of GL and corn silage uninoculated (GLC), or grass/legume and corn silage both inoculated (GLICI). Forage and milk samples collected in the fall and spring from 24 dairy farms were analyzed using 16S rRNA gene high-throughput sequencing following a treatment with propidium monoazide to account for viable cells. Three community types separating H, GL, and GLICI forage were identified. While the H community was co-dominated by Enterobacteriaceae, Microbacteriaceae, Beijerinckiaceae, and Sphingomonadaceae, the GL and GLICI communities showed high proportions of Leuconostocaceae and Acetobacteraceae, respectively. Most of the GLC and GLICI rations were similar, suggesting that in the mixed forage rations involving grass/legume and corn silage, the addition of inoculant in one or both types of feed does not considerably change the microbiota. Raw milk samples were not grouped in the same way, as the GLC milk was phylogenetically different from that of GLICI across sampling periods. Raw milk communities, including the GLICI group for which cows were fed inoculated forage, were differentiated by Enterobacteriaceae and other Proteobacteria, instead of by lactic acid bacteria. Of the 113 amplicon sequence variants (ASVs) shared between forage rations and corresponding raw milk, bacterial transfer rates were estimated at 18 to 31%. Silage-based forage rations, particularly those including corn, share more ASVs with raw milk produced on corresponding farms compared to that observed in the milk from cows fed hay. These results show the relevance of cow forage rations as sources of bacteria that contaminate milk and serve to advance our knowledge of on-farm raw milk contamination.

Associations between the Bacterial Composition of Farm Bulk Milk and the Microbiota in the Resulting Swedish Long-Ripened Cheese.

The maturation of a traditional Swedish long-ripened cheese has shown increasing variation in recent years and the ripening time is now generally longer than in the past. While the cheese is reliant on non-starter lactic acid bacteria for the development of its characteristic flavour, we hypothesised that the observed changes could be due to variations in the microbiota composition and number of bacteria in the raw milk used for production of the cheese. To evaluate associations between microbiota in the raw milk and the resulting cheese, three clusters of commercial farms were created to increase variation in the microbiota of dairy silo milk used for cheese production. Cheese production was performed in three periods over one year. Within each period, milk from the three farm clusters was collected separately and transported to the cheese production facility. Following pasteurisation, the milk was processed into the granular-eyed cheese and matured at a dedicated cheese-ripening facility. For each cheese batch, farm bulk and dairy silo milk samples, a starter culture, early process samples and cheese samples from different stages of maturation (7-20 months) were collected and their microbiota characterised using 16S rRNA amplicon sequencing. The microbiota in the farm bulk milk differed significantly between periods and clusters. Differences in microbiota in dairy silo milk were observed between periods, but not between farm clusters, while the cheese microbiota differed between periods and clusters. The top 13 amplicon sequence variants were dominant in early process samples and the resulting cheese, making up at least 93.3% of the relative abundance (RA). Lactococcus was the dominant genus in the early process samples and, together with Leuconostoc, also dominated in the cheese samples. Contradicting expectations, the RA of the aroma-producing genus Lactobacillus was low in cheese during ripening and there was an unexpected dominance of starter lactic acid bacteria even at the later stages of cheese ripening. To identify factors behind the recent variations in ripening time of this cheese, future studies should address the effects of process variables and the dairy environment.

Effect of Thermostable Enzymes Produced by Psychrotrophic Bacteria in Raw Milk on the Quality of Ultra-High Temperature Sterilized Milk.

Ultra-high temperature sterilized milk (UHT) is a popular dairy product known for its long shelf life and convenience. However, protein gel aging and fat quality defects like creaming and flavor deterioration may arise during storage. These problems are primarily caused by thermostable enzymes produced by psychrotrophic bacteria. In this study, four representative psychrotrophic bacteria strains which can produce thermostable enzymes were selected to contaminate UHT milk artificially. After 11, 11, 13, and 17 weeks of storage, the milk samples, which were contaminated with Pseudomonas fluorescens, Chryseobacterium carnipullorum, Lactococcus raffinolactis and Acinetobacter guillouiae, respectively, demonstrated notable whey separation. The investigation included analyzing the protein and fat content in the upper and bottom layers of the milk, as well as examining the particle size, Zeta potential, and pH in four sample groups, indicating that the stability of UHT milk decreases over time. Moreover, the spoiled milk samples exhibited a bitter taste, with the dominant odor being attributed to ketones and acids. The metabolomics analysis revealed that three key metabolic pathways, namely ABC transporters, butanoate metabolism, and alanine, aspartate, and glutamate metabolism, were found to be involved in the production of thermostable enzymes by psychrotrophic bacteria. These enzymes greatly impact the taste and nutrient content of UHT milk. This finding provides a theoretical basis for further investigation into the mechanism of spoilage.

Occurrence of some probiotic bacteria in ewe's and goat's milk in Assuit City with regarding to genes responsible for production of bacteriocins.

Occurrence of some probiotic bacteria in ewe's and goat's milk in Assuit City with regarding to genes responsible for production of bacteriocins.

Identification, proteolytic activity quantification and biofilm-forming characterization of Gram-positive, proteolytic, psychrotrophic bacteria isolated from cold raw milk.

Psychrotrophic bacteria of raw milk face the dairy industry with significant spoilage and technological problems due to their ability to produce heat-resistant enzymes and biofilms. Despite extensive information about Gram-negative psychrotrophic bacteria in milk, little is known about Gram-positive psychrotrophic bacteria in milk, and their proteolytic activity and biofilm-forming characteristics. In the present study, Gram-positive, proteolytic, psychrotrophic bacteria of cold raw milk were identified, and their proteolytic activity and biofilm-forming capacity were quantified. In total, 12 genera and 22 species were represented among the bacterial isolates, however 50% belonged to three genera, namely Staphylococcus (19.4%), Bacillus (16.7%), and Enterococcus (13.9%). Different levels of proteolytic activity were detected in the identified isolates, even among the strains belonging to the same species. In addition, proteolytic activity was significantly higher at 25°C than at 7°C for all isolates. The crystal violet staining assay in polystyrene microtitre plates revealed a high level of variation in the biofilm-forming capacity at 7°C. After 72 hours of incubation, 11.1% of the strains did not produce a biofilm, while 27.8%, 52.8%, and 8.3% produced low, moderate, and high amounts of biofilm on polystyrene, respectively. The psychrotrophic bacteria were also able to produce biofilms on the surface of stainless steel coupons in ultra-high temperature milk after 72 h of incubation at 7°C; the number of attached cells ranged from 1.34 to 5.11 log cfu/cm2. These results expand the knowledge related to the proteolytic activity and biofilm-forming capacity of Gram-positive psychrotrophic milk bacteria.

The interaction among Kluyveromyces marxianus G-Y4, Lacticaseibacillus paracasei GL1, and Lactobacillus helveticus SNA12 and signaling molecule AI-2 participate in the formation of biofilm

In this study, two strains of lactic acid bacteria (Lacticaseibacillus paracasei GL1 and Lactobacillus helveticus SNA12) and one yeast strain of Kluyveromyces marxianus G-Y4 (G-Y4) isolated from Tibetan kefir grains were co-cultured. It was found that the addition of G-Y4 could not only promote the growth of lactic acid bacteria, but also increase the release of metabolites (lactic acid, ethanol, and amino nitrogen). Furthermore, the addition of live cells and cell-free fermentation supernatant (CFS) of G-Y4 could increase the ability of biofilm formation. Morever, the surface characteristics results showed that the addition of G-Y4 live cells could enhance the aggregation ability and hydrophobicity of LAB. Meanwhile, adding live cells and CFS of G-Y4 could promote the release of signaling molecule AI-2 and enhance the expression of the LuxS gene related to biofilm formation. In addition, Fourier-transform infrared spectroscopy and chemical composition analysis were used to investigate the composition of the biofilm, and the results indicated that the biofilm was mainly composed of a small amount of protein but it was rich in polysaccharides including glucose, galactose, and mannose with different ratios. Finally, the formation of biofilm could delay the decline of the number of viable bacteria in storage fermented milk.

Dual Role of Probiotic Lactic Acid Bacteria Cultures for Fermentation and Control Pathogenic Bacteria in Fruit-Enriched Fermented Milk.

The food industry has been developing new products with health benefits, extended shelf life, and without chemical preservation. Bacteriocin-producing lactic acid bacteria (LAB) strains have been evaluated for food fermentation to prevent contamination and increase shelf life. In this study, potentially probiotic LAB strains, Lactiplantibacillus (Lb.) plantarum ST8Sh, Lacticaseibacillus (Lb.) casei SJRP38, and commercial starter Streptococcus (St.) thermophilus ST080, were evaluated for their production of antimicrobial compounds, lactic acid and enzyme production, carbohydrate assimilation, and susceptibility to antibiotics. The characterization of antimicrobial compounds, the proteolytic activity, and its inhibitory property against Listeria (List.) monocytogenes and Staphylococcus (Staph.) spp. was evaluated in buriti and passion fruit-supplemented fermented milk formulations (FMF) produced with LAB strains. Lb. plantarum ST8Sh was found to inhibit List. monocytogenes through bacteriocin production and produced both L(+) and D(-) lactic acid isomers, while Lb. casei SJRP38 mainly produced L(+) lactic acid. The carbohydrate assimilation profiles were compatible with those usually found in LAB. The potentially probiotic strains were susceptible to streptomycin and tobramycin, while Lb. plantarum ST8Sh was also susceptible to ciprofloxacin. All FMF produced high amounts of L(+) lactic acid and the viability of total lactobacilli remained higher than 8.5 log CFU/mL during monitored storage period. Staph. aureus ATCC 43300 in fermented milk with passion fruit pulp (FMFP) and fermented milk with buritipulp (FMB), and Staph. epidermidis KACC 13234 in all formulations were completely inhibited after 14days of storage. The combination of Lb. plantarum ST8Sh and Lb. casei SJRP38 and fruit pulps can provide increased safety and shelf-life for fermented products, and natural food preservation meets the trends of the food market.

Antioxidant, antimicrobial and emulsification properties of exopolysaccharides from lactic acid bacteria of bovine milk: Insights from biochemical and genomic analysis

Exopolysaccharides (EPSs) are versatile organic thickeners derived from microbes, offering beneficial bioactive properties. This study focused on isolating new bacterial strains from bovine milk capable of EPS biosynthesis. Out of 63 isolates, four strains, Lactiplantibacillus sp. ME2b, Lactococcus sp. ME7, Lacticaseibacillus sp. ME17, and Lactobacillus sp. ME27a, exhibited high EPS production, ranging from 0.98 g/L to 2.68 g/L. EPS extracted from these strains had higher carbohydrate and lower protein contents. The EPS extracts showed strong antioxidant activity, demonstrated by DPPH (∼82–89%) and hydroxyl radical scavenging (∼88–91%), and reducing power capacity (3.28–3.65). They also displayed potent antimicrobial activity against Gram-negative and Gram-positive pathogens, including Bacillus cereus, Salmonella typhi, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, with stronger inhibition against Gram-positive strains. Additionally, the EPS showed emulsification activity with ME17 showing the highest emulsification index (60%). The EPS extracts exhibited significant anti-inflammatory effects (∼81–84%). Whole genome sequencing of the prominent strain ME2b revealed a 3,281,666 kb genome carrying 3,101 coding sequences including EPS biosynthesis-related genes epsABCDE, wxz, wzy, and glycosyltransferases. These findings underscore significant potential of the EPS in diverse applications, particularly in food and biotechnology sectors, with future efforts aimed at maximizing EPS capacities for industries and driving innovation across multiple sectors.

Modelling and Predicting the Growth of Escherichia coli and Staphylococcus aureus in Co-Culture with Geotrichum candidum and Lactic Acid Bacteria in Milk

The growth of two pairs of co-cultures (Escherichia coli/Geotrichum candidum and Staphylococcus aureus/Geotrichum candidum) with a starter culture of lactic acid bacteria was studied in milk at temperatures ranging from 15 °C to 21 °C, related to the ripening of artisanal cheese. For an inoculum of approximately 106 CFU/mL, LAB not only induced an early stationary phase of E. coli (two isolates BR and PS2) and S. aureus (isolates 2064 and 14733) but also affected their death phase. In co-cultures with LAB and G. candidum, the numbers of E. coli and S. aureus increased in 2 logs and 1 log, respectively, reaching maximum population densities (MPDs) of less than 5 and 4 logs, respectively. After that, the populations of both bacteria represented with two isolates decreased in more than 2 logs and 3 logs within 2 days compared to their MPDs, respectively. G. candidum was found to be the subject of interactions with LAB within a given temperature range only partially. To develop a tertiary model for the growth curves of the populations, a one-step approach was used, combining the Huang-Gimenez and Dalgaard primary model with secondary square-root models for growth rate and lag time. Furthermore, the reparametrized Gompertz-inspired function with the Bigelow secondary model was used to describe the death phase of the E. coli and S. aureus isolates. The prediction ability of the growth of the H-GD tertiary model for co-cultures was cross-validated within the isolates and datasets in milk and milk medium with 1% NaCl. The study can be used as knowledge support for the hygiene guidelines of short-ripened raw milk cheeses, as was our case in Slovakia.

The Enzymatic Hydrolysis of Human Milk Oligosaccharides and Prebiotic Sugars from LAB Isolated from Breast Milk.

Breastfeeding is essential in the first months of a newborn's life. Breast milk is a source of crucial macronutrients, prebiotic oligosaccharides, and potential probiotic strains of bacteria. Oligosaccharides from breast milk (HMOs) are a significant part of the composition of breast milk and represent a complex of digestible sugars. This study aims to elucidate the enzymatic hydrolysis of these oligosaccharides and other prebiotics by the bacteria present in breast milk. We used modified methods to isolate oligosaccharides (HMOs) from human milk. Using unique techniques, we isolated and identified different bacteria from breast milk, mainly Lactobacillus fermentum. Using enzymatic analyses, we established the participation of α-fucosidase, α-glucosidase, β-galactosidase, and β-glucosidase from breast milk bacteria in the hydrolysis of prebiotic sugars. We also optimized the scheme for isolating oligosaccharides from breast milk by putting the lyophilized product into different food media. We found that the oligosaccharides from breast milk (HMOs) are a potent inducer for the secretion of the studied bacterial enzymes. Also, we found that all the lactobacilli strains we studied in detail could digest mucin-linked glycans. The degradation of these sugars is perhaps a built-in defense mechanism in cases where other sugars are lacking in the environment. We also determined fucosidase activity in some of the isolated strains. We recorded the highest values (2.5 U/mg in L. fermentum ss8) when the medium's oligosaccharides isolated from breast milk were present. Lactobacilli and Bifidobacteria supplied with breast milk are the first colonizers in most cases in the gastrointestinal tract of the newborn. The presence and study of different genes for synthesizing other enzyme systems and transporters of various sugars in this type of bacteria are essential.

Combined treatment of ε-polylysine and thermosonication to inactivate Bacillus cereus and their application in pasteurized milk

At present, most liquid milk products use high-temperature short-time heat treatment (HTST). However, HTST treatment cannot completely inactivate the bacteria in milk, which seriously affects the quality, safety, and shelf life of the product. Therefore, exploring the combined use of HTST and other technologies is of great importance to the production of high-quality pasteurized milk. In this study, we compared thermosonication (TS), ε-Polylysine (ε-PL), ε- PL combined with TS (ε-PL-TS), and HTST to inactivate B. cereus in pasteurized milk. Based on this comparison, we further discussed the inactivation ability and potential mechanism of ε-PL-TS treatment on B. cereus. The experimental result indicated that ε-PL-TS treatment had an obvious inactivation impact on B. cereus. The cell architecture was damaged by ε-PL-TS treatment, causing the leakage of proteins and DNA from the cell. ε-PL-TS treatment caused metabolic disorders by reducing the activities of key enzymes such as succinate dehydrogenase (SDH) (72.5%), malate dehydrogenase (MDH) (70.5%), and ATPase (80.3%). Meanwhile, the activity of CAT and POD decreased by 42.5% and 48.7%, respectively, suggesting that oxidative stress may be involved in the bactericidal mechanism. In addition, the combined treatment effectively inhibits microbial proliferation of pasteurized milk during storage at 4 °C. Therefore, ε-PL-TS treatment can be used as a potential new inactivated microorganism technology that is helpful to obtain high-quality pasteurized milk with a long shelf life.

High-Pressure Processing of Human Milk: A Balance between Microbial Inactivation and Bioactive Protein Preservation

BackgroundDonor human milk banks use Holder pasteurization (HoP; 62.5°C, 30 min) to reduce pathogens in donor human milk, but this process damages some bioactive milk proteins. ObjectivesWe aimed to determine minimal parameters for high-pressure processing (HPP) to achieve >5-log reductions of relevant bacteria in human milk and how these parameters affect an array of bioactive proteins. MethodsPooled raw human milk inoculated with relevant pathogens (Enterococcus faecium, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii) or microbial quality indicators (Bacillus subtilis and Paenibacillus spp. spores) at 7 log CFU/mL was processed at 300–500 MPa at 16–19°C (due to adiabatic heating) for 1–9 min. Surviving microbes were enumerated using standard plate counting methods. For raw milk, and HPP-treated and HoP-treated milk, the immunoreactivity of an array of bioactive proteins was assessed via ELISA and the activity of bile salt-stimulated lipase (BSSL) was determined via a colorimetric substrate assay. ResultsTreatment at 500 MPa for 9 min resulted in >5-log reductions of all vegetative bacteria, but <1-log reduction in B. subtilis and Paenibacillus spores. HoP decreased immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G, lactoferrin, elastase and polymeric immunoglobulin receptor (PIGR) concentrations, and BSSL activity. The treatment at 500 MPa for 9 min preserved more IgA, IgM, elastase, lactoferrin, PIGR, and BSSL than HoP. HoP and HPP treatments up to 500 MPa for 9 min caused no losses in osteopontin, lysozyme, α-lactalbumin and vascular endothelial growth factor. ConclusionCompared with HoP, HPP at 500 MPa for 9 min provides >5-log reduction of tested vegetative neonatal pathogens with improved retention of IgA, IgM, lactoferrin, elastase, PIGR, and BSSL in human milk.

Survival of lactic acid bacteria species in Filipino donor milk subjected to different heating temperatures

This study aimed to characterize the behaviors of a cocktail of lactic acid bacteria (LAB) isolated from human donor milk after subjecting to processing at different temperatures. The isolates (n = 12) were identified using 16S rRNA sequencing prior to the propagation and challenge studies. BLAST comparisons revealed that the isolates were Streptococcus spp., Lactobacillus spp., and Enterococcus spp. (% Identity 91.20–98.67%). At an initial inoculation level (8.0 log CFU/ml), the isolates exhibited a non-linear inactivation curve in all temperatures. In the initial logarithmic-linear phase, a decreasing D-value that ranged from 1.70 to 0.20 min was observed with increasing heating temperatures (55 °C, 57 °C, 60 °C, and 63 °C). Microbial load reduction in this initial phase (LR1) also increased (3.31–5.42 log CFU/ml). Moreover, the time necessary to reach LR1 (tLR1), prior to the inactivation curve inflection to tailing, generally decreased. During tailing, minimal to no additional microbial load changes were observed. Total reduction (TLR) increased (3.32–5.42 log CFU/ml), the time it took to reach TLR (tTLR) decreased (60.00–40 min), and the survivor population decreased (4.83–2.63 log CFU/ml) with increasing temperature. The study was able to establish the thermal challenging behaviors and kinetic parameters of a cocktail of lactic acid bacteria (LAB) in human donor milk with known composition and physicochemical properties. The inactivation kinetic parameters were determined from the curves and significantly varied with increasing heating temperature. This study provides a basis for evaluation and possible improvement of the Holder pasteurization (HoP) protocols for human donor milk, specifically temperatures below HoP that allow for keeping the milk biologically active without compromising quality.

Community diversity of psychrophilic bacteria in dairy farm raw milk and its characteristic enzyme production at different temperature

Psychrophilic bacteria and their heat-resistant enzymes are important risk factors affecting the quality of shelf life of UHT milk, and it is important for the development of the industry to uncover their action pattern. In these study, 40 raw milk samples were taken from dairy farms in Hebei (northern China) and Guangdong (southern China) in four seasons. The structure and composition of psychrophilic bacteria by PCR targeting 16S rRNA, as well as its extracellular protease, lipase activity and heat resistance was analyzed. The results showed that there was a significant temperature dependence but not geographical dependence in the distribution of the psychrophilic bacteria community. Secondly, P. fluorescens, P. azotoformans and P. paralactis were not only the dominant bacterium, but also had higher proteolytic and lipolytic activities. Lastly, Analysis of the enzyme-producing heat resistance of a typical psychrophilic bacteria, P. azotoformans B15-1. At 135 °C, the average residual rate of protease only 3.08%, however, lipase was more heat resistant and the average residual rate was still 19.16%. The present study investigated the psychrophilic bacteria and its hydrolytic enzymes activity in raw milk from different regions and seasons, in order to assess the baseline of psychrophilic bacteria contamination in the dairy farm.

Clinical aspects of treatment of breast skin wounds in cats

Injuries to the skin of the mammary gland are one of the leading causes of mastitis in lactating cats. Given the progressive nature of the problem of mastitis in cats, the important role of mammary skin injuries in their occurrence and the limited range of drugs for their treatment, we conducted studies aimed at studying bacterial contamination of the mammary gland skin in lactating cats and the effectiveness of its correction with pharmacological agents with different mechanisms of action. . In the conditions of a shelter for homeless animals located in the Voronezh region, studies were conducted to study the effectiveness of several pharmacological agents for the treatment of mammary gland injuries in lactating cats.A comparative analysis of the pharmacological action of Bepanthen plus ointment, 1% dioxidine solution and its combination with PEG-400 showed that these agents have a pronounced sanitizing effect on the skin surface. However, with extensive and deep damage, skin treatment with agents that do not have or with mild transdermal properties does not significantly affect the degree of contamination of the deep layers of the mammary gland parenchyma and the number of bacteria in milk. Strengthening the penetrating ability of dioxidine, in particular with the help of PEG-400, increased the efficiency of milk sanitation, and hence the mammary gland parenchyma, by 48%. The results of the studies have shown that, depending on the degree of damage to the skin of the breast, there is a corresponding risk of progression of wound infection, contamination of breast tissues and the development of mastitis.

Ultrasensitive Nonenzymatic Real-Time Hydrogen Peroxide Monitoring Using Gold Nanoparticle-Decorated Titanium Dioxide Nanotube Electrodes.

An amperometric enzyme-free hydrogen peroxide (H2O2) sensor was developed by catalytically stabilizing active gold nanoparticles (Au NPs) of 4-5 nm on a porous titanium dioxide nanotube (TiO2 NTs) electrode. The Au NPs were homogeneously distributed on anatase TiO2 NTs with an outer diameter of ~102 nm, an inner diameter of ~60 nm, and a wall of thickness of ~40 nm. The cyclic voltammogram of the composite electrode showed a pair of redox peaks characterizing the electrocatalytic reduction of H2O2. The entrapping of Au NPs on TiO2 NTs prevented aggregation and facilitated good electrical conductivity and electron transfer rate, thus generating a wide linear range, a low detection limit of ~104 nM, and high sensitivity of ~519 µA/mM, as well as excellent selectivity, reproducibility, repeatability, and stability over 60 days. Furthermore, excellent recovery and relative standard deviation (RSD) were achieved in real samples, which were tap water, milk, and Lactobacillus plantarum bacteria, thereby verifying the accuracy and potentiality of the developed nonenzymatic sensor.

Effects of carao (Cassia grandis L.) on physico-chemical, microbiological and rheological characteristics of yogurt

Yogurt fortification with functional ingredients can increase health benefits. Carao fruit has bioactive compounds which could exert antidiabetic potential. Objective was to study the influence of carao powder on physicochemical, microbiological, and rheological properties of yogurt. Carao powder was added at 0, 1.3, 2.65, and 5.3 g/L to milk to produce yogurt, which was stored for 42 days. The pH, titratable acidity (TA), syneresis, color, Streptococcus thermophilus (ST) and Lactobacillus delbrueckii spp. bulgaricus (LB) counts, viscosity, steady shear flow, thixotropic analysis, and frequency sweep measurements were evaluated over yogurt shelf life. The protease activity of yogurt starter culture bacteria and particle size of the carao were also measured. Particle size distribution of carao powder varied. Carao did not affect the syneresis and the growth of ST and LB. Carao yogurt decreased pH and L* values and increased a*, b*, n’, TA, and viscosity values. At 42 days, thixotropic behavior increased with the addition of carao. Carao also increased the protease activity of yogurt starter culture bacteria in skim milk. Carao could be added to yogurt without producing major changes to its characteristics.

Bioluminescence resonance energy transfer biosensor for measuring activity of a protease secreted by Pseudomonas fluorescens growing in milk

Bacterial proteases are sporadic contributors to milk spoilage, reducing the quality of ultra-heat treated (UHT) milk and other dairy products. Current methods for measuring bacterial protease activity in milk are insensitive and too slow to be used in routine testing in dairy processing plants. We have designed a novel bioluminescence resonance energy transfer (BRET)-based biosensor to measure the activity of proteases secreted by bacteria in milk. The BRET-based biosensor is highly selective for bacterial protease activity compared with other proteases tested, notably including plasmin, which is abundant in milk. It incorporates a novel peptide linker that is selectively cleaved by P. fluorescens AprX proteases. The peptide linker is flanked by green fluorescent protein (GFP2) at the N-terminus and a variant Renilla luciferase (RLuc2) at the C-terminus. Complete cleavage of the linker by bacterial proteases from Pseudomonas fluorescens strain 65, leads to a 95% decrease in the BRET ratio. We applied an azocasein-based calibration method to the AprX biosensor using standard international enzyme activity units. In a 10-min assay, the detection limit for AprX protease activity in buffer was equivalent to 40 pg/mL (≈0.8 pM, 22 μU/mL) and 100 pg/mL (≈2pM, 54 μU/mL) in 50% (v/v) full fat milk. The EC50 values were 1.1 ± 0.3 ng/mL (87 μU/mL) and 6.8 ± 0.2 ng/mL (540 μU/mL), respectively. The biosensor was approximately 800x more sensitive than the established FITC-Casein method in a 2-h assay, the shortest feasible time for the latter method. The protease biosensor is sensitive and fast enough to be used in production settings. It is suitable for measuring bacterial protease activity in raw and processed milk, to inform efforts to mitigate the effects of heat-stable bacterial proteases and maximise the shelf-life of dairy products.

Characterization of Physicochemical, Biological, and Chemical Changes Associated with Coconut Milk Fermentation and Correlation Revealed by 1H NMR-Based Metabolomics.

Fermentation of milk enhances its nutritional and biological activity through the improvement of the bioavailability of nutrients and the production of bioactive compounds. Coconut milk was fermented with Lactiplantibacillus plantarum ngue16. The aim of this study was to evaluate the effect of fermentation and cold storage for 28 days on physicochemical characteristics, shelf life, and antioxidant and antibacterial activities of coconut milk as well as its proximate and chemical compositions. The pH of fermented milk decreased from 4.26 to 3.92 on the 28th day during cold storage. The viable cell count of lactic acid bacteria (LAB) in fermented coconut milk was significantly increased during fermentation and cold storage period (1 to 14 days), reaching 6.4 × 108 CFU/mL, and then decreased significantly after 14 days to 1.6 × 108 CFU/mL at 28 days. Yeast and molds in fermented coconut milk were only detected on the 21st and 28th days of cold storage, which ranged from 1.7 × 102 to 1.2 × 104 CFU/mL, respectively. However, the growth of coliforms and E. coli was observed on the 14th until the 28th day of cold storage. The fermented coconut milk demonstrated strong antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Cronobacter sakazakii, Bacillus cereus, and Salmonella typhimurium compared to fresh coconut milk. Fermented coconut milk had the greatest 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) values, with 67.1% and 61.961 mmol/g at day 14 of cold storage, respectively. Forty metabolites were detected in fermented and pasteurized coconut milk by proton nuclear magnetic resonance (1H NMR) metabolomics. The principal component analysis (PCA) showed clear difference between the fermented and pasteurized coconut milk as well as the studied cold storage days. The metabolites responsible for this variation were ethanol, valine, GABA, arginine, lactic acid, acetoin, alanine, phenylalanine, acetic acid, methionine, acetone, pyruvate, succinic acid, malic acid, tryptophan, uridine, uracil, and cytosin, which were higher in fermented coconut milk. However, sugars and other identified compounds were higher in fresh coconut milk. The findings of this study show that fermentation of coconut milk with L. plantarum ngue16 had high potential benefits to extending its shelf life and improved biological activities as well as other beneficial nutrients.

Coordinated response of milk bacterial and metabolic profiles to subacute ruminal acidosis in lactating dairy cows

BackgroundBovine milk is an important source of nutrition for human consumption, and its quality is closely associated with the microbiota and metabolites in it. But there is limited knowledge about the milk microbiome and metabolome in cows with subacute ruminal acidosis.MethodsEight ruminally cannulated Holstein cows in mid lactation were selected for a 3-week experiment. The cows were randomly allocated into 2 groups, fed either a conventional diet (CON; 40% concentrate; dry matter basis) or a high-concentrate diet (HC; 60% concentrate; dry matter basis).ResultsThe results showed that there was a decreased milk fat percentage in the HC group compared to the CON group. The amplicon sequencing results indicated that the alpha diversity indices were not affected by the HC feeding. At the phylum level, the milk bacteria were dominated by Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes both in the CON and HC groups. At the genus level, the HC cows displayed an improved proportion of Labrys (P = 0.015) compared with the CON cows. Results of both the principal components analysis and partial least squares of discriminant analysis of milk metabolome revealed that samples of the CON and HC groups clustered separately. A total of 31 differential metabolites were identified between the two groups. Of these, the levels of 11 metabolites decreased (α-linolenic acid, prostaglandin E2, L-lactic acid, L-malic acid, 3-hydroxysebacic acid, succinyladenosine, guanosine, pyridoxal, L-glutamic acid, hippuric acid, and trigonelline), whereas the levels of the other 20 metabolites increased in the HC group with respect to the CON group (P < 0.05).ConclusionThese results suggested that subacute ruminal acidosis less impacted the diversity and composition of milk microbiota, but altered the milk metabolic profiles, which led to the decline of the milk quality.