Milk Casein Research Articles

Comparative analysis of milk casein hydrolysates from cow, water buffalo, goat and sheep: effects of flavourzyme and neutrase on formation of bioactive peptides

Comparative analysis of milk casein hydrolysates from cow, water buffalo, goat and sheep: effects of flavourzyme and neutrase on formation of bioactive peptides

Combination of Milk Polar Lipids and Casein Hydrolysate as a Healthy Emulsifier for Ice Cream

Combination of Milk Polar Lipids and Casein Hydrolysate as a Healthy Emulsifier for Ice Cream

Unveiling the Potential Bioactive Peptides Derived From Goat Casein Hydrolysates Based on In Silico Analyses

ABSTRACTBioactive peptides (BAPs) have attracted considerable interest in scientific research due to their heterogeneity in sequence and structure, which underpins various biological functionalities. In this context, goat casein, an abundant by‐product of the dairy industry, emerges as a valuable source of BAPs. The present study undertook a meticulous evaluation of the bioactive potentials of goat casein‐derived peptides through an integrated approach combining computational simulations, high‐throughput screening, and molecular docking techniques. The initial phase involved the enzymatic digestion of goat milk casein using trypsin, followed by the identification of peptides via liquid chromatography–tandem mass spectrometry (LC–MS/MS), uncovering a total of 597 peptides. Subsequent prioritization using the PeptideRanker algorithm identified 70 peptides exhibiting potential bioactivity, as denoted by scores above 0.8. Advanced screening employing the BIOPEP database and the AutoDock and CAMPR4 tools facilitated the elucidation of 16 antioxidant, 59 hypotensive, 63 hypoglycemic, 70 hypolipidemic, and 25 antimicrobial peptides. Molecular docking studies further elucidated the spontaneous nature of the interactions between the peptides and their respective receptors, predominantly mediated by hydrogen bonding and hydrophobic interactions. Four peptides specifying all activities simultaneously were synthesized, and their activities were verified by in vitro experiments. These results not only highlight an effective strategy for the high‐throughput screening of goat casein‐derived peptides but also underscore the potential of utilizing casein as a viable source of functional food ingredients. This study thereby contributes significantly to the expanding field of functional food research, suggesting a sustainable approach to explore the potential of dairy by‐products.

Linseed oil supplementation alters milk fatty acid profile, mitigates heat stress, and improves summer milk yield in grazing dairy cows.

Dietary supplementation of fat can be an important source of energy to compensate for the reduction in dry matter intake in dairy cows during heat stress periods. Studies have reported that supplementing dairy cow diets with linseed oil (LO) can increase milk yield and enhance the levels of beneficial fatty acids, such as omega-3 fatty acids, in the milk. The objective of this research was to evaluate the effect of LO supplementation on milk fatty acids profile, milk yield and composition, and physiological parameters of grazing cows. The study was conducted in two seasons, one in spring and one in summer. A 2 × 2 Latin square design was used in each experiment. Twelve Holstein and crossbred Holstein x Jersey cows were involved in each season. Cows were divided into two groups: control (TC) with no supplementation and treatment (TL) supplemented with 400g/day of LO. The results showed that LO supplementation altered the milk fatty acid profile: decreased concentrations of short and medium-chain fatty acids (C10:0 - C17:1) except for C13:0 and increased concentrations of long-chain fatty acids (C18, C18:1 (both trans and cis isomers), C18:2 (specific conjugated linoleic acid - CLA isomers), and C18:3 n3 (omega-3)). Additionally, milk yield increased by 1.5l per day during summer in LO-supplemented cows, while milk fat, protein, and casein content decreased. Milk stability increased by 2.2% in the LO-supplemented group. LO-supplemented cows reduced internal body temperature and heart frequency in the afternoon and increased daily rumination time by 20min. In conclusion, LO supplementation can be an effective strategy to improve the nutritional profile of milk by altering fatty acid composition towards potentially healthier fats, mitigate the negative effects of heat stress on grazing cows during summer, as evidenced by reduced body temperature and heart frequency and increase milk yield.

Investigating the effect of whey and casein proteins on drug solubility from a paediatric drug absorption perspective

Considering the predominantly milk-based diet of neonates and infants and their immature gastrointestinal digestion, milk proteins may affect drug behaviour and absorption in this population. Using in vitro models, this study investigated the impact of the representative milk proteins, whey and casein, on the solubility and permeation of the lipophilic model drugs spironolactone, clopidogrel and ritonavir. Drug solubility experiments revealed that the presence of milk proteins increased drug solubility. Next, permeation studies demonstrated that the same milk proteins reduced drug permeation across an artificial membrane. These results highlight the importance of the solubility-permeability interplay and indicate the effect of these proteins may be considered during (paediatric) drug development. Lastly, the findings underscore the importance of considering milk protein-drug interactions to optimize drug delivery strategies during (paediatric) drug development and especially for the youngest and most vulnerable part of this population.

Impact of preacidification on the functionality and insoluble calcium levels of Low-Moisture Part-Skim Mozzarella made from high-casein milk

The use of low-concentration factor ultrafiltered (LCF-UF) milk for cheesemaking has become popular in recent years. Research on using LCF-UF milk to make low-moisture part-skim (LMPS) Mozzarella manufacture is limited due to concerns about the negative impact of the higher casein content on functional properties, such as melt and stretch. Pre-acidification (PA) (reflects acid added to milk before the addition of the starter culture) has been used in low-fat and nonfat Mozzarella cheeses to lower calcium levels and improve their texture and melting properties. We explored the use of PA to reduce the insoluble calcium content of cheese (calcium that is a crosslinking material for the casein proteins) and its impact on the functionality of LMPS Mozzarella made from high casein (4.2%) milk. Seven vats of LMPS Mozzarella, with a control (no PA), 2 pH values for PA (6.40 and 6.00) and 3 different acidification methods (use of acetic, citric, or carbonic acid from carbon dioxide) were evaluated for their impact on solubilization of colloidal calcium phosphate during LMPS manufacture. Composition, proteolysis, microstructural and functional changes in the LMPS cheeses were monitored during 90 d of refrigerated storage. All cheeses had similar composition except for the cheese made with milk PA with carbonic acid, which had a lower fat-in-dry matter probably due to foaming observed in this sample during the gas sparging. The cheese made with milk PA with citric acid to pH 6.00 had the lowest amount of total and insoluble calcium compared with other cheeses at all steps during cheese manufacture and storage, likely due to the calcium chelating ability of citric acid. The cheese that was made with milk PA with citric acid to pH 6.40 had a trend of slightly lower (not significant) total and insoluble calcium during cheese manufacture compared with other cheeses that were made with milk PA to pH 6.40. The pH value in all the cheeses remained similar throughout 90 d of storage, whereas the cheese that was made with milk PA with citric acid to pH 6.00 had the lowest pH values during storage. Treatment significantly (P < 0.05) impacted instrumental hardness, the maximum loss tangent during heating, the melting temperature and the storage modulus values measured at 70°C. Proteolysis was not impacted by treatment suggesting that typical aging should occur. Cheese that was PA with citric acid to pH 6.00 initially had lower maximum loss tangent values and higher hardness, possibly due to the lower pH value of this cheese. The performance of cheeses on pizza was assessed using Sensory Spectrum® method and quantitative descriptive analysis. The PA of cheese milk with citric acid helped to reduce the insoluble Ca level and at 30 and 90 d of storage, these cheeses had lower first chew hardness, lower chewiness, and lower strand thickness values compared with other cheeses. The use of PA could help improve the functionality of LMPS Mozzarella made from milk high in casein with citric acid being the most efficient in dissolving the insoluble calcium from casein.

Study on Single Nucleotide Polymorphism of LAP3 Gene and Its Correlation with Dairy Quality Traits of Gannan Yak.

This study explored the polymorphism of the leucine aminopeptidase (LAP3) gene and its relationship with milk quality characteristics in Gannan yak. A cohort of 162 Gannan yak was genotyped utilizing the Illumina Yak cGPS 7K BeadChip, and the identified single nucleotide polymorphisms (SNPs) were evaluated for their association with milk protein, casein, lactose, and fat concentrations. The results showed that four SNPs (g.4494G > A, g.5919A > G, g.8033G > C, and g.15,615A > G) in the LAP3 gene exhibited polymorphism with information content values of 0.267, 0.267, 0.293, and 0.114, respectively. All four SNPs were in Hardy-Weinberg equilibrium (p > 0.05). The g.4494G > A and g.5919A > G SNPs were significantly associated with protein content (p < 0.05), with homozygous genotypes showing significantly higher protein content than heterozygous genotypes (p < 0.05). The g.8033G > C SNP was significantly associated with casein content, protein content, non-fat solids, and acidity (p < 0.05), with the CC genotype having significantly higher casein, protein, and non-fat solids content than the GG and GC genotypes (p < 0.05). The g.15,615A > G SNP was significantly associated with average fat globule diameter (p < 0.05). In general, the mutations within the LAP3 gene demonstrated a positive impact on milk quality traits in Gannan yak, with mutated genotypes correlating with enhanced milk quality. These results indicate that the LAP3 gene could be a significant or candidate gene affecting milk quality traits in Gannan yak and offer potential genetic markers for molecular breeding programs in this species.

An Overview of Interactions between Goat Milk Casein and Other Food Components: Polysaccharides, Polyphenols, and Metal Ions.

Casein is among the most abundant proteins in milk and has high nutritional value. Casein's interactions with polysaccharides, polyphenols, and metal ions are important for regulating the functional properties and textural quality of dairy foods. To improve the functional properties of casein-based foods, a deep understanding of the interaction mechanisms and the influencing factors between casein and other food components is required. This review started by elucidating the interaction mechanism of casein with polysaccharides, polyphenols, and metal ions. Thermodynamic incompatibility and attraction are the fundamental factors in determining the interaction types between casein and polysaccharides, which leads to different phase behaviors and microstructural types in casein-based foods. Additionally, the interaction of casein with polyphenols primarily occurs through non-covalent (hydrogen bonding, hydrophobic interactions, van der Waals forces, and ionic bonding) or covalent interaction (primarily based on the oxidation of proteins or polyphenols by enzymatic or non-enzymatic (alkaline or free radical grafting) approaches). Moreover, the selectivity of casein to specific metal ions is also introduced. Factors affecting the binding of casein to the above three components, such as temperature, pH, the mixing ratio, and the fine structure of these components, are also summarized to provide a good foundation for casein-based food applications.

Functional Peptides from Yak Milk Casein: Biological Activities and Structural Characteristics.

The average content of casein in yak milk is 40.2 g/L. Casein can be degraded by enzymatic digestion or food processing to produce abundant degradation peptides. International researchers have studied the degradation peptides of yak milk casein by using multiple techniques and methods, such as in vitro activity tests, cellular experiments, proteomics, bioinformatics, etc., and found that the degradation peptides have a wide range of functional activities that are beneficial to the human body, such as angiotensin-converting enzyme (ACE) inhibitory, antioxidant, anti-inflammatory, antidiabetic, antimicrobial, anticancer, and immunomodulatory activities, etc., and it has been proved that the types and strengths of functional activities are closely related to the structural characteristics of the peptides. This paper describes the characteristics of yak milk proteins, the functional activities, and mechanism of action of degraded peptides. Based on the types of functional activities of yak milk casein degradation peptides, we classified and elucidated the effects of structural factors, such as peptide molecular weight, peptide length, amino acid sequence, physicochemical properties, electrical charge, hydrophobicity, spatial conformation, chain length, and the type of enzyme on these activities. It reveals the great potential of yak milk casein degradation peptides as functional active peptide resources and as auxiliary treatments for diseases. It also provides important insights for analyzing yak casein degradation peptide activity and exploring high-value utilization.

Exploring structural features of sleep-enhancing peptides derived from casein hydrolysates by chemometrics and random forest methodology

Milk casein is regarded as source to release potential sleep-enhancing peptides. Although various casein hydrolysates exhibited sleep-enhancing activity, the underlying reason remains unclear. This study firstly revealed the structural features of potential sleep-enhancing peptides from casein hydrolysates analyzed through peptidomics and multivariate analysis. Additionally, a random forest model and a potential Tyr-based peptide library were established, and then those peptides were quantified to facilitate rapidly-screening. Our findings indicated that YP-, YI/L, and YQ-type peptides with 4–10 amino acids contributed more to higher sleep-enhancing activity of casein hydrolysates, due to their crucial structural features and abundant numbers. Furthermore, three novel strong sleep-enhancing peptides, YQKFPQY, YPFPGPIPN, and YIPIQY were screened, and their activities were validated in vivo. Molecular docking results elucidated the importance of the YP/I/L/Q- structure at the N-terminus of casein peptides in forming crucial hydrogen bond and π-alkyl interactions with His-102 and Asn-60, respectively in the GABAA receptor for activation.

Fe3O4@silica-thermolysin: A robust, advantageous, and reusable microbial nanobiocatalyst for proteolysis and milk-clotting

Thermolysin (TLN) is a microbial highly-priced thermostable metallo-endoprotease with complementary substrate specificity to those of proteases widely used in science and industry for protein digestion and milk-clotting. This study is the first to immobilize TLN on aminated superparamagnetic nanoparticles (Fe3O4@silica-NH2) aiming for higher stability, recoverability, reusability, and applicability in proteolysis and as a microbial rennet-like milk-clotting enzyme. The nanobiocatalyst developed (Fe3O4@silica-TLN) displays hydrolytic activity on a synthetic TLN substrate and, apparently, was fully recovered from reaction media by magnetic decantation. More importantly, Fe3O4@silica-TLN retains TLN catalytic properties in the presence of calcium ions even after exposure to 60 °C for 48 h, storage at 4 °C for 80 days and room temperature for 42 days, use in proteolyses, and in milk-clotting for up to 11 cycles. Its proteolytic activity on bovine milk casein in 24 h furnished 84 peptides, of which 29 are potentially bioactive. Also, Fe3O4@silica-TLN catalyzed the digestion of bovine serum albumin. In conclusion, Fe3O4@silica-TLN showed to be a new, less autolytic, thermostable, non-toxic, magnetically-separable, and reusable nanobiocatalyst with highly attractive properties for both science (peptide/protein chemistry and structure, proteomic studies, and the search for new bioactive peptides) and food industry (cheese manufacture).

ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY OF PEPTIDES FORMED DURING THE HYDROLYSIS OF CASEIN BY PROTEASES OF THE ENTEROCOCCUS FAECALIS AN1

Much attention is paid to the study of biologically active peptides from milk caseins, which are usually obtained by partial hydrolysis by the proteolytic system of lactic acid bacteria. These peptides have a wide range of beneficial properties, including mineral binding, antidiabetic, satiating, immunomodulating, opioid, antimicrobial and antioxidant properties. In this work, we investigated the antimicrobial and antioxidant activity of peptides obtained during the hydrolysis of milk caseins by Enterococcus faecalis AN1 proteases. SDS PAAG electrophoretic analysis of peptide fractions after 8 hours of milk casein proteolysis showed that proteases of this strain cleave all casein fractions (αs1-, αs2 and β-casein). RP-HPLC profile elution revealed a large amount of small, medium and long cationic and anionic peptides. Some fractions of these peptides have antimicrobial activity against Listeria monocytogenes EGDe 107776, Bacillus cereus, Escherichia coli CCM 4517 and Aspergillus niger CCM 8189. Peptide fractions obtained during the hydrolysis of caseins possessed strong antioxidant activity and surpassed native casein by 4.5 times. These data stimulate further research in the field of biomedicine and food technology with the aim of developing new and effective preparations and products for maintaining the health and safety of consumers.

Characterization of lactic acid bacteria isolated from human breast milk and their bioactive metabolites with potential application as a probiotic food supplement.

The probiotic properties of twenty-five lactic acid bacteria (LAB) isolated from human breast milk were investigated considering their resistance to gastrointestinal conditions and proteolytic activity. Seven LAB were identified and assessed for auto- and co-aggregation capacity, antibiotic resistance, and behavior during in vitro gastrointestinal digestion. Three Lacticaseibacillus strains were further evaluated for antifungal activity, metabolite production (HPLC-Q-TOF-MS/MS and GC-MS/MS) and proteolytic profiles (SDS-PAGE and HPLC-DAD) in fermented milk, whey, and soy beverage. All strains resisted in vitro gastrointestinal digestion with viable counts higher than 7.9 log10 CFU mL-1 after the colonic phase. Remarkable proteolytic activity was observed for 18/25 strains. Bacterial auto- and co-aggregation of 7 selected strains reached values up to 23 and 20%, respectively. L. rhamnosus B5H2, L. rhamnosus B9H2 and L. paracasei B10L2 inhibited P. verrucosum, F. verticillioides and F. graminearum fungal growth, highlighting L. rhamnosus B5H2. Several metabolites were identified, including antifungal compounds such as phenylacetic acid and 3-phenyllactic acid, and volatile organic compounds produced in fermented milk, whey, and soy beverage. SDS-PAGE demonstrated bacterial hydrolysis of the main milk (caseins) and soy (glycines and beta-conglycines) proteins, with no apparent hydrolysis of whey proteins. However, HPLC-DAD revealed alpha-lactoglobulin reduction up to 82% and 54% in milk and whey, respectively, with L. rhamnosus B5H2 showing the highest proteolytic activity. Overall, the three selected Lacticaseibacillus strains demonstrated probiotic capacity highlighting L. rhamnosus B5H2 with remarkable potential for generating bioactive metabolites and peptides which are capable of promoting human health.

Age-specific composition of milk microbiota in Tibetan sheep and goats

This study investigates the dynamic changes in milk nutritional composition and microbial communities in Tibetan sheep and goats during the first 56 days of lactation. Milk samples were systematically collected at five time points (D0, D7, D14, D28, D56) post-delivery. In Tibetan sheep, milk fat, protein, and casein contents were highest on D0, gradually decreased, and stabilized after D14, while lactose and galactose levels showed the opposite trend. Goat milk exhibited similar initial peaks, with significant changes particularly between D0, D7, D14, and D56. 16S rRNA gene sequencing revealed increasing microbial diversity in both species over the lactation period. Principal coordinates analysis identified distinct microbial clusters corresponding to early (D0–D7), transitional (D14–D28), and mature (D56) stages. Core phyla, including Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, dominated the milk microbiota, with significant temporal shifts. Core microbes like Lactobacillus, Leuconostoc, and Streptococcus were common in both species, with species-specific taxa observed (e.g., Pediococcus in sheep, Shewanella in goats). Furthermore, we observed a highly shared core microbiota in sheep and goat milk, including Lactobacillus, Leuconostoc, and Streptococcus. Spearman correlation analysis highlighted significant relationships between specific microbial genera and milk nutrients. For instance, Lactobacillus positively correlated with total solids, non-fat milk solids, protein, and casein, while Mannheimia negatively correlated with protein content. This study underscores the complex interplay between milk composition and microbial dynamics in Tibetan sheep and goats, informing strategies for livestock management and nutritional enhancement.Key points• The milk can be classified into three types based on the microbiota composition• The changes of milk microbiota are closely related to the variations in nutrition• Filter out microbiota with species specificity and age specificity in the milk

Valorization of cheese-making residues in biorefineries using different combinations of dark fermentation, hydrothermal carbonization and anaerobic digestion

Dark fermentation (DF), hydrothermal carbonization (HTC) and anaerobic digestion (AD) are applied, in different combinations, to cheese whey (CW), which is the liquid effluent from the precipitation and removal of milk casein during the cheese-making process. The aim and novelty of this research is to investigate the production of various biofuels (H2-rich gas, hydrochar and biogas) in cascade, according to the waste biorefinery concept. The simplest case is the direct AD of CW. The second investigated possibility is the preliminary HTC of CW, producing hydrochar, followed by the AD of the process water from which hydrochar is separated by filtration. The third possibility is based on DF of CW, followed by the AD of the fermentate (F) from DF. The final possibility is based on DF of CW, followed by HTC of the F, and then AD of the process water. Accordingly, the physical and chemical properties of CW, F, resulting hydrochar and process water (PW), and biomethane potentials of CW, F, and process waters are studied to determine the energy and carbon balances of all variants. In brief, the first variant, direct AD of CW, is believed to be the most energy efficient method.

Assessment of isolation strategies to remove caseins for high-quality milk-derived extracellular vesicles

Increasing studies have highlighted the significance of milk-derived extracellular vesicles (MEVs) in mother-newborn integration, as well as their application as novel drug delivery systems and diagnostic biomarkers. However, conventional ultracentrifugation (UC) often results in the co-precipitation of casein micelles in MEV pellets. In this study, we compared methods with different principles to screen the optimal pretreatment in caseins removal, and found that isoelectric precipitation by hydrochloric acid (HA) could most effectively remove caseins in porcine milk. We further characterized MEV populations isolated by UC and HA/UC from diverse aspects, including particle methodology via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), RNA and protein contents, and purity analysis. Importantly, the proliferative and anti-inflammatory effects of MEVs were evaluated in vitro, showing the superiority of MEVs via HA/UC in functionality compared with UC. Our results suggest that HA pretreatment before ultracentrifugation could effectively remove caseins and other protein complexes, leading to MEVs via HA/UC with higher purity and more significant effects in vitro. This study provides valuable insights for the advancement of MEVs isolation techniques across different species and accurate function analysis of MEVs.

The Severity of Gastrointestinal Disorders and Autistic-Like Behaviors Could Be Associated with a Selective Humoral Response to Bovine Milk Caseins: A Case Series

The Severity of Gastrointestinal Disorders and Autistic-Like Behaviors Could Be Associated with a Selective Humoral Response to Bovine Milk Caseins: A Case Series

Cloning and expression of recombinant arazyme with anti-inflammatory and anti-breast cancer potential.

Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC50 were 16.2µg/ml and 13.2mg/ml after 24h and 48h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.

Enhanced Protease Production from Diabetic Ulcer-Derived E. coli

This study explored a local Escherichia coli isolate from diabetic foot ulcer patients for protease enzyme production. Qualitative screening on skim milk agar showed a 6 mm hydrolysis halo, while quantitative screening with casein revealed a specific activity of 144.55 U/mg protein. Purification via ion exchange chromatography on DEAE-cellulose increased the specific activity to 767.5 U/mg, with a 5.3-fold enhancement and a 29.5% yield. The findings suggest E. coli as a viable source for protease production and demonstrate the efficiency of ion-exchange chromatography for enzyme purification, with potential significant industrial applications. Highlight: E. coli sourced from diabetic ulcers shows promise for protease production. Skim milk agar and casein used for qualitative and quantitative screening. Ion exchange chromatography significantly enhances enzyme specific activity. Keywoard: Escherichia coli, protease production, diabetic foot ulcer, enzyme purification, ion exchange chromatography

Virulence properties of nisin-resistant bacteria isolated from raw milk and cheese samples

AbstractNisin is a bacteriocin produced by Lactococcus lactis and it is generally recognised as safe by the U.S. Food and Drug Administration. The study aimed to determine the presence of nisin-resistant Staphylococcus, Enterococcus, and Listeria spp. in 157 raw milk and 125 cheese samples. As a result of the isolation procedure, a total of 282 bacterial strains were isolated. 83/282 strains showed resistance to 300 ppm nisin concentration and they were identified by 16S rRNA sequencing. The two most common species were Enterococcus faecalis and Staphylococcus aureus. In both raw milk and cheese samples, nisin-resistant Listeria could not be detected. Generally, enterococci (59/65) showed higher biofilm formation ability than staphylococci (13/18). 38% (25/65) of enterococci and 88% (16/18) of staphylococci were determined to have protease activity in at least one of Skim Milk Agar, Casein Agar, or Milk Agar. In addition, the most common antibiotic resistance in nisin-resistant enterococci was against chloramphenicol. This study revealed that nisin-resistant staphylococci and enterococci are present in raw milk and cheese. In addition, the presence of some virulence factors such as biofilm production, protease activity, and antibiotic resistance in resistant isolates needs to be drawn attention to. Consumption of nisin-resistant microorganisms with virulence factors through food is a food safety risk.