Αs1 Casein Research Articles

Efficient Protein Expression and Biosynthetic Gene Cluster Regulation in Bacillus subtilis Driven by a T7-BOOST System.

Bacillus subtilis is a generally recognized as safe microorganism that is widely used for protein expression and chemical production, but has a limited number of genetic regulatory components compared with the Gram-negative model microorganism Escherichia coli. In this study, a two-module plug-and-play T7-based optimized output strategy for transcription (T7-BOOST) systems with low leakage expression and a wide dynamic range was constructed based on the inducible promoters Phy-spank and PxylA. The first T7 RNA polymerase-driven module was seamlessly integrated into the genome based on the CRISPR/Cpf1 system, while the second expression control module was introduced into low, medium, and high copy plasmids for characterization. As a proof of concept, the T7-BOOST systems were successfully employed for whole-cell catalysis production of γ-aminobutyric acid (109.8 g/L with a 98.0% conversion rate), expression of human αS1 casein and human lactoferrin, and regulation of exogenous lycopene biosynthetic gene cluster and endogenous riboflavin biosynthetic gene cluster. Overall, the T7-BOOST system serves as a stringent, controllable, and effective tool for regulating gene expression in B. subtilis.

Quantitative determination of αs1–casein in goat’s milk using reversed-phase high performance liquid chromatography (RP–HPLC)

The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Despite indications that more than 90% of cow’s milk protein allergy (CMPA) children reacted similarly to goat's milk in typical IgE-mediated forms, the availability of goat's milk without αs1-casein appeared to be a potential alternative for patients with cow's milk allergy. In Malaysia, there is a lack of quantitative data on αs1-casein in raw and commercial goat’s milk. To address this problem, this study aimed to quantify αs1-casein in goat’s milk using reversed-phase high performance liquid chromatography (RP–HPLC). The procedure was developed using a total of twenty samples comprised of commercial and raw goat’s milk. For the calibration curve, a linear relationship with R² > 0.999 was obtained using cow’s α-casein standard and peak areas were observed over the concentration range, with a limit of detection (LOD) of 0.025 mg/ mL and limit of quantification (LOQ) of 0.075 mg/mL, respectively. The amount of αs1- casein in the twenty samples ranged from 0.08±0.01 to 1.45±0.17 mg/mL. Three samples which were claimed as goat’s milk infant formula were not found to contain αs1-casein. Repeatability and reproducibility were satisfactory for both retention times and peak areas. The RSD of peak areas ranged from 0.01 - 8.07% within an analytical day and from 0.61 - 1.77% across analytical days. This study will contribute as a reference method for analysis and surveillance data of αs1-casein, in private or national laboratories, as well as milk and milk product manufacturers.

Chymosin pretreatment accelerated papain catalysed hydrolysis for decreasing casein antigenicity by exposing the cleavage site at tyrosine residues

Cow milk protein allergy (CMPA) induced by casein poses major health challenges that hinders the consumption of milk-based formulas. In this study, a novel sequential enzymatic hydrolysis catalysed by chymosin and papain was proposed to reduce casein antigenicity. Its effects on reducing casein antigenicity, structural properties and peptide profiles were evaluated by ELISA, multispectral techniques and peptidome analysis. It was revealed that the sequential enzymatic hydrolysis obtained a similarly residual antigenicity level in a shorter time (60 min) compared to papain-hydrolysis for 360 min. The hydrolysis-site at Tyr residues accessibility of papain was increased to 36.84 % by the chymosin pretreatment and it was significantly higher than 26.93 % obtained by only papain for 60 min. Moreover, the sequential enzymatic hydrolysis led to decrease in the large fragment peptides from αs1 casein. These findings suggested that the proposed sequential enzymatic hydrolysis can be exploited in the development of CMPA-free formulas.

Insight into the molecular-level details of αs1 casein interactions with IgG: Combining with LC-MS/MS and molecular modelling techniques

αs1-Casein (αs1-CN) is a major cow milk allergen, while the tertiary structure of αs1-CN and conformational epitopes of αs1-CN have not been clarified. Here, a reasonable three-dimensional structure of αs1-CN was established using ab initio methods, and hot-spot residues and epitopes were investigated by combining molecular dynamics simulation, peptides synthesis, and ELISA. Obtained results demonstrated that the binding mechanism between αs1-CN and IgG was located on three main regions: a helical structure zone (E77-Q97), the flexible loop zone (Y154-T174), and a flexible C-terminal (N190-L198), mainly connecting via hydrogen bond and ionic bonds. The hydrolysates produced by papain with lowest antigenicity (12.43%), which could considerably destroy the essential epitopes of αs1-CN confirmed by epitope synthesis, and LC-MS/MS. The results reported herein would provide novel insights into the interface interactions between αs1-CN and IgG, and prove valuable for developing hypoallergenic infant-formula and peptide vaccines for allergen-specific immunotherapy.

Determination of genetic variability at αs1-casein gene in Indian dromedary

The analysis of genetic variability at αs1-casein gene in 112 Indian dromedary (Camelus dromedarius) was carried out. The 930 bp fragment of exon 5 of αs1 casein gene was successfully amplified by PCR. The nucleotide substitution at g.942G>T SNP (GenBank ID: JF429140) was detected by PCR-RFLP using SmlI restriction enzyme and verified by sequencing. The GT genotype was not observed in Bikaneri and Kachchhi camels but was observed in Jaisalmeri and Mewari camels at the frequency of 0.143 and 0.071, respectively. Though, the frequency of GT genotype was quite low but still it was enough to document the dynamic nature of the locus g.942G>T SNP in Indian dromedary. Due to the existence of full agreement between genotyping by PCR-RFLP and Isoelectric Focusing at αs1-casein, as envisaged by earlier researchers, the existence of two protein patterns corresponding to αs1-casein alleles, A and C at the same frequency values in the Indian dromedary breeds may also be inferred from the present study. Hence, this DNA based test can be used for typing camel αs1-casein gene variability in Indian dromedary.

Fast Anxiolytic-Like Effect Observed in the Rat Conditioned Defensive Burying Test, after a Single Oral Dose of Natural Protein Extract Products.

Anxiety appears among the most frequent psychiatric disorders. During recent years, a growing incidence of anxiety disorders can be attributed, at least in part, to the modification of our eating habits. To treat anxiety disorders, clinicians use benzodiazepines, which unfortunately display many side effects. Herein, the anxiolytic-like properties of two natural products (αS1–casein hydrolysate and Gabolysat®) were investigated in rats and compared to the efficacy of benzodiazepine (diazepam). Thus, the conditioned defensive burying test was performed after a unique oral dose of 15 mg/kg, at two time-points (60 min and then 30 min post oral gavage) to show potential fast-onset of anxiolytic effect. Both natural products proved to be as efficient as diazepam to reduce the time rats spent burying the probe (anxiety level). Additionally, when investigated as early as 30 min post oral gavage, Gabolysat® also revealed a fast-anxiolytic activity. To date, identification of bioactive peptide, as well as how they interact with the gut–brain axis to sustain such anxiolytic effect, still remains poorly understood. Regardless, this observational investigation argues for the consideration of natural compounds in care pathway.

Post translational modifications of milk proteins in geographically diverse goat breeds

Goat milk is a source of nutrition in difficult areas and has lesser allerginicity than cow milk. It is leading in the area for nutraceutical formulation and drug development using goat mammary gland as a bioreactor. Post translational modifications of a protein regulate protein function, biological activity, stabilization and interactions. The protein variants of goat milk from 10 breeds were studied for the post translational modifications by combining highly sensitive 2DE and Q-Exactive LC-MS/MS. Here we observed high levels of post translational modifications in 201 peptides of 120 goat milk proteins. The phosphosites observed for CSN2, CSN1S1, CSN1S2, CSN3 were 11P, 13P, 17P and 6P, respectively in 105 casein phosphopeptides. Whey proteins BLG and LALBA showed 19 and 4 phosphosites respectively. Post translational modification was observed in 45 low abundant non-casein milk proteins mainly associated with signal transduction, immune system, developmental biology and metabolism pathways. Pasp is reported for the first time in 47 sites. The rare conserved peptide sequence of (SSSEE) was observed in αS1 and αS2 casein. The functional roles of identified phosphopeptides included anti-microbial, DPP-IV inhibitory, anti-inflammatory and ACE inhibitory. This is first report from tropics, investigating post translational modifications in casein and non-casein goat milk proteins and studies their interactions.

Vers une sélection génomique chez les caprins laitiers

La sélection génomique, qui a révolutionné la sélection génétique des bovins laitiers notamment, est désormais envisagée dans d’autres filières animales. Chez les caprins laitiers français, le gain de précision attendu des valeurs génomiques était un des questionnements de la filière en raison de la petite taille de la population de référence disponible (825 mâles et 1945 femelles génotypés sur une puce SNP 50K). Le but de cette étude est de tester différentes techniques d’évaluation génomique afin d’obtenir les évaluations génomiques les plus précises possibles. Une étude de la structure génétique de la population de référence caprine constituée d’animaux de races Saanen et Alpine, a révélé de faibles niveaux de déséquilibre de liaison (0,17 entre deux SNP consécutifs), de consanguinité et de parenté au sein de la population, ce qui n’est pas favorable à une bonne précision des évaluations génomiques. Les méthodes d’évaluations génomiques (GBLUP ou Bayésiennes), basées sur des performances pré-corrigées n’ont pas permis une amélioration significative des précisions des évaluations génomiques pour les caractères évalués en routine (caractères de production, de morphologie et comptages de cellules somatiques). Cependant les évaluations génomiques basées sur les performances propres des femelles ont permis d’obtenir des précisions supérieures à celles obtenues sur ascendance. La sélection génomique est donc envisageable chez les caprins laitiers français. Ces précisions peuvent également être légèrement augmentées par l’inclusion de gènes majeurs tels que celui de la caséine αs1.

Expression and Purification of Hydrophobic PEP‐Inhibiting Peptides from Bovine αS1 Casein

According to previous studies, there is a prolyl endopeptidase‐inhibiting sequence (LNENLLRFFVAPFPEVFG) within a relatively hydrophobic region of αS1 casein (Juillerat‐Jeanneret et al., 2011). Recently, sixteen recombinant peptides were designed in assorted lengths, each containing the inhibitory sequence at varied positions within the αS1 casein sequence. The corresponding cDNA sequences were originally ligated into the pET15b vector, which provides an N‐terminal poly‐histidine tag for purification. The vector was in turn transformed into a BL21(DE3)pLysS E. coli expression host (Vishram et al., 2016). However, the system proved unsuccessful in adequate expression of the hydrophobic peptide. The intention of this study was to assemble a new pET28a expression system, kindly provided by Dr. X. Cheng (MD Anderson in Houston, TX) that utilizes a SUMO (small ubiquitin‐like modifier) fusion tag. The SUMO tag is known for enhancing expression levels of potentially toxic or insoluble recombinant peptides (Bommarius et al., 2010). The expressed peptides were purified via Ni‐NTA column chromatography, with subsequent UlpI cleavage. This was followed by reversed‐phase high performance liquid chromatography (RP‐HPLC) to further purify the peptides. Tricine‐SDS‐PAGE, a useful screening tool for hydrophobic peptides smaller than 30kDa, was used to analyze expression and purification. A comparison was made between SUMO‐fused expression and non‐fused expression of the hydrophobic casein peptides.Support or Funding InformationOffice of Research and Sponsored Projects, SFASU and Ed & Gwen ColeThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Milk authenticity by ion-trap proteomics following multi-enzyme digestion

The practice of adding adulterating substances in milk in order to raise profits is unfortunately worldwide. In addition, higher priced milk, coming from minor dairy species, is often illegally integrated with the lower priced cow milk. The presence of species-specific proteins, different from those declared in label, may be a serious problem for people with allergies. The development of proper analytical methods is therefore essential to protect consumer benefits and product authenticity.In this study, a proteomic approach for the detection of adulteration processes of specific milks in mixtures is proposed. Few microliters of milk samples have been digested with trypsin and chymotrypsin and analyzed by nanoLC-ESI-IT-MS/MS. A post-database processing was performed to obtain confident peptide sequence assignments, allowing the detection of milk adulteration at a level lower than 1%. Species-specific peptides from bovine β-lactoglobulin and αS1 casein were identified as suitable peptide markers of milk authenticity.

Antioxidant and antibacterial activities of bioactive peptides in buffalo's yoghurt fermented with different starter cultures.

The effect of Lactobacillus acidophilus 20552 ATCC (T2) or Lactobacillus helveticus CH 5 (T3) in combination with yoghurt starter (1:1) on the antioxidant and antibacterial activities of the bioactive peptides present in buffalo's yoghurt was studied. The SDS-PAGE results indicate that all caseins were completely hydrolyzed by both strains, whereas whey protein fractions were still present. All starter cultures have the ability to produce low-molecular-weight bioactive peptides, most of which were originated from β-casein and fewer from αs1 casein. The antioxidant activity (%) of the water-soluble peptide extract from yoghurt samples increased in all samples during storage. Samples containing Lb. helveticus CH 5 showed the highest values. All yoghurt treatments displayed antibacterial activity against Escherichia coli. Control yoghurt and T3 showed higher antibacterial activity on E. coli, Staphylococcus aureus, and Bacillus cereus as compared to T2.

In silico Designing of Protein Rich in Large Neutral Amino Acids Using Bovine αs1 Casein for Treatment of Phenylketonuria

Phenylketonuria (PKU) is a genetically inherited disease where the body fails to convert Phenylalanine (Phe) to Tyrosine (Tyr) due to the defective Phenylalanine Hydroxylase (PAH) enzyme, resulting in the elevated blood Phe level causing neurological damage. Of all therapies, Large Neutral Amino Acid (LNAA) supplementation has become a promising approach to the dietary treatment of PKU, where the LNAA compete with Phe for the same L-Type Large Neutral Amino Acid Transporter (LAT1, SLC7A5) across the blood-brain barrier, decreasing brain Phe level. In this study we have designed an easily digestible protein enriched with LNAA (except Phe), using bovine αs1 casein as template by homology modeling. Our challenge was to maximize the LNAAs (except Phe) in the protein model by finding a suitable scaffold for hosting LNAAs, thereby turning the usual concept of homology modeling. Bioinformatics tools like SWISS-MODEL, EXPASY, PROFUNC, I-TASSER, RaptorX, and SAVeS Server were used for the structure prediction, and validation of the designed protein. Out of 63 different models designed, Protein Model-54 was selected based on sequence similarity to template (61.4%), compact 3D structure containing only α-helices and coils without β-sheets (QMEAN score of 0.567), and good in silico digestibility. The molecular weight of protein was 12,094.6 Da. Ramachandran plot revealed that the designed protein contained 89.9% amino acid residues in the favored region with ERRAT score of 88.04. Based on these evaluations, the Protein Model-54 was found to be the best, stable and reliable model which may be of high nutritional significance for PKU patients..

Influence of chymosin type and curd scalding temperature on proteolysis of hard cooked cheeses.

In this work, we studied the influence of the type of coagulant enzyme and the curd scalding temperature on the proteolysis and residual coagulant and plasmin activities of a cooked cheese, Reggianito, in the interest of reducing ripening time. A two-factor experimental design was applied in two levels: type of coagulant enzyme, bovine chymosin or camel chymosin, and curd scalding temperature, 50 or 56 °C. The experimental treatments were applied in Reggianito cheese making experiments, and the samples were ripened for 90 d at 12 °C. Scalding temperature influenced residual coagulant activity; the cheeses cooked at 50 °C had significantly higher activity than those treated at 56 °C. In contrast, scalding temperature did not modify plasmin activity. Proteolysis was primarily affected by curd cooking temperature because chymosin-mediated hydrolysis of αs1 casein was slower in cheeses treated at 56 °C. Additionally, the nitrogen content in the cheese soluble fractions was consistently lower in the cheeses scalded at 56 °C than those cooked at 50 °C. A significant influence of the type of coagulant enzyme was observed, especially in the nitrogen fractions and peptide profiles, which demonstrated that camel chymosin was slightly less proteolytic; however, these differences were lower than those caused by the scalding temperature.

Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells.

This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on αs1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium-F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117μg/ml) was substituted partly with peptide-bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl-phenylalanyl-phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide-bound Phe at 10% (11.7μg/ml) significantly enhanced αs1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide-bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids.

Genetic polymorphism of α S1 casein in Guéra and Sahel goat

Summary The αS1 casein gene is one of the lacto protein genes, which are involved in the milk synthesis. The objective of this study was to identify genotypes of B and E alleles at the locus αS1 casein in the Guera and Sahel goat herds in Mali. These herds were located in the Regional Center of Agronomic Research and the Segala village in Kayes, Mali. A total of 101 blood samples (53 for Guera goat and 48 for Sahel goats) were collected and analysed. Alleles B and/or E, located on exon 19, of the αS1 casein gene were amplified using allele specific amplification polymerase chain reaction and analysed using 2 percent agarose gel electrophoresis. Results from this analysis identified three genotypes: BB, BE and EE with frequencies 0.77, 0.17 and 0.06 for the Guera goats. However, only the BB genotype was identified in the Sahel goats. The frequencies of alleles B and E in the Guera goats were 0.86 and 0.14, respectively, with an observed and expected heterozygotes of 0.08 and 0.12, respectively.

Detection of αs1-I casein in mozzarella Fiordilatte: A possible tool to reveal the use of stored curd in cheesemaking

The assessment of the origin and quality of raw materials is pivotal for the protection and valorization of typical dairy products. Italian high moisture mozzarella cheese, also called “Fiordilatte”, is manufactured by stretching in hot water the curd, which can be produced inside the dairy (fresh curd) or purchased as semi-finished product from specialized companies (pre-made stored curd). The employment of stored curd allows cheese manufacturers to obtain economic profit thanks to the reduction of the production costs. The use of the semi-finished product has not to be mentioned in the label according to the actual EU regulation, and this has given rise to competition between local milk farmers and curd-producing companies causing misinformation among the consumers, and unfair competition to the disadvantage of the traditional dairies. In the present research, a proteomic approach was used to investigate “Fiordilatte” produced with or without the employment of stored curd. The results obtained allowed us to identify a molecular marker which is present in very small amounts in cheese made from fresh curd and at high levels in cheese made from stored curd. The marker is αs1-I casein (fragment 24–199), the main product of primary proteolysis formed by the action of chymosin on αs1 casein. It can be easily detected by electrophoresis in the presence of urea (urea-PAGE) and is measured by image analysis. Even though the kinetic of formation of this proteolytic product has been well known for several decades, in mozzarella it has a peculiar behavior compared to other cheeses. As a matter of fact, it originates during the first part of cheesemaking, and has a very slow increase during storage of the cheese, due to the denaturation of the clotting enzyme by the hot water stretching process. The results obtained demonstrated that the quantification of αs1-I fragment should be recommended to guarantee the production of “Fiordilatte” obtained from fresh milk under normal conditions, and could be the basis for the development of a method able to fully protect the traditional cheesemaking procedure.

Association between single nucleotide polymorphisms (SNPs) and milk production traits in Italian Brown cattle

Italian Brown is a cattle breed largely exploited in the production of many dairy products in Italy, including typical and traditional cheeses. For this reason, the improvement of selection methods is of economic relevance while a deeper understanding of the genetic mechanisms regulating milk production is of general scientific interest. We selected a total of 561 samples, representing virtually all Italian Brown bull population, to test for association between milk production traits and 29 known genes harbouring 106 single nucleotide polymorphisms (SNPs). After filtering, a total of 31 SNPs in 22 candidate genes and 473 bulls were retained. Associations between each SNP and milk traits were tested by a mixed model approach, obtaining seven significantly associated SNPs, two of which (in β-Lactoglobulin) associated with all traits, and four (in Chemokin receptor I, αs1 casein, k casein, fatty acid synthase, thyroid hormone responsive and Oxytocin prepropetide genes) associated with at least one trait.

Regulation of milk protein solubility by a whey-derived proline-rich peptide product

The effects of a bovine whey peptide product enriched in proline (wPRP) on the solubility of milk proteins were tested under ambient conditions or following heat treatment at 75 and 100°C, for 1 and 15min, followed by post-incubation storage at either ambient temperature or 4°C for up to 7d. wPRP promoted solubilisation of milk proteins in a concentration-dependent manner without heat treatment and also after heat treatment at 75 and 100°C, and the effect was enhanced after storage under either ambient or refrigerated storage conditions. Interactions of wPRP and milk proteins were monitored by particle size analysis and tryptic digestion and specifically linked with solubilisation of αS1 casein (αS1-Cn), which supported observed changes in milk protein solubility. The results suggested that wPRP preferably prevented or reversed physical versus covalent protein aggregation, with the relaxation of hydrophobic interactions at 4°C providing an additive effect. This application of wPRP represents a novel approach to stabilisation of dairy proteins following thermal processing with industrial usefulness yet to be explored.

Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits

C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12g/l) harboring genomic human C1INH sequences fused to 5′ bovine αS1 casein promoter sequences. Recombinant hC1INH was isolated from milk to a specific activity of 6.1U/mg and a purity of 99%; by size-exclusion chromatography the 1% impurities consisted of multimers and N-terminal cleaved C1INH species. Mass spectrometric analysis of purified rhC1INH revealed a relative molecular mass (Mr) of 67,200. Differences in Mr on SDS PAGE and mass spectrometric analysis between rhC1INH and pd-hC1INH are explained by differential glycosylation (calculated carbohydrate contents of 21% and 28%, respectively), since protein sequencing analysis of rhC1INH revealed intact N- and C-termini. Host-related impurity analysis by ELISA revealed trace amounts of rabbit protein (approximately 10ppm) in purified batches, but not endogenous rabbit C1INH. The kinetics of inhibition of the target proteases C1s, Factor XIIa, kallikrein and Factor XIa by rhC1INH and pd-hC1INH, indicated comparable inhibitory potency and specificity. Recently, rhC1INH (Ruconest®) has been approved by the European Medicines Agency for the treatment of acute attacks of HAE.

Comparison of quality characteristics of Çökelek and Lor cheeses

The objective of this study was to compare some quality characteristics of Cokelek (cottage cheeselike) and Lor (whey cheese) cheeses during a storage period of 21 days. Cokelek and Lor cheeses significantly (P < 0.05 , P < 0.01) showed differences in terms of the examined parameters and storage period. Mean value of lipolysis in Cokelek cheese was higher (P < 0.01) than that of Lor cheese. Also storage time affected (P < 0.01) the values of lipolysis in two experiments. It was found that αs1 casein was hydrolyzed much faster than those of γ-casein and β-casein in the Cokelek and Lor cheeses, the γ-, β-, and αs1 casein levels in two cheeses remained relatively constant during storage. There were no significant differences in water-soluble N (WSN), trichloroacetic acid soluble N (TCA-SN) and phosphotungstic acid soluble N (PTA-SN) contents of the two experiments, but the storage time significantly (P < 0.01) affected these parameters. The counts of coliforms, lactic acid, proteolytic, psycrotrophic bacteria and yeasts-molds between Cokelek and Lor cheese samples were found to be significant (P < 0.01), but total aerobic mesophilic bacteria (TAMB) and Salmonella thermophilus counts were not significant statistically. Storage time significantly (P < 0.01) affected the examined microbiological parameters. Sensory evaluations of the experimental cheeses showed differences in terms of cheese types and storage period.