Genetic variability in the kappa‐casein gene in Sahiwal,Cholistani and Red Sindhi cattle breeds

Polymorphism of kappa‐casein (κ‐CN) gene in three Bos indicus cattle breeds was investigated using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique. Only genotypes AA and AB were observed, and no BB, AE, BE, EE, AC or BC genotypes were detected in the three cattle breeds. In the Sahiwal, Cholistani and Red Sindhi breeds, the frequencies of the allele A were 0.69, 0.90 and 0.86, and the frequencies of the allele B were 0.31, 0.10 and 0.14, respectively. The Sahiwal breed was found to have a higher frequency of the AB genotype as compared to the Cholistani and Red Sindhi breeds. The frequency of the κ‐CN alleles noted in the three cattle breeds was similar to those observed in other cattle breeds of B. indicus origin.     

Κ‐CN gene polymorphism in Nili‐ravi buffalo,Achai and Sahiwal cattle of Pakistan

Kappa‐casein (κ‐CN) is the subtype of casein protein, an important constituent of bovine milk protein. The current study was undertaken to investigate the genetic polymorphism in κ‐CN gene of Nili‐ravi buffalo, Achai and Sahiwal cattle of Pakistan using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) technique. The Nili‐ravi buffalo was found to be monomorphic (genotype BB only) for κ‐CN gene. Achai cattle were polymorphic for κ‐CN (having three genotypes AA, AB and BB) with a frequency of 0.70, 0.18 and 0.12, respectively, while in Sahiwal cattle, both the genotypes AA and AB were found with genotypic frequencies of 0.92 and 0.08, respectively. The presence of genotype BB in Achai cattle is surprising as it is absent in most of the cattle breeds worldwide.     

Milk protein polymorphisms in Holstein cattle†

Milk samples from 203 Holstein cows were phenotyped for genetic variants α_s1‐casein, β‐casein, κ‐casein and β‐lactoglobulin using starch‐gel electrophoresis. All of the four milk protein loci exhibited polymorphism with allele frequencies of 0.862 ± 0.017 for α_s1‐casein B, 0.966 ± 0.0009 for β‐casein A, 0.712 ± 0.0224 for κ‐casein A and 0.567 ± 0.0245 for β‐lactoglobulin B. The mean heterozygosity estimated over all the four milk protein loci was 0.3015. Genetic equilibrium was observed among all of the loci investigated, except κ‐casein. Chi‐squared tests revealed that there was no significant linkage among studied milk protein phenotypes.     

Influence of whey peptides on the surface activity of κ‐casein and β‐lactoglobulin A

Whey protein hydrolysate (WPH) was fractionated by reverse‐phase chromatography to obtain fractions of varying surface‐hydrophobicities. A model oil–water interface (MI) was pre‐coated with the WPH or fractions thereof. Contact angle (θ) of sessile drops of κ‐casein (κ‐CN) or β‐lactoglobulin A (β‐LGA) were measured on the MI. Pre‐coating of MI with un‐fractionated WPH decreased θ, that is, increased surface activity, of both κ‐CN (35–8.3°) and β‐LGA (38–21.3°). Conversely, pre‐coating of MI with the fractions significantly increased θ of both proteins as a function of hydrophobicity. Data provide insight into variability of whey protein functionality in food applications.     

Associations between milk protein polymorphism and first lactation milk production traits in Finnish Ayrshire cows.

Genotypic effects of beta-casein (CN), kappa-CN, and beta-lactoglobulin (LG) on milk, fat, and protein production and fat and protein percentages were estimated for 18,686 Finnish Ayrshire cows in first lactation using an animal model. Casein genotype effects were estimated including individual beta-CN and kappa-CN simultaneously in a model and then as composite beta-kappa-CN. The A2 allele of beta-CN and the A allele of kappa-CN, as well as the A1 allele of beta-CN and the B or E allele of kappa-CN, appeared together more frequently than was expected. Because of linkage disequilibrium in the casein loci and, consequently, unbalanced data, some contradictory effects of casein genotypes were obtained with the two models. A well-founded way to estimate the effects of casein genotypes was to use beta-kappa-CN genotypes. Composite casein genotypes including the A2 allele of beta-CN were associated with the highest milk and protein production and the lowest fat content, those including the B allele of kappa-CN with the highest protein content, and those including the E allele of kappa-CN with the lowest protein content. The effect of the beta-kappa-CN genotypes on protein content was moderately strong, and the effect was somewhat smaller for other traits. The AA genotype of beta-LG had a favorable effect on milk and protein production, and the BB genotype had a favorable effect on fat content.     

Determination of allele frequencies of growth hormone AluI polymorphism in Brown Swiss,Holstein, native East Anatolian Red and Turkish Grey breeds

A total of 177 cattle of four breeds were genotyped for the bovine growth hormone (BGH)‐AluI polymorphism by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP). The genotype and gene frequencies for each breed were determined and tested to be in Hardy–Weinberg equilibrium. According to breeds, frequencies of allele L gene were 0.905 for Brown Swiss, 0.898 for Holstein, 0.976 for East Anatolian Red and 0.893 for Turkish Grey Breeds. The allele L was predominant and variant VV was not detected in the breeds studied. BGH‐AluI genotypes were found to be in equilibrium within and among breeds.     

A comparison of the physico‐chemical properties of low‐cholesterol ghee with standard ghee from cow and buffalo creams

Low‐cholesterol ghee with 90% less cholesterol was prepared using β‐cyclodextrin. The physico‐chemical properties such as Reichert‐Meissl (RM) value, Polenske value, Butyro‐refractometer (BR) reading at 40°C, Iodine value and free fatty acids (FFA) as oleic acid in cow standard ghee and the corresponding low‐cholesterol ghee remained almost unaltered. A similar trend was also observed in buffalo ghee. Fat soluble vitamins (β‐carotene, A and E) in both cow and buffalo low‐cholesterol ghee were very similar to that of respective standard ghee samples. However, 65 to 70% loss of vitamin D was observed in low‐cholesterol ghee.     

Properties of casein concentrates containing various levels of beta‐casein

Microfiltration (MF) of milk was used to produce casein (CN) concentrates (80% protein) with reduced whey protein levels. By varying temperature of MF, we altered the proportion of β‐CN to αs‐CN in CN concentrates and compared them to milk protein concentrate (MPC). Casein content as a % of protein was approximately 90% for CN concentrates and approximately 80% for MPC. Smaller micelles and weaker rennet gels were observed for CN concentrates with low β‐CN level. Foam stability and yield stress values were higher for CN concentrates with a high β‐CN level. Modified CN concentrates can be produced by altering the proportions of individual CNs.     

Effect of pH on the formation of serum heat‐induced protein aggregates in heated yak milk

Serum heat‐induced protein aggregates at various pH levels were isolated from heated yak milk by size‐exclusion fast protein liquid chromatography. Analysis by reversed‐phase high‐performance liquid chromatography showed that β‐lactoglobulin and κ‐casein were the two main proteins in these aggregates. The amount of serum heat‐induced protein aggregates increased from pH 6.6 to 7.4, whereas the content of β‐lactoglobulin in aggregates decreased from 61.9% at pH 6.6 to 50.4% at pH 7.2, and then increased to 51.3% at pH 7.4. The content of κ‐casein, αs₁‐casein and β‐casein increased from pH 6.6 to pH 7.4. These results show that serum heat‐induced protein aggregates are pH dependent.     

Identification of Conformational Antigenic Epitopes and Dominant Amino Acids of Buffalo β‐Lactoglobulin

Major allergen β‐lactoglobulin exists in many mammalian types of milk except human breast. Buffalo milk also contains this major allergen but the detailed information on its epitopes is not available. The aim of this work was to map and characterize its conformational antigenic epitopes. Sixty mimotopes of buffalo β‐lactoglobulin were produced by biopanning of phage display peptide library and then 2 mimotopes, specific for sera from rabbit 1 and 2, respectively, were predicted to be conformational epitope candidates by the use of DNAStar and web tool of MIMOX. On the basis of bioinformation analysis, 5 conserved amino acid residues PL‐ENK were identified in 2 conformational epitope sequences and 7 conformational epitopes were derived from 2 mimotopes by molecular modeling. The result showed that these conformational epitopes were located in the 2 regions on buffalo β‐lactoglobulin and composed of 5 hydrophilic and 2 hydrophobic amino acids.     

The effect of glycosylation on emulsifying and structural properties of bovine β‐casein

A number of attempts have been made to improve the functional properties of milk proteins by chemical modifications. One of such modifications is glycosylation which was carried out to determine the effect of covalent binding of glucose molecules to β‐casein (βCN) on its emulsifying properties. It was found that up to six molecules of glucose were bound to one molecule of βCN. Glycosylated βCN produced smaller emulsion droplets than the intact βCN. Increases in emulsion‐forming and ‐stabilizing properties were observed. A prerequiste of proteins to form emulsions is their adsorption onto oil/water interface. Therefore the secondary structure of intact and glycosylated βCN, both in solution and adsorbed onto a hydrophobic teflon/water interface also were studied by far‐ultra violet circular dichroism (CD). It appeared, that after glycosylation the degree of helicity of intact βCN decreased and the incorporation of glucose moieties most likely resulted in a type β‐turn formation after adsorption onto the interface.     

Studies on the interaction of ‐epigallocatechin‐3‐gallate from green tea with bovine β‐lactoglobulin by spectroscopic methods and docking

The binding interaction between‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐lactoglobulin (βLG) was thoroughly studied by fluorescence, circular dichroism (CD) and protein–ligand docking. Fluorescence data revealed that the fluorescence quenching of βLG by EGCG was the result of the formation of a complex of βLG–EGCG. The binding constants and thermodynamic parameters at two different temperatures and the binding force were determined. The binding interaction between EGCG and βLG was mainly hydrophobic and the complex was stabilised by hydrogen bonding. The results suggested that βLG in complex with EGCG changes its native conformation. Furthermore, preheat treatment (90 °C, 120 °C) and emulsifier (sucrose fatty acid ester) all boosted the binding constants (K_a) and the binding site values (n) of the βLG‐EGCG complex. This study provided important insight into the mechanism of binding interactions of green tea flavonoids with milk protein.     

A survey of the occurrence and properties of methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus intermedius in water buffalo milk and dairy products in Turkey

Antimicrobial resistance, β‐lactamase activity and mecA gene of Staphylococcus aureus and Staphylococcus intermedius isolated from raw water buffalo milk and dairy products in Turkey were determined. All strains showed resistance to at least one antibiotic but none was resistant to vancomycin. Of the 97 S. aureus and 35 S. intermedius strains, 9 (9.2%) and 2 (5.7%) were resistant to oxacillin and harboured mecA gene. β‐lactamase activity of 13.4% and 5.7% of S. aureus and S. intermedius strains was positive, respectively. Overall, 2.5% and 0.55% of the samples were contaminated with methicillin‐resistant S. aureus and S. intermedius, respectively.     

Proteolysis,protein distribution and stability of UHT milk during storage at room temperature

Proteolytic degradation and distribution of caseins and whey proteins between the soluble and colloidal phases were studied in six batches of commercial UHT milk (three skim and three whole milks) during storage at 25 ± 2 °C. For that purpose, at 30 day intervals, milk samples were ultracentrifuged and the pellets and supernatants analysed by capillary electrophoresis and SDS‐PAGE. Samples were also visually examined for signs of gelation. Extensive proteolytic degradation of the micellar fractions and severe changes in the electrophoretic pattern of the proteins present in the serum fractions were observed in all the batches. A higher proportion of denatured whey proteins not attached to the micelle surface was found in the skim milk samples as compared with the whole milk samples that could provide less resistance against gelation. In addition to β‐Lg, para‐κ‐casein was also found in the serum fraction. A high proteolytic activity against κ‐casein could be responsible for the hydrolysis of serum‐liberated κ‐casein or could have enhanced the liberation of β‐Lg–para‐κ‐casein complexes through proteolysis of micellar κ‐casein. © 1999 Society of Chemical Industry     

Physical‐Mechanical Properties of Agar/κ‐Carrageenan Blend Film and Derived Clay Nanocomposite Film

Abstract: Binary blend films with different mixing ratio of agar and κ‐carrageenan were prepared using a solution casting method with and without nanoclay and the effect of their composition on the mechanical, water vapor barrier, and water resistance properties was tested. The tensile strength (TS) of the κ‐carrageenan film was greater than that of agar film. The water vapor permeability (WVP) of the agar film was lower than that of κ‐carrageenan film, the swelling ratio (SR) and water solubility (WS) of κ‐carrageenan film were higher than those of agar film. Each property of the binary blend films varied proportionately depending on the mixing ratio of each component. The XRD result indicated that the nanocomposite with agar/κ‐carrageenan/clay (Cloisite^® Na⁺) was intercalated. Consequently, the mechanical strength, water vapor barrier properties, and water contact angle (CA) were significantly (P < 0.05) improved through nanocomposite formation.     

The (193–209) 17‐residues peptide of bovine β‐casein is transported through Caco‐2 monolayer

Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine residues. β‐casein (β‐CN, 193–209) is a long and hydrophobic peptide composed of 17 amino acid residues (molecular mass 1881 Da) with immunomodulatory activity. The present work examined the transport of the β‐CN (193–209) peptide across Caco‐2 cell monolayer. In addition, we evaluated the possible routes of the β‐CN (193–209) peptide transport, using selective inhibitors of the different routes for peptide transfer through the intestinal barrier. The results showed that the β‐CN (193–209) peptide resisted the action of brush‐border membrane peptidases, and that it was transported through the Caco‐2 cell monolayer. The main route involved in transepithelial transport of the β‐CN (193–209) peptide was transcytosis via internalized vesicles, although the paracellular transport via tight‐junctions could not be excluded. Our results demonstrated the transport of an intact long‐chain bioactive peptide in an in vitro model of intestinal epithelium, as an important step to prove the evidence for bioavailability of this peptide.     

A Survey of the Peptide Profile in Prato Cheese as Measured by MALDI‐MS and Capillary Electrophoresis

In this study, we describe the characterization of the peptide profile in commercial Prato cheese by matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) and capillary electrophoresis (CE). Ten commercial Prato cheese brands were characterized via their physicochemical composition and subjected to fractionation according to solubility at pH 4.6. The pH 4.6 insoluble fraction was evaluated by CE, whereas MALDI‐MS was applied to the fraction soluble at pH 4.6 and in 70% ethanol. CE revealed a characteristic pattern of hydrolysis, with formation of para‐κ‐casein, hydrolysis of α_s1‐casein at the Phe₂₃ ‐ Phe₂₄ bond, and hydrolysis of β‐casein. For the MALDI‐MS data, a complex peptide profile was observed, with the identification of 44 peptides previously reported (24 peptides from α_s1‐casein, 14 from β‐casein, 3 from κ‐casein, and 3 from α_s2‐casein). It was also observed that cheeses with salt‐in‐moisture content greater than 5% showed an accumulation of a bitter‐tasting peptide (m/z 1536, α_s1‐CN f1‐13), suggesting a relationship between the higher salt concentration and the abundance of this peptide. In conclusion, the results showed that even commercial cheeses produced with different raw material and processing conditions showed very similar peptide profiles when assessed at the molecular level, and only 9 peptides were responsible for discrimination of cheeses.     

Angiotensin I‐converting enzyme inhibitory peptides in skim milk fermented with Lactobacillus helveticus 130B4 from camel milk in Inner Mongolia,China

BACKGROUND: Angiotensin I‐converting enzyme (ACE) is a dipeptidyl carboxypeptidase associated with the regulation of blood pressure. ACE inhibition results in a lowering of blood pressure. Lactic acid bacteria are known to produce ACE inhibitors during fermentation. Fermented camel milk is the main traditionally fermented dairy food for desert nomads. The beneficial effects of fermented camel milk, which include the prevention of such diseases and conditions as gastroenteritis, tuberculosis and hypertension, have been demonstrated experimentally. RESULTS: ACE inhibitory activity was observed in fermented milk containing Lactobacillus helveticus 130B4, a strain isolated from traditionally fermented camel milk. The peptide that inhibited ACE was purified from the fermented milk by reverse‐phase high‐performance liquid chromatography. The amino acid sequence of the peptide was identified as Ala‐Ile‐Pro‐Pro‐Lys‐Lys‐Asn‐Gln‐Asp (IC₅₀ = 19.9 µmol L^?1). The same Ala‐Ile‐Pro‐Pro‐Lys‐Lys‐Asn‐Gln‐Asp sequence was found in κ‐casein (κ‐CN) f107–115 from milk. The inhibitory activity of this nonapeptide (κ‐CN f107–115) was almost preserved even after successive digestion with pepsin, trypsin and chymotrypsin. Furthermore, the inhibitory activity of the purified peptide was completely preserved after heat treatment at 100 °C for 20 min. CONCLUSION: The fermented milk prepared with Lactobacillus helveticus 130B4 contained an ACE inhibitory peptide, κ‐CN 107–115. This fermented milk was expected to have anti‐hypertensive effect after ingestion because the peptide was stable to digestive protease and heat treatment in vitro. Copyright © 2008 Society of Chemical Industry     

Low‐field NMR: A tool for studying protein aggregation

Low‐field nuclear magnetic resonance (NMR) spin–spin relaxation (T₂) measurements were used to study the denaturation and aggregation of β‐lactoglobulin (β‐LG) solutions of varying concentrations (1–80 g L^?1) as they were heated at temperatures ranging from ambient up to 90 °C. For concentrations of 1–10 g L^?1, the T₂ of β‐LG solutions did not change, even after heating to 90 °C. A decrease in T₂ was only observed when solutions having higher concentrations (20–80 g L^?1) were heated. Circular dichroism (CD) spectroscopy and fluorescence tests using the dye 1‐anilino‐8‐naphthalene sulfonate (ANS) on 0.2 and 1 g L^?1 solutions, respectively, indicated there were changes in the protein's secondary and tertiary conformations when the β‐LG solutions reached 70 °C and above. In addition, dynamic light scattering (DLS) showed that protein aggregation occurred only at concentrations above 10 g L^?1 and for heating at 70 °C and above. The hydrodynamic radius increased as T₂ decreased. When excess 2‐mercaptoethanol was added, the changes in both T₂ and the hydrodynamic radius followed the same trend for all β‐LG protein concentrations between 1 and 40 g L^?1. These observations led to the conclusion that the changes in T₂ were due to protein aggregation, not protein unfolding. Copyright © 2007 Society of Chemical Industry     

Primary structure of water buffalo alpha-lactalbumin variants A and B

A novel electrophoretic alpha-lactalbumin (alpha-la) variant was detected in the Italian water buffalo breed. The isoelectric point of the variant, labelled A, was lower than the most frequent variant B. It presented an allelic frequency of 0.5% compared with the 97.1% of the BB allele. From Liquid Chromatography-Electrospray Ionization/Mass spectrometry, the molecular mass of the two alpha-la A and B variants were measured as 14,235.1+/-0.8 and 14,236.1+/-0.9 Da, respectively. The two proteins were sequenced and differentiated from one another by a single amino acid substitution, Asn45(B)-->Asp45(A). As this amino acid substitution altered the N-glycosylation sequence consensus Asn45-X-Ser46 it may be deduced that the protein glycosylation level of the alpha-la A would decrease.