Goat Casein Research Articles

Moleculer analysis of genotype kappa casein and composition of goat milk Etawah grade, Saanen and their crossbred.

Polymorphism of goat casein gene closely linked to the quality of milk protein. κ-casein is one of the casein fractions that influence the shape and stability of grain milk. This study is aimed to identify the variation of genotype κ-casein and related with milk quality from Etawah grade, Saanen and their crossbreed (PE-SA). The number of dairy goats used in this study was 150 animals consisted of Etawah grade (48 animals), Saanen (51 animals) and PE-SA (51 animals). Steps of experiment were: blood and milk sampling collection, DNA amplification by PCR and the product digestion using Pst1 enzyme, κ-casein gene sequencing and analyzing the quality of fresh milk. The results showed that κ-casein gene is monomorphic by PCR-RFLP (Pst1) for all the goat breeds, but DNA sequencing indicated 38 point of mutation. Observation on goat milk quality showed that Etawah grade milk had highest (P < 0.05) density value (1.033 ± 0.002) and solid non fat (9.577 ± 0.704%) than those of Saanen and PE-SA fresh milk goat. Key Words: Dairy Goats, Κ-Casein Gene, PCR-RFLP, Milk

Detection of cow’s milk in Shaanxi goat’s milk with an ELISA assay

The aim of this work was to develop an ELISA assay capable of detecting adulteration of goat milk and its products with bovine milk in China. Polyclonal antibodies against bovine β-casein were used and modified by mixing them with lyophilized Saanen goat β-casein (blocking) to improve the specificity of the antibodies. The blocked antibodies recognize some specific epitopes of bovine β-casein with no cross-activity with bovine α-casein, κ-casein and whey protein. The absorbance values of the indirect ELISA showed a linear relationship to the adulteration concentration of bovine milk in the range of 2%–50%, which can be applied to quantify adulteration of bovine milk. There was no significant difference in titration curves between Saanen and Guanzhong goat milk, and the detection limits were all 2% for goat caseins, raw milk and heat-treated milk. Low coefficients of variation (＜10%) of the experiments indicated good repeatability. The ELISA assay is likely suitable for routine detection of cow’s milk adulteration of Shaanxi goat’s milk.

Fast screening and quantitative evaluation of internally deleted goat αs1‐casein variants by mass spectrometric detection of the signature peptides

Currently, the internally deleted caprine alphas1-casein (alphas1-CN) variants F and G, associated with low casein expression, are detected by means of ordinary descriptive techniques. No relevant procedure is available to detect internally deleted goat alphas1-CN in bulk milks. The availability of full-length and alphas1-CN F and G variants allowed us to further investigate this issue. Using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and high-performance liquid chromatography (HPLC)/electrospray ionization (ESI)-MS and ESI-MS/MS, tryptic signature peptides alphas1-CN F f59-63/f43-63, alphas1-CN G f4-20/f4-21, alphas1-CN B2 f4-22 Pro16 and alphas1-CN A f4-22 Leu16 were identified. This also helped to solve the interesting question of how the casein variants contribute to the composition of goat's bulk milk. Synthetic peptide analogues with ionization efficiency equivalent to that of tryptic junction peptides were used as internal standards to evaluate alphas1-CN variants, either individually or globally, using bulk milk from a single goat breed as a model system. Here, alphas1-CN F accounted for 0.15+/-0.08% and the alphas1-CN G variant was missing or below the 0.10% detection limit. The analysis of six samples confirmed that alphas1-CN G was missing and that alphas1-CN F occurred at a low frequency in hybrid and local breed bulk milks from Mediterranean areas. In conclusion, a quantitative MS-based application of the signature peptides for full-length and internally deleted variants in goat's casein is provided. The strategy is also suggested for the determination of splice variants in any biological sample.

Focusing on casein gene cluster and protein profile in Garganica goat milk

A survey was carried out in eight goat dairy farms, a total of 71 individual Garganica goat milk samples were collected for genomic DNA extraction. Casein alleles and haplotype frequencies of Garganica population were estimated. Individual milks were also analysed for chemical composition, rheological properties, and protein profile. The strong A* allele of CSN1S1 was predominant in the population investigated, the weak allele F of CSN1S1 showed a relatively high frequency and the null alleles N and 01 were first observed in this breed. At CSN1S2 locus the strong A* allele was the most frequent, followed by the F allele and the null allele. The strong A* allele was predominant at CSN2 locus, and relatively high incidence of null allele 0 was observed. CSN3 locus was monomorphic for B* allele. The exact test of sample differentiation based on haplotype frequencies discriminate the farms into two groups characterized by the highest frequency of strong (S-CSN1S1) or weak (W-CSN1S1) alleles at CSN1S1. Protein and casein contents were higher in the group characterized by strong allele than in the group with weak allele at CSN1S1. The 2D electrophoresis technique was performed to screen goat casein variability at the protein level and to evaluate global casein genotype (alphas1, alphas2, beta and kappa-CN). Gels displayed the protein profile associated with casein genotype, and demonstrated differences in the protein expression deriving from interactions between loci. The variability of goat casein loci in Garganica goat breed could be exploited to differentiate the population on the basis of milk utilization and could represent a strategy to preserve the genotype of this autochthonous breed.

Technical Note: Simultaneous Identification of CSN1S2 A, B, C, and E Alleles in Goats by Polymerase Chain Reaction-Single Strand Conformation Polymorphism

Most variability in goat caseins originates from the high number of genetic polymorphisms often affecting the specific protein expression, with strong effects on milk composition traits and technological properties. At least 7 alleles have been found in the goat αS2-CN gene (CSN1S2). Five of them (CSN1S2*A, CSN1S2*B, CSN1S2*C, CSN1S2*E, and CSN1S2*F) are widespread in most breeds, whereas the other 2 (CSN1S2*D and CSN1S2*0) are rarer alleles. Four different PCR-RFLP tests are needed to detect all of these variants at the DNA level. The objective of this study was to develop and validate a rapid method for typing 4 of the 5 most-common goat CSN1S2 alleles by means of PCR-single strand conformation polymorphism (SSCP). The method was validated by analyzing 37 goat samples at the protein and DNA level, respectively, by milk isoelectrofocusing and PCR-RFLP methods already described. The genotypes obtained using the PCR-SSCP approach were in full agreement with those obtained by the validation analyses. The newly developed PCR-SSCP approach provides an accurate and inexpensive assay highly suitable for genotyping goat CSN1S2.

Casein polymorphism in goat's milk

In this review we summarize the current knowledge on the extensive goat casein poly- morphism. Gene structure, and allelic and non-allelic protein variants, characterized by genomic and proteomic analysis, have been described. The effect of genetic casein polymorphism on techno- logical and nutritional properties of goat's milk has been briefly reported. Goat casein genes show a range of different post-translational modifications and complex patterns of splicing, leading to a great heterogeneity of this fraction. In the light of some recent findings, a genomic and proteomic combined approach appears to be the tool of choice in order to gain an exhaustive characterization of these milk proteins. goat's milk / casein fraction / heterogeneity / genetics / proteomics

Polymerase Chain Reaction - Single Strand Conformation Polymorphism (PCR-SSCP) analysis of goat αs2-casein

Goat caseins show high genetic polymorphism, with several mutations affecting the specific protein expression. To date at least seven alleles have been found at goat α gene (CSN1S2). Five of them, CSN1S2*A, CSN1S2*B, CSN1S2*C, CSN1S2*E, and CSN1S2*F, code for a normal content of α (about 2.5 g/l), whereas CSN1S2*D and CSN1S2*0 are associated respectively with a reduced α content and with the absence of this specific protein in the milk. Four different Restriction Fragment Length Polymorphism (RFLP) - Polymerase Chain Reaction (PCR) tests are needed to detect all these seven alleles at the DNA level. The aim of this study was to develop a new tool for the genetic study of CSN1S2 variability in goat.A protocol for the simultaneous genotyping of CSN1S2*A, CSN1S2*B, CSN1S2*C, and CSN1S2*E alleles was developed by PCR – Single Strand Conformation Polymorphism (SSCP) technique. A 247 bp fragment containing exon 9 and a 273 bp fragment containing exon 16 of the goat CSN1S2 gene were simultaneously amplified by PCR, and subsequently analysed by SSCP. The test was validated by screening the CSN1S2 variability in the following goat breeds: Orobica (n = 81), Camosciata (n = 112), and Saanen (n = 76).DNA was extracted from blood or milk by a commercial kit. Milk samples were analysed by isoelectric focusing (IEF) for α typing at the protein level. The DNA samples were analysed both by the novel PCR-SCCP protocol as well as by two PCR-RFLP tests already available, respectively for the detection of CSN1S2*A, CSN1S2*B, CSN1S2*C and for the detection of CSN1S2*E. Full agreement was found in the typing results obtained by the PCR-SSCP protocol developed, the two PCR-RFLP methods, and the IEF analysis which allows the simultaneous detection of the four variants at the protein level.In all cases, a specific DNA test is needed to detect the mutation characterizing the CSN1S2*F allele. The PCR-SSCP technique developed is a cheep alternative to the two PCR-RFLP methods for CSN1S2 typing at the DNA level, allowing to reduce to three the number of tests necessary to identify the seven CSN1S2 alleles described.

The challenge of cow milk protein allergy

Hypersensitivity to cow milk proteins is one of the main food allergies and affects mostly but not exclusively infants, while it may also persist through adulthood and can be very severe. Different clinical symptoms of milk allergy have been established. The diagnosis of milk allergy differs widely due to the multiplicity and degrees of symptoms, and can be achieved by skin or blood tests. Cow milk contains more than 20 proteins (allergens), that can cause allergic reactions. Casein fractions and β-lactoglobulin are the most common cow milk allergens. Human milk is free of β-lg, similar to camel milk. On the contrary, β-lg is a major whey protein in cow, buffalo, sheep, goat, mare and donkey milk. Caseins in milk of the different species differ in fraction number, amino acid composition, and their peptide mappings. β-Casein is the major fraction in goat casein, which is similar to human casein and different from cow casein. The peptide mappings of goat α-la and β-lg are completely different from those of cow milk. Different procedures can reduce the allergenicity of cow milk proteins by heat or enzymatic treatment to some degree. Allergies to milk proteins of non-bovine mammals have also been documented due to cross-reactivity between cow milk proteins and their counterpart in other species, and even between goat and sheep caseins. Genetic polymorphisms of milk proteins play an important role in eliciting different degrees of allergic reactions. Goat milk lacking α-s1-casein, which is the main casein in cow milk, is less allergenic than goat milk with α-s2-casein, which is more typical for many goat breeds. Several studies have reported real and dramatic benefits from using goat, camel, mare or even soy milk as alternatives in cases of cow milk allergy and they can be considered hypoallergenic. However, therapeutic benefits vary with the degree of severity of the allergy and may be only around 60% of all cases, since other studies revealed allergenicity to occur also for any of those other milks.

Allergy To Sheep's And Goat's Milk Cheese With Good Tolerance to Cow's Milk

RATIONALE: Cow's milk (CM) allergy is common in children, although there are few reports of allergy to sheep's (SM) and goat's milk (GM) and with good tolerance to CM. METHODS: Patients: 2 y.o. female and a 10 y.o. male who presented inmediate systemic allergic reactions after ingestion of sheep's cheese and another cheese of unknown origin, respectively. Both patients have always tolerated cow's milk.Skin prick test, CAP-FEIA and immunoblotting were performed. RESULTS: Patient 1:Prick test (PT):CM, BSA, ALA and BLG: negative. Prick-prick (PPmm): SM (15), GM (13), cow's casein (CC) (3), sheep's casein (SC) (8), goat's casein (GC) (5). Specific IgE (CAP kU/L): CM:0.53, CC:0.79, SM:81.90; GM >100; BSA, ALA and BLG:<0,35. . Patient 2: PT: CM(3), BSA, BLG and ALA: negative. PP: SM(18), GM(12), CC(3), SC(8), GC(8). CAP: CM:2,75, CC:2.94, SM >100, GM>100; ALA, and BLG< 0.35. Both patients had strong IgE-binding to a protein with a MW about 31 kDa in SM, GM, SC and GC extracts. Both patients had weak IgE-binding to other proteins in a range of 19-60 kDa in SM and GM. Patient 2 show weak IgE-binding to other proteins in a range of 19- 60 kDa in CM, SC and GC. Serum from patient 1 did not recognize any band in CM or CC. CONCLUSIONS: An IgE-mediated allergy to sheep's and goat's milk was demonstrated in two patients without allergy to cow's milk. Sheep's and goat's casein seem to be the main allergen in these cases.

Casein and Cheese Peptide Degradation by Enterococcus durans FC12 Isolated from Feta Cheese

The growth of Enterococcus durans FC12 isolated from Feta cheese in broths containing casein, casein hydrolysate or cheese water-soluble extract as sole nitrogen source was studied. FC12 assimilated casein hydrolysate and casein, the former at a higher extent, while it also grew in the presence of 4% NaCl. It assimilated at a similar extent cow, ewe or goat casein, and it grew rapidly using cheese soluble nitrogenous materials. The cell-envelope enzyme preparation of E. durans FC12 hydrolysed both α-casein and β-caseins, the latter more rapidly, producing three main peptides. It also hydrolysed two peptides analog to the cheese peptide αs1-CNf1-23 that is a key peptide in the first steps of cheese proteolysis. FC12 cell-envelope enzyme preparation degraded the above caseins and peptides in the presence of 4% NaCl. The ability of E. durans FC12 to assimilate casein and cheese soluble nitrogenous materials along with the ability of its cell-envelope enzyme preparation to hydrolyse caseins and peptides analog to the αs1-CNf1-23 indicates that the FC12 strain may be useful in cheese proteolysis as an adjunct culture.

Casein genetic polymorphisms in goat breeds of Lombardy

RiassuntoPolimorfismo genetico delle caseine in razze caprine allevate in Lombardia. E’ stato condotto uno studio della variabilità genetica delle caseine in sei razze caprine allevate in Lombardia. La tipizzazione, condotta a livello proteico mediante isoelettrofocalizzazione, è preliminare ad un approccio molecolare dettagliato su un numero più limitato di soggetti, scelti in base a particolari fenotipi caseinici.

Selective allergy to sheep’s and goat’s milk proteins

BackgroundCow’s milk proteins are amongst the most common causes of food allergy in infants, and caseins are probably the main allergens. The existence of a high degree of cross-reactivity between milk caseins from different animals has been reported. We describe a 2-year-old boy who experienced allergic reactions after eating and touching sheep’s cheese, but who tolerated cow’s milk and cow’s milk dairy products. He had never ingested milk or milk derivatives from sheep or goat MethodsSkin prick tests were carried out using whey fractions of cow’s milk, whole milk and casein from goat, sheep and cow. We also performed skin prick tests with enzymes used in cheese production. Prick-by-prick tests with cheese made from cow, sheep and goat and their corresponding whole milk were also performed. Total serum IgE and specific IgE to cow’s milk proteins, whole cow’s milk and sheep’s milk were determined. Specific IgE against casein and whole milk from the three different species were determined by ELISA. Inhibition of IgE binding to bovine casein was tested for casein and whole milk from all three species. The proteins of three types of casein and whole milk from cow, sheep and goat were separated by SDS-PAGE and were incubated with the patient’s serum ResultsSkin tests were positive to sheep’s milk and goat and sheep casein and were negative to all cow’s milk proteins and whole cow’s and goat’s milk. Prick-by-prick tests were positive to goat’s and sheep’s cheese and were negative to cow’s cheese. In ELISA-inhibition, sheep’s milk and goat and sheep casein were able to inhibit > 50 % of specific IgE binding to sheep casein. The results of immunoblotting showed that the patient’s circulating IgEs recognized only one band in the lanes corresponding to sheep and goat casein ConclusionsWe report a patient with allergy to sheep’s and goat’s milk proteins but not to cow’s milk proteins. Sheep casein was probably the main allergen causing sensitization in this patient. The results suggest that sheep casein shows a high degree of cross-reactivity with goat casein but not with cow casein. Our patient presented allergic symptoms caused by sheep and goat milk and cheese proteins. However, he was able to tolerate cow’s milk and cow’s milk dairy products without any ill effects

IgA anti-gliadin antibody immunoreactivity to food proteins

We developed a competitive enzyme-linked immunosorbent assay (cELISA) based on bread wheat flour gliadin as coated antigen and human serum IgA as primary antibody in order to quantify the immunoreactivity of untreated celiac patient IgA antigliadin antibodies (AGAs) to different food proteins. No significant AGA immunoreactivity to bovine serum albumin or zeins was detected, but there was considerable immunoreactivity to caseins: 39% to bovine caseins and 65% to goat caseins (base gliadin reactivity: 100%). We also investigated AGA immunoreactivity to the same proteins after peptic-pancreatic digestion. Taken together, the results suggest that AGA immunoreactivity to gliadins and caseins could be due to the β-turn content in these proteins and that a minor portion of serum AGAs from untreated celiac patients is represented by antibodies recognizing linear epitopes.

Whole-Body Nitrogen and Splanchnic Amino Acid Metabolism Differ in Rats Fed Mixed Diets Containing Casein or Its Corresponding Amino Acid Mixture

Whole-body and splanchnic metabolism of dietary amino acids derived from casein (CAS) or the corresponding crystalline L-amino acid mixture (AA) were compared. Male adult rats were adapted for 9 d to two isoenergetic, isonitrogenous diets (15 g/100 g protein, 5 g/100 g fat) containing either CAS or AA. On d 10, the rats were fed a single mixed meal (3 g dry mass) containing either intrinsically (13)C-labeled goat casein or the amino acid mixture containing [U-(13)C(6)] leucine and [alpha-(15)N] lysine. Rats were killed before and 1, 3, 5 and 7 h after meal ingestion and samples of plasma, stomach wall and contents, small intestine and liver were collected. (13)C and (15)N enrichments of free and protein-bound amino acids in plasma and tissues were analyzed by gas chromatography-combustion isotope ratio mass spectrometry. Urinary nitrogen excretion was higher (P < 0.05) and weight gain lower (P < 0.05) in rats given the AA diet, indicating a lower whole-body net protein synthesis. Free (13)C-leucine from the AA diet appeared in the intestinal mucosa free pool more rapidly (P < 0.05) than the CAS-(13)C-leucine, probably due to the faster transit through the stomach of the AA group. However, the incorporation of dietary leucine into plasma and liver proteins was higher in the CAS group 7 h after the meal (P < 0.05), whereas lysine incorporation into liver protein was higher in the AA group (P < 0.05). We conclude that whole-body protein homeostasis is better supported by dietary casein-bound than crystalline free amino acids, and that protein-bound leucine, but not lysine, is used more efficiently for liver protein synthesis than dietary free leucine.

Structural organization of the goat casein micelle: effect of the physico-chemical environment (pH, temperature, ionic strength) on its mineral and protein composition

The structure of casein micelles in caprine milk has been studied. Although there were marked differences in casein composition, the basic structure of caprine milks is dependent on the participation of colloidal calcium phosphate in an analogous way to that found in bovine milk.

Type I (insulin-dependent) diabetes mellitus and cow milk: casein variant consumption

Previously published Type I (insulin-dependent) diabetes mellitus incidence in 0 to 14-year-old children from 10 countries or areas was compared with the national annual cow milk protein consumption. Countries which were selected for study had appropriate milk protein polymorphism studies, herd breed composition information and low dairy imports from other countries. Total protein consumption did not correlate with diabetes incidence (r = +0.402), but consumption of the beta-casein A1 variant did (r = +0.726). Even more pronounced was the relation between beta-casein (A1+B) consumption and diabetes (r = +0.982). These latter two cow caseins yield a bioactive peptide beta-casomorphin-7 after in vitro digestion with intestinal enzymes whereas the common A2 variant or the corresponding human or goat caseins do not. beta-casomorphin-7 has opioid properties including immunosuppression, which could account for the specificity of the relation between the consumption of some but not all beta-casein variants and diabetes incidence.

Proteolytic Activities of Chymosin and Porcine Pepsin on Buffalo, Cow, and Goat Whole and β-Casein Fractions

The proteolytic specificity and activity of a recombinant chymosin (Maxiren) and porcine pepsin on buffalo, cow, and goat whole casein (CN) and β-CN were studied by analyzing the degradation products. The results suggest that the hydrolysis of whole casein of buffalo and goat by chymosin was similar to that of cow casein resulting in αs1-I and β-I, -II, and -III as degradation fragments of αs1- and β-CN. The exception was goat β-I which was resistant to further hydrolysis by chymosin but not to porcine pepsin at pH 5.4−6.2. Increasing NaCl concentration to ≥5% reduced the proteolysis of β-CN in all three species, but not that of αs1-CN. The fragments of β-I, -II, and -III produced from β-CN of the three species gave identical results with PAGE. αs1-I and its degradation fragments had in all three species, regardless of the different electrophoretic mobilities on PAGE, the same sequence of appearance. The results indicate that chymosin and porcine pepsin attacked in buffalo and goat caseins the same region...

Proteolysis of goat casein by calf rennet

Abstract The proteolytic activity of calf rennet on goat casein (CN) was studied under various conditions which affect cheesemaking and ripening processes (pH, salt, calf rennet concentration and α s 1 -CN polymorphism). Electrophoretic studies showed that goat casein was hydrolysed to give characteristic breakdown products derived from individual caseins (β-I to β-V from β-CN, primary hydrolysis product from α s 1 -CN, para-κ-CN from κ-CN and other degradation products from α s 2 -CN). Both goat β-CN and α s 1 -CN were more sensitive to hydrolysis than their bovine counterparts under the same conditions. The pH did not influence the proteolytic specificity of rennet on β-CN, but rennet activity was highly dependent on the pH. However, in the case of α s 1 -CN, the rate of proteolysis and the nature of breakdown products were pH-dependent. NaCl affected only the rate of proteolysis. After prolonged incubation times, the electrophoretic patterns of hydrolysates produced from whole goat casein by rennet were similar to those produced from isolated β-CN, showing that β-CN and its degradation products are quite resistant to proteolysis compared with α s 1 -CN and its primary hydrolysis product. Rennet hydrolysates of goat casein, containing different α s 1 -CN genetic variants, showed slight differences in the electrophoretic profiles which tended to disappear as hydrolysis advanced.

Immunoreactivity of Goat's Milk Casein Fractionated by Ion-Exchange Chromatography

Goat's milk casein was fractionated by anion-exchange fast protein liquid chromatography (FPLC) on a Mono Q HR 5/5 column and by cation-exchange chromatography on a column containing an S-Sepharose Fast Flow matrix. The identification of the resulting fractions was performed by SDS-PAGE using a PhastSystem electrophoresis unit. Three fractions (β-casein, κ-casein, and α s -casein) were separated by anion-exchange FPLC, while cation-exchange chromatography allowed the identification of four fractions (β-casein, κ-casein, α s1 -casein, and α s2 -casein). The purified fractions were characterized immunologically by an indirect ELISA using polyclonal antibodies raised in rabbits against goat's casein and further purified by affinity chromatography in a column containing immobilized goat's caseins, biotinylated and blocked with lyophilized ovine and bovine caseins. The most immunoreactive caprine casein fraction was the α s2 -casein purified by cation-exchange chromatography, meaning this is the fraction with the largest number of epitopes specific of goat's milk casein.

Sandwich ELISA for detection of goats’ milk in ewes’ milk and cheese

A sandwich ELISA has been developed for the detection of defined amounts of goats’ milk (1–100%) in ewes’ milk and cheese. Polyclonal antibodies were raised in rabbits against goat caseins (GC). The resultant antibodies were affinity purified by immunoadsorption of the crude antiserum on to columns containing immobilized cow, ewe and goat caseins, followed by elution of the goats’ milk‐specific antibodies (anti‐GC) from the column containing the goat caseins. The anti‐GC antibodies bound to the wells of a microtiter plate were used to capture the goats’ caseins from milk or cheese mixtures. Further immunorecognition of the captured proteins was achieved with the same antibodies conjugated to biotin. ExtrAvidin‐peroxidase was used to detect biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of the substrate gave clear absorbance differences when assaying mixtures of ewes’ milk and cheese containing variable amounts of goats’ milk.