Use of batch anion exchange technique for separation of κ-casein glycomacropeptide from bovine whey fraction.

Bovine κ-casein glycomacropeptide (GMP) is a sialic acid containing glycopeptide, which is considered as a health promoting compound found in cheese whey. The study described in this research communication was undertaken to determine whether GMP with undetectable level of contaminating protein or phenylalanine can be isolated from bovine whey fraction using batch anion exchange technique with chitin as an adsorbent. A soluble whey fraction (SWF) prepared from 1 g whey protein isolate (WPI) was mixed with a slurry of 1 g chitin, and the mixture was incubated at pH 3.0. After incubation, the mixture was filtered, and the residue obtained (containing chitin-GMP complex) was washed with water and eluted stepwise with 0.5 M NaCl and 2.0 M NaCl. Most of GMP (corresponding to 75.8% of total sialic acid recovered) was eluted with 0.5 M NaCl. The recovered GMP accounted for 5.4% dry weight of WPI (or 18.9% dry weight of SWF). Amino acid analysis showed that there was no detectable level of contaminating amino acids including phenylalanine, histidine, arginine and tyrosine in the GMP fraction. It was concluded that the batch anion exchange method with chitin developed in this study can be used for the isolation of high purity GMP from bovine SWF.

Production of sialic acid rich glycopeptide from bovine κ-casein glycomacropeptide by hydrolyzing with papain.

Bovine κ-casein glycomacropeptide (GMP) is a sialic acid containing glycopeptide having many biological activities. The study described in this research communication was undertaken to determine whether sialic acid rich glycopeptide can be produced from GMP by proteinase treatment. A sample of GMP was hydrolyzed with papain, and the obtained hydrolysate was chromatographed on a column of diethylaminoethyl-Sephacel to obtain a glycopeptide fraction (GPF). This product accounted for average 48.1% dry weight of GMP or 81.1% total recovered sialic acid from GMP. The content of sialic acid (expressed as % dry weight) was 1.7 times higher in GPF (22.6) than in unhydrolyzed GMP (13.4). Major differences in amino acid composition between GPF and GMP were found in the contents (mol%) of: lysine (<1 and 4.5, respectively), serine (20.3 and 10.3, approximately twice higher in GPF), asparagine/aspartic acid and isoleucine. The contents of the last two amino acids were approximately twice lower in GPF. On gel filtration chromatography with Sephacryl S-100, GMP was eluted as a single peak with elution volume similar to that of dimeric ß-lactoglobulin (36.6 kDa) whereas GPF was eluted in two peaks both with elution volumes greater than that of α-lactalbumin (14.2 kDa). These peak fractions containing high (fraction I) and low (fraction II) molecular size glycopeptides gave different sialic acid to peptide ratio, which was 1.7 times higher in fraction I than in fraction II. Results of size exclusion HPLC on Superdex-75 were consistent with those of gel filtlation chromatography. On cellulose acetate electrophoresis, the mobility of GPF relative to that of GMP as 1.0 was found to average 1.2, suggesting a higher negative charge density in GPF than in GMP. It was concluded that papain digestion of GMP is an efficient method to produce glycopeptide with high sialic acid content.

Isolation of κ-casein glycomacropeptide from bovine whey fraction using food grade anion exchange resin and chitin as an adsorbent.

Bovine κ-casein glycomacropeptide (GMP) found in cheese whey is a sialylated phosphorylated peptide which is thought to be a potential ingredient for functional food as well as dietetic food. This study was undertaken to determine whether high purity GMP can be isolated from soluble whey fraction (SWF) using column chromatography on food grade anion exchange resin and chitin as an adsorbent. Samples of commercially available anion exchange resin (resin A, resin B and resin C) and those of chitin (chitin A, chitin B and chitin C) were examined in this experiment. The GMP fraction obtained from each column was analyzed for amino acid composition which reflects the purity of the peptide. Major findings for commercial anion exchange resin were that: (1) the proportion of GMP monitored as sialic acid in total recovered sialic acid was similar among the three samples of resin accounting for average 78% of recovered sialic acid; (2) the GMP fraction from resin A or resin B contained undetectable level of contaminating amino acids including phenylalanine, histidine, arginine and tyrosine; (3) the GMP fraction from resin C contained small amounts (<1 mol%) of contaminating amino acids, arginine, phenylalanine and tyrosine; and (4) the GMP binding capacity expressed as mg/100 mg dry weight of resin was more than 2.5 times higher in resin C (average 22.9) than in resin A or resin B with no difference between resin A and resin B averaging 8.7. Results obtained for chitin A, chitin B and chitin C were in general similar to those found with resin A and resin B. Since chitin has a remarkable GMP binding capacity averaging 8.6 mg/100 mg dry weight of chitin, it may be a useful adsorbent for whey fractionation. Further research is needed to develop an efficient inexpensive method to purify GMP.

Extraction of aggrecan-peptide from cartilage by tissue autolysis.

Aggrecan is a cartilage specific proteoglycan containing chondroitin sulfate (CS) and keratan sulfate (KS). CS is an acidic polysaccharide having wide range of applications in pharmaceutical, cosmetic, and food industries. CS is extracted from cartilage by tissue proteolysis with an exogenous proteinase or by activating endogenous proteinases (autolysis) to release aggrecan-peptides from the tissue. This review is focused on the latter technique. Bovine nasal and tracheal cartilages, and broiler chicken sternum cartilage have been used for autolysis studies. To extract aggrecan-peptide, cartilage tissues are cut into small pieces, and incubated in a monovalent or divalent salt solution (e.g., 0.1 M sodium or calcium acetate) at pH 4.5 and 37 °C for 7 - 24 h. Most (~80% or more) of total tissue uronic acid, a constituent sugar of aggrecan, is extracted and released into the salt solution during incubation. Reextraction of the tissue residue results in release of a small amount of uronic acid. Aggrecan-peptides purified using anion exchange chromatography are large compounds containing CS and KS. On gel chromatography, they are excluded from the column of Sephacryl S-300. Chemical composition analysis demonstrated that aggrecan-peptides from either bovine or chicken cartilage contain >90% CS with small amount (< 10%) of either KS or peptide. Patent information included production of aggrecan-peptide substantially free of DNA. The bovine aggrecan-peptide prepared by tissue autolysis has been used as a plate coating antigen in enzyme- linked immunosorbent assay (ELISA) to determine KS.

A sialic acid assay in isolation and purification of bovine k-casein glycomacropeptide: a review.

Sialic acid is a carbohydrate moiety of k-casein glycomacropeptide (GMP), which is a 64 amino acid residue C-terminal sialylated phosphorylated glycopeptide released from k-casein by the action of chymosin during cheese making. GMP lacks aromatic amino acids including phenylalanine, tyrosine, and tryptophan. Because of its unique amino acid composition and various biological activities, GMP is thought to be a potential ingredient for dietetic foods (e.g., a food for PKU patients) and pharmaceuticals. Thus, increased attention has been given to the development of techniques to purify GMP. In this review, techniques of GMP purification described in patents and scientific research papers were introduced. A sialic acid assay is the important method to track GMP isolation and purification processes, for which the thiobarbituric acid reaction with 1-propanol as a chromophore extracting solvent is an inexpensive, practical and specific technique. Sephacryl S-200 gel filtration chromatography, cellulose acetate electrophoresis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis are the major techniques to identify sialic acid specific to GMP. Sephacryl S-200 chromatography and cellulose acetate electrophoresis are also used to detect GMP sialic acid in whey pearmeate and whey added commercial margarine samples. Future research includes development of an economical industrial scale method to produce high purity GMP.

Detection of keratan sulfate by immunological methods in commercial chondroitin sulfate preparations.

Chondroitin sulfate (CS), a well known nutraceutical, and keratan sulfate (KS) are glycosaminoglycans involved in the structure of cartilage proteoglycan, aggrecan. Since CS is extracted from cartilage, there may be a possibility that purified CS is contaminated with small amount of KS. A total of 15 samples, including four samples of CS as laboratory reagents, one sample of CS as a food additive and ten samples of dietary supplements containing CS were examined to detect KS in these samples by using immunodiffusion and enzyme-linked immunosorbent assay (ELISA) with anti-KS monoclonal antibody (IgM). With the exception of three samples of CS as laboratory reagents, all samples were found to contain varying amounts of KS. It was concluded that both the immunodiffusion, a quick one-step method, and ELISA for quantification, are reliable methods to detect KS contamination in CS products.

Immunological detection of keratan sulfate in meat products with and without mechanically separated chicken meat.

Keratan sulfate is a glycosaminoglycan found in the structure of cartilage proteoglycans, aggrecan and fibromodulin. This study was undertaken to detect this glycosaminoglycan in meat products containing mechanically separated chicken meat (MSCM) having cartilage particles. Dry-defatted samples of MSCM and meat products with or without MSCM were digested with papain, and a non-dialyzable fraction from each papain digest was examined by immunodiffusion analysis using anti-keratan sulfate monoclonal antibody (IgM). No precipitine line was formed with the antibody for all samples of meat products without MSCM, while a sample of MSCM and all samples of meat products with MSCM gave clear precipitine lines with the antibody. The immunodiffusion test described here appears to be a simple sensitive specific method for qualitative analysis of keratan sulfate, which in combination with other methods may be useful for detection of MSCM in meat products.

Extraction, isolation and analysis of chondroitin sulfate glycosaminoglycans.

Glycosaminoglycans (GAGs) including chondroitin sulfate (CS) and chondroitin sulfate/dermatan sulfate (CS/DS) copolymers are anionic straight chain polysaccharides. They are galactosamine containing GAGs (galactosaminoglycans) having wide range of applications in pharmaceutical, cosmetic and food industries. This article reviews techniques to isolate and characterize these galactosaminoglycans from animal and poultry tissues. Patent based information is also discussed. Cartilaginous tissues are the major source of CS consisting entirely of D-glucuronosyl-N-acetylgalactosamine repeating disaccharide units, in which the galactosamine is sulfated at C4 or C6. In contrast, most galactosaminoglycans in non-cartilaginous connective tissues (e.g. skin and tendon) are CS/DS copolymers comprised of varying proportions of D-glucuronosyl-N-acetylgalactosamine and L-iduronosyl-N-acetylgalactosamine. Tissues are digested with proteinase (e.g. papain) to liberate GAGs, which are fractionated to isolate and purify galactosaminoglycans. Common techniques used for fractionation of GAGs include: precipitation with different concentrations of ethanol; solubilization of GAG precipitated as GAG-quarternary ammonium compound complexes with different concentrations of NaCl; anion exchange chromatography and gel filtration chromatography. Purified galactosaminoglycans are examined by various methods including chondroitinase digestion, high performance liquid chromatography and electrophoresis. Histological methods are used to localize galactosaminoglycans in tissues. The patent information on the CS hydrolase and ultraviolet irradiation may be useful for the preparation of CS oligosaccharide.

[High conjugated linoleic acid (CLA) content in milk and dairy products using a dietary supplementation of sunflower seed in cows. Thrombogenic/atherogenic risk issues]. / Alto contenido de ácido linoleico conjugado (CLA) en leche y productos derivados al incorporar semillas de girasol a la dieta vacuna. Implicaciones sobre el riesgo trombo/aterogénico.

This study was undertaken to determine the effect of dietary supplementation of sunflower seed in cows on the chemical composition of milk and dairy products. Cream, butter and butter oil were prepared from milk produced by cows fed a control diet (control products) or diet supplemented with 11.2% sunflour seed (CLA-rich products). Milk samples collected were determined for lactose. A sample of CLA-rich or control product was determined for fatty acid profile as well as fat, protein and ash contents. The index of atherogenicity (IA) and the index of thrombogenicity (IT) were also calculated. Results revealed that there was no effect of the inclusion of sunflower seed in the diet on the lactose content in milk and total fat, protein and ash contents in the dairy products. Average contents of conjugated linoleic acid (CLA) and transvaccenic acid (TVA), expressed as g/100g total fatty acid were 0.54 and 1.6, respectively in the control products, and 2 and 6.4, respectively in the CLA-rich products. The content of either CLA or TVA was approximately four fold higher in the latter products. Moreover, CLA-rich products showed considerably low IA and IT, which were, respectively, 38.4 and 25.0% less than those from control products. Fatty acid profiles were unaffected during processing, which demonstrates that CLA is a stable component in the dairy products analyzed. It was concluded that dietary supplementation of sunflower seed in cows increases the CLA and TVA contents in milk, which may contribute to the reduction of the risk of cardiovascular diseases in humans.

Alto contenido de ácido linoleico conjugado (CLA) en leche y productos derivados al incorporar semillas de girasol a la dieta vacuna: implicaciones sobre el riesgo trombo/aterogénico / High conjugated linoleic acid (CLA) content in milk and dairy products using a dietary supplementation of sunflower seed in cows: thrombogenic/atherogenic risk issues

Con la finalidad de determinar el perfil de ácidos grasos y la composición química de productos lácteos enriquecidos con ácido linoleico conjugado (CLA) de manera natural, se elaboraron crema, mantequilla y grasa butírica con leche obtenida de vacas que recibieron una dieta control o suplementación con semilla de girasol en un 11.2%. El análisis químico incluyó el perfil de ácidos grasos,materia grasa, proteína y cenizas; en la leche se determinó además el contenido de lactosa. Se calcularon los índices de aterogenicidad (IA) y trombogenicidad (IT) en la leche y productos elaborados. Los resultados indicaron que los contenidos de grasa, proteína, lactosa y ceniza no fueron afectados por la incorporación de semilla de girasol en la dieta de los animales. El contenido promedio de CLA y ácidotrans vaccénico (TVA) expresados en g/100 g de lípidos totales fue, para los productos control, 0.54 y 1.6; mientras que para los productos ricos en CLA fueron 2 y 6.4, lo cual representa un incremento de cuatro veces. Además, en los productos ricos en CLA los IA e IT disminuyeron considerablemente (38.4 y 25% menos, respectivamente). Se observó que los perfiles de ácidos grasos no se modificaron durante el procesamiento, indicando que el CLA es un componente estable en los productos lácteos analizados. El uso de semilla de girasol en la dieta de las vacas, incrementa el contenido de CLA y TVA en los productos lácteos y disminuye el riesgo de enfermedades cardiovasculares en humanos sin afectar la proporción de los componentes mayoritarios.

High conjugated linoleic acid (CLA) content in milk and dairy products using a dietary supplementation of sunflower seed in cows. Thrombogenic/atherogenic risk issues. This studywas undertaken to determine the effect of dietary supplementation of sunflower seed in cows on the chemical composition of milk and dairy products. Cream, butter and butter oil were prepared from milk produced by cows fed a control diet (control products) or diet supplemented with 11.2% sunflour seed (CLA-rich products). Milk samples collected were determined for lactose. A sample of CLArich or control product was determined for fatty acid profile as well as fat, protein and ash contents. The index of atherogenicity (IA) and the index of thrombogenicity (IT) were also calculated. Results revealed that there was no effect of the inclusion of sunflower seed in the diet on the lactose content in milk and total fat, protein and ash contents in the dairy products. Average contents of conjugated linoleic acid (CLA) and transvaccenic acid (TVA), expressed as g/ 100g total fatty acid were 0.54 and 1.6, respectively in the control products, and 2 and 6.4, respectively in the CLA-rich products. The content of either CLA or TVA was approximately four fold higher in the latter products. Moreover, CLA-rich products showed considerably low IA and IT, which were, respectively, 38.4 and 25.0% less than those from control products. Fatty acid profiles were unaffected during processing, which demonstrates that CLA is a stable component in the dairy products analyzed. It was concluded that dietary supplementation of sunflower seed in cows increases the CLA and TVA contents in milk, which may contribute to the reduction of the risk of cardiovascular diseases in humans.

Detection of sialylated phosphorylated kappa-casein glycomacropeptide electrophoresed on polyacrylamide gels and cellulose acetate strips by the thiobarbituric acid and malachite green dye reactions.

A 64 amino acid residue sialylated phosphorylated glycomacropeptide (GMP) from bovine sweet whey can be detected as a Coomassie blue-staining peptide by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. There is, however, limited information available concerning detection of GMP as a sialylated phosphorylated compound. Samples of GMP were electrophoresed on SDS-polyacrylamide gels or cellulose acetate strips (CAS). Immediately following electrophoresis, fractions obtained by cutting gels or strips were subjected to sialic acid determination by the thiobarbituric acid reaction and phosphorus determination by the malachite green dye reaction. Both determinations were found to be sensitive enough to detect approximately 20 and 40 microg of GMP in CAS and SDS gels, respectively. Further studies demonstrated that sialylated phosphorylated GMP can be detected on either SDS gels or CAS loaded with whey products or whey-added margarine residues.

Chemical composition of the infrapatellar fat pad of swine.

The porcine infrapatellar fat pad is a structure composed of adipocytes and adipose connective tissues. Limited information is available concerning its chemical composition. Samples of the fat pad collected from young hogs were dissected into two portions: a relatively hard core of the pad with cushioning properties (inner tissue), and a soft adipose tissue surrounding the core (outer tissue). The inner tissue contained less moisture and nitrogen than did the outer tissue. The yield of dry-delipidated tissue was also lower in the inner tissue, indicating a higher content of lipid in this tissue. Fatty acid analysis showed that the proportions of C18: 1, C16: 1 and C18: 2n-6 are higher, and the proportion of C16: 0 is lower in the inner than in the outer tissue. Collagen is the major protein, with relatively small amounts of glycosaminoglycans in both tissues. The content of hyaluronic acid relative to sulphated galactosaminoglycan was lower in the inner than in the outer tissue. The electrophoresis pattern of sulphated galactosaminoglycan was also different between the two tissues. These results suggest that chemical composition varies between adipose tissues with different biomechanical function.

Use of epichlorohydrin-treated chitosan resin as an adsorbent to isolate kappa-casein glycomacropeptide from sweet whey.

This study was undertaken to develop a method to isolate glycomacropeptide (GMP), a bioactive compound, from sweet whey by using chitosan resins as anion exchangers. Shrimp shells were used to prepare two chitosan (polyglucosamine) resins, one with the primary amine (-NH(2)) (resin A) and the other with the secondary amine (-NH-) (resin B) as the major functional group. These resins were tested as adsorbents for the isolation of GMP from sweet whey, and the results obtained were compared with those obtained with commercial anion exchangers. The most important finding in this experiment was that the GMP binding capacity of resin A was much higher than that of resin B. Resin A may be the anion exchanger to be tested for industrial scale production of GMP. Amino acid analysis of the GMP-depleted whey fraction suggests that this product can replace sweet whey as an ingredient in various food products including infant formulas, bakery products, and beverages.

Purification of kappa-casien glycomacropeptide from sweet whey with undetectable level of phenylalanine.

Glycomacropeptide (GMP) found in sweet whey is a biologically active compound released from kappa-casein by the action of chymosin during cheese making. This study was undertaken to purify GMP from sweet whey as a research chemical on a laboratory scale. Glycomacropeptide was isolated from proteins and other non-GMP compounds by deproteinization with trichloroacetic acid and gel chromatography on Sephacryl S-200. The purified GMP accounted for 0.12% of dry sweet whey powder and contained 107.0, 50.9, 61.2 and 4.3 microg, respectively, of sialic acid, galactose, galactosamine and phosphorus per mg dry weight. The GMP was of high purity, with its amino acid composition showing undetectable levels of phenylalanine, tyrosine and arginine, the amino acids that do not occur in bovine GMP. On gel electrophoresis, the GMP showed a single broad band with an average mobility faster than that of carbonic anhydrase (molecular weight = 31 kDa). The purified GMP may be used as a standard glycopeptide in chromatography and electrophoresis and may also be used to test various known or unknown properties and biological activities of this compound.

Isolation and analysis of kappa-casein glycomacropeptide from goat sweet whey.

Glycomacropeptide (GMP) was purified from goat sweet whey by anion-exchange and hydrophobic interaction chromatography. Approximately 0.06% (w/v) of sweet whey was recovered as GMP. Amino acid analysis of the GMP preparation showed that the content of phenylalanine (an amino acid that does not occur in goat GMP) was negligible, indicating that the GMP was of high purity. The goat GMP contained 25 microg sialic acid per mg of dry weight. This was approximately 3-fold lower than the sialic acid concentration in bovine GMP reported in the literature. Gel electrophoretic results demonstrated that most of the goat GMP occurs as a dimer. The GMP was intensely stained with Coomassie blue in 50% methanol containing 12.5% (w/v) trichloroacetic acid, but showed very weak metachromasia with the same dye in 45% methanol containing 10% acetic acid, a preparation commonly used to stain protein.