Intact Casein Research Articles

L -Homoarginine suppresses exocrine pancreas in rats

Previously, we found that guanidinated casein, a L-homoarginine-containing protein, was a more potent stimulator of pancreatic enzyme secretion than intact casein in rats. In this study, we examined secretory response and adaptation of the exocrine pancreas to the administration of free L-homoarginine in normal and bile-pancreatic juice (BPJ)-diverted rats. An intraperitoneal injection of L-homoarginine (10 mg/rats) produced immediate and transient reduction in pancreatic secretion in BPJ-diverted rats, but not in normal rats. The BPJ-diverted rats were fed with either a 25% casein, 45% casein, or 45% casein diet supplemented with L-homoarginine (19 g/kg diet) for 4 days. Feeding of a diet containing L-homoarginine inhibited the pancreatic adaptation induced by the high-protein diet. These results indicate that L-homoarginine has an inhibitory effect on the secretion and production of exocrine pancreatic enzyme in BPJ-diverted rats, and L-homoarginine may have an antagonistic effect on CCK receptors.

Structure development in a soft cheese curd model during manufacture in relation to its biochemical characteristics.

The structure development of a soft cheese curd model has been studied in relationship to its rheological properties and its biochemical characteristics (pH, amount and partition of minerals, casein proteolysis) at different technical steps including cutting, drawing, three turns and demoulding. Scanning electron microscopy was used to observe structural changes during the drainage of a fat-free soft cheese. The micrographs provided visual evidence of changes in the casein matrix from casein particles aggregated in clusters to uniform strands observed at the demoulding. The initial increase of loss tangent and of the exponent of the power law between G' and G" and frequency (that were maximal at the second turn) was related to the solubilization of micellar calcium phosphate, while intact caseins and large casein fragments accumulated in the curd. After the second turn, the strength, Youngs' and loss moduli of the curd increased greatly. The hydrolysis of alpha(s1)-casein into alpha(s1)-I-CN f(24-199) may facilitate the rearrangement of casein particles within the curd. The pH-induced solubilization of calcium phosphate continued throughout the manufacture process but was unexpectedly incomplete at the end of the drainage. Combination of electron microscopic observations with dynamic rheological measurements and chemical and biochemical assessments provided increased knowledge about the structure of soft cheese during drainage, an important but poorly understood cheese making stage.

Effects of Added Plasmin on the Formation and Rheological Properties of Rennet-Induced Skim Milk Gels

Elevated plasmin enzyme activity has been suggested as a likely cause of impaired functional properties that occur in milk from cows either in their latelactational period or that are experiencing mastitis. However, there are conflicting reports on the impact of plasmin on rennet coagulation properties of milk. The effects of added plasmin on the rheological properties, at small and large deformation, of rennet-induced gels were investigated. The microstructure of rennet-induced gels was studied, using confocal scanning laser microscopy. Porcine plasmin was added to reconstituted milk, and samples were incubated at 37°C for between 0.5 to 8h. The hydrolysis reaction was terminated using soybean trypsin inhibitor. The extent of degradation of caseins was determined with SDS-PAGE. The extent of breakdown of αs- and β-caseins increased with incubation time with plasmin. Storage modulus of rennet gels decreased linearly with increasing degradation of caseins. There was an increase in the loss tangent parameter of the gels with increasing casein degradation, indicating a more liquid-like gel character. Gelation time decreased until approximately 3h of incubation with plasmin (when the amounts of intact αs- and β-caseins were ∼46 and 50%, respectively); thereafter, gelation time increased considerably. Yield stress of rennet-induced gels decreased with increasing casein breakdown. When the level of casein hydrolysis was high (<40% of intact caseins), the microstructure of rennet-induced gels was drastically altered. Even when there were low levels of casein hydrolysis, the rheological properties of rennet gels were altered, which could have negative impacts on cheese yield and texture.

Correlations between biochemical characteristics and foam-forming and -stabilizing ability of whey and casein hydrolysates.

Whey protein and casein were hydrolyzed with 11 commercially available enzymes. Foam properties of 44 samples were measured and were related to biochemical properties of the hydrolysates using statistical data analysis. All casein hydrolysates formed high initial foam levels, whereas whey hydrolysates differed in their foam-forming abilities. Regression analysis using the molecular weight distribution of whey hydrolysates as predictors showed that the hydrolysate fraction containing peptides of 3-5 kDa was most strongly related to foam formation. Foam stability of whey hydrolysates and of most casein hydrolysates was inferior to that of the intact proteins. The foam stability of casein hydrolysate foams was correlated to the molecular weight distribution of the hydrolysates; a high proportion of peptides >7 kDa, composed of both intact casein and high molecular weight peptides, was positively related to foam stability.

Emulsion properties of casein and whey protein hydrolysates and the relation with other hydrolysate characteristics.

Casein and whey protein were hydrolyzed using 11 different commercially available enzyme preparations. Emulsion-forming ability and emulsion stability of the digests were measured as well as biochemical properties with the objective to study the relations between hydrolysate characteristics and emulsion properties. All whey protein hydrolysates formed emulsions with bimodal droplet size distributions, signifying poor emulsion-forming ability. Emulsion-forming ability of some casein hydrolysates was comparable to that of intact casein. Emulsion instability was caused by creaming and coalescence. Creaming occurred mainly in whey hydrolysate emulsions and in casein hydrolysate emulsions containing large emulsion droplets. Coalescence was dominant in casein emulsions with a broad particle size distribution. Emulsion instability due to coalescence was related to apparent molecular weight distribution of hydrolysates; a relative high amount of peptides larger than 2 kDa positively influences emulsion stability.

Effect of ripening temperature on low moisture Mozzarella cheese: 2. Texture and functionality

Low-moisture Mozzarella cheeses were made in duplicate and ripened for 70 d at 0, 4, 10 or 15 o C. For all temperatures, advance in ripening time resulted in significant ( P < 0.05) decreases in the concentration of intact casein, firmness, melt time and apparent viscosity. In contrast, the flowability and stretchability of the molten cheeses increased significantly during storage. Increasing the ripening temperature from 0 to 15 o C resulted in a significant decrease in the mean concentration of intact casein and a decrease in the level of serum expressed on centrifugation. The latter changes were paralleled by a significant decrease in the mean melt time (time required for shred fusion) of the cheese over the 70 d ripening period, and a significant increase in the mean flowability and reduction in the mean apparent viscosity of the molten cheese. The effect of elevation of storage temperature on the functional attributes of the molten cheese was attributed mainly to the concomitant reduction in the content of intact casein which was negatively correlated with flowability and positively with ap- parent viscosity and melt time. Ripening / temperature / Mozzarella / functionality / texture

Mechanism of pressure-induced gelation of milk.

The pressure-induced gelation of concentrated skimmed milk and milk-sugar mixtures was studied to discover the main components responsible for gelation. The major protein component responsible for gelation is micellar casein. Gelation occurs at similar pressures to casein micelle disintegration in dilute milk, and both can be prevented by inclusion of excess calcium chloride. Transmission electron micrographs show that the protein network is formed from particles with diameters approximately an order of magnitude smaller than those of intact casein micelles. Gelation occurs on decompression and is found to be baroreversible. Concentrations of sugar up to 30% reduce the critical concentration of casein required for gelation, but higher sugar concentrations inhibit gelation. A mechanism of gelation based on the aggregation of casein submicelles formed by pressure-induced disintegration of casein micelles is proposed. Observations on the effect of sucrose on gelation are discussed in terms of the influence of sugars on the solvent quality in aqueous casein systems.

Characterization of binding between the rat small intestinal brush-border membrane and dietary proteins in the sensory mechanism of luminal dietary proteins.

Dietary proteins are recognized by the gastrointestinal tract to display physiological functions, however, the sensory mechanism of the intestinal mucosa is not known. We examined binding properties between the rat small intestinal brush-border membrane (BBM) and proteins by using a surface plasmon resonance biosensor. BBM and solubilized BBM prepared from the rat jejunum bound to casein immobilized on the sensor surface, but not to bovine serum albumin. The ileal BBM showed less binding to casein than the jejunal BBM. Solubilized BBM binding to immobilized alpha-casein was slightly inhibited by aminopeptidase inhibitors, but still more inhibited by addition of casein with the inhibitors. Guanidinated casein inhibited the solubilized BBM binding to alpha-casein more strongly than casein (casein sodium and alpha-casein) inhibited. Trypsinization of solubilized BBM abolished its binding activity to alpha-casein. These results indicate that some membrane protein, but not aminopeptidases, contained in BBM interacts with dietary proteins, and that guanidinated casein has a higher affinity for BBM than intact casein. These binding intensities for proteins were closely correlated to physiological responsiveness, and are possibly involved in a sensory system for dietary protein in the intestine.

PRELIMINARY OBSERVATIONS ON EFFECTS OF FAT CONTENT AND DEGREE OF FAT EMULSIFICATION ON THE STRUCTURE‐FUNCTIONAL RELATIONSHIP OF CHEDDAR‐TYPE CHEESE

Cheddar type cheeses of different fat contents were produced and denoted: full‐fat (FFC), 306g/kg; half‐fat (HFC), 174 g/kg; and low fat (LFC, 13 g/kg). Full‐fat Cheddar cheese (FFCH) was also prepared from milk which had been homogenized at first and second stage pressures of 25 and 5 MPa, respectively. The cheeses were held at 4C for 30 days and at 7C for the remainder of the 190‐day ripening period. Reducing the fat level from 174 to 13 g/kg resulted in decreases in contents of moisture in nonfat substance and pH 4.6 soluble N as a percentage of total N (pH4.6SN), and increases in the contents of moisture, protein and intact casein. Homogenization of cheesemilk resulted in a slight increase in moisture content and an increase in pH4.6SN. Confocal laser scanning microscopy revealed that the extent of fat globule clumping and coalescence in both the unheated and heated (to 95C) cheeses decreased with homogenization of the cheesemilk and with fat reduction. Homogenization of the cheesemilk and reducing the fat content of the cheese resulted in a decrease in the flowability and stretchability of the melted cheese. Dynamic measurement of the viscoelastic changes on heating the cheese from 20 to 90C showed that reduction of fat content resulted in a decrease in the magnitude of the phase angle, δ, at temperatures &gt;50C. At temperatures&lt;∼60C, the storage modulus, G', increased on reducing the fat content from 306–174 g/kg to 13 g/kg. Homogenization resulted in a marked decrease in δ at temperatures&gt;45–50C, with δmax typically decreasing from ∼65–70° in the FFC to ∼35° in the FFCH.

Kinetics of L-[1-(13)C]leucine when ingested with free amino acids, unlabeled or intrinsically labeled casein.

In two groups of five adults, each adapted to two different dietary regimens for 6 days, the metabolic fate of dietary [1-(13)C]leucine was examined when ingested either together with a mixture of free amino acids simulating casein (extrinsically labeled; condition A), along with the intact casein (extrinsically labeled; condition B), or bound to casein (intrinsically labeled; condition C). Fed state leucine oxidation (Ox), nonoxidative leucine disposal (NOLD), protein breakdown, and splanchnic uptake have been compared using an 8-h oral [1-(13)C]leucine and intravenous [(2)H(3)]leucine tracer protocol while giving eight equal hourly mixed meals. Lower leucine Ox, increased NOLD, and net protein synthesis were found with condition C compared with condition A (19.3 vs. 24.9; 77 vs. 55.8; 18.9 vs. 12.3 micromol. kg(-1). 30 min(-1); P < 0.05). Ox and NOLD did not differ between conditions B and C. Splanchnic leucine uptake calculated from [1-(13)C]- and [(2)H(3)]leucine plasma enrichments was between 24 and 35%. These findings indicate that the form in which leucine is consumed affects its immediate metabolic fate and retention by the body; the implications of these findings for the tracer balance technique and estimation of amino acid requirements are discussed.

Primary proteolysis and textural changes during ripening in Cheddar cheeses manufactured to different fat contents

Abstract The effects of varying fat content in Cheddar cheese, from 6.3 to 32.5 g 100 g −1 , on changes in pH, primary proteolysis and texture were monitored over a 225 d ripening period. Reduction in the fat content resulted in significant ( P α s1 -casein was degraded more slowly, and β -casein more rapidly, as the fat content was reduced. The slower degradation of α s1 -casein with decreased fat content coincided with a decrease in the ratio of residual chymosin activity to protein in the cheese. At most ripening times, reduction in the fat content resulted in significant increases in the concentration of intact casein, fracture stress, fracture strain, and cheese firmness. The effects of fat reduction on proteolysis and rheology are probably due to the interactive effects of the concomitant changes in composition.

Addition of Arginine but Not Glycine to Lysine Plus Methionine–Enriched Diets Modulates Serum Cholesterol and Liver Phospholipids in Rabbits

Previous experiments from our laboratory showed that in rabbits fed an amino acid diet corresponding to 30% casein, enrichment of the diet with L-lysine and L-methionine caused a marked increase in serum total and LDL cholesterol levels as well as a substantial body weight loss. Both effects were partially prevented by supplementation with L-arginine. The present studies were designed to extend this earlier observation by assessing the role of different dietary amino acids in modulation of cholesterolemic responses and body weights. In the first experiment, the original lysine and methionine-enriched diet was supplemented with glycine in an attempt to modify methionine metabolism, and thus to reduce body weight loss. In addition, the mechanism of action of lysine and methionine was investigated by quantitation of major liver phospholipids. The results showed that glycine addition had no effect on weight loss or hypercholesterolemia, nor did it alter plasma levels of homocyst(e)ine, an intermediate in methionine metabolism. However, enrichment of the diet with lysine and methionine (with or without glycine) significantly increased liver levels of phosphatidylcholine and the ratio of phosphatidylcholine to phosphatidylethanolamine, apparently through increased enzymatic conversion. These changes were consistent with higher lipoprotein levels and thus may explain the hypercholesterolemia. A second experiment showed that similar effects on body weights and serum cholesterol could be obtained by adding lysine and methionine to a diet containing amino acids equivalent to only 15% casein, or 15% intact casein. This approach is more physiologic and also reduces the expense of experiments designed to study the effects of lysine and methionine in more detail.

Guanidino group is involved in the stimulation of exocrine pancreatic secretion by protamine in normal and chronic bile-pancreatic juice-diverted rats.

We previously demonstrated that the feeding of guanidinated casein, whose lysine residues are converted to homoarginine, stimulates pancreatic secretion much higher than that of intact casein in chronic bile-pancreatic juice (BPJ)-diverted rats, which suggests that the guanidino group is involved in BPJ-independent enhancement of pancreatic secretion. However, the role of the guanidino group in the protein for the enhancement of pancreatic secretion has not been clarified. In this study, we examined the stimulation of pancreatic secretion by a arginine rich dietary protein, protamine (25, 50 mg/ml), and then determined whether the guanidino group in protamine was responsible for the secretory responses in normal and BPJ-diverted rat by comparison with pancreatic secretion between intact and deguanidinated protamine. The deguanidinated protamine was prepared by converting arginine residues of salmon protamine to ornithine using heated hydrazine (conversion rate of arginine residue was 87%). In normal rats, pancreatic protein and chymotrypsin secretion were stimulated in dose-response fashion after a duodenal instillation of native protamine solution (25, 50 mg in 1 ml). In chronic BPJ-diverted rats, native protamine (25 mg) maximally stimulated pancreatic protein and protease secretion. In contrast, deguanidinated protamine (50 mg in 1 ml) did not stimulate pancreatic secretion in both normal and BPJ-diverted rats. In addition, the duodenal administration of arginine, which is equal to the amount contained in 50 mg of native protamine, had no effect on pancreatic secretion in both rats. These results suggest that a naturally occurring protein, protamine, stimulates pancreatic secretion by a luminal BPJ-independent mechanism and that the guanidino group in this protein is responsible for stimulating pancreatic secretion in BPJ-diverted rats.

Guanidinated casein hydrolysate stimulation of cholecystokinin release via pancreatic enzyme- and cholinergic-independent mechanisms in rats.

We had demonstrated that a peptic hydrolysate of guanidinated casein that is made from casein by the conversion of lysine to homoarginine stimulated pancreatic exocrine secretion in rats with chronic bile-pancreatic juice (BPJ) diversion from the proximal small intestine. This modified protein also stimulated cholecystokinin (CCK) release from dispersed rat intestinal cells. In this study, we found that guanidinated casein hydrolysate stimulates CCK release in chronic BPJ-diverted rats with cholinergic control blocked by atropine. Intraduodenal guanidinated casein hydrolysate increased portal plasma CCK concentration and pancreatic secretion in atropine-treated BPJ-diverted rats. In contrast, the portal plasma CCK concentration was not increased by intact casein hydrolysate. We conclude that guanidinated casein hydrolysate directly stimulates CCK release from the intestine via some cholinergic-independent mechanism, and an increase of the pancreatic exocrine secretion is regulated by CCK released by guanidinated casein hydrolysate. A guanidyl residue is likely to be involved in this control.

Guanidinated casein hydrolysate stimulates pancreatic secretagogue release by direct action to the intestine in rats.

Previously, we observed that pancreatic exocrine secretion was strongly enhanced after duodenal administration of guanidinated casein peptic hydrolysate (HGC) in rats with chronic bile-pancreatic juice (BPJ) diversion. Using a perifusion system of isolated dispersed rat intestinal mucosal cells, we investigated whether this phenomenon depends on a direct effect of protein on the intestine to release pancreatic secretagogues, such as cholecystokinin (CCK). Amylase release from isolated pancreatic acini was used as an assay to measure CCK or other pancreatic secretagogues in the effluents. Mucosal effluent with HGC stimulated amylase release from acini with or without soybean trypsin inhibitor (SBTI). Perifused effluent with low-concentrated SBTI did not stimulate amylase release. These results indicate that HGC stimulates release of pancreatic secretagogue from the intestinal mucosal cells independent of remaining trypsin activity in the isolated mucosa. Effluents with intact casein, its peptic digest, and homoarginine, which is a unique amino acid contained in HGC, were unable to stimulate amylase release from acini. Effluent with a high concentration of SBTI, which is rich in arginine residues, stimulated amylase release, but not with the same tryptic inhibitory effect of lima bean trypsin inhibitor, which is poor in arginine residues. These findings suggest that guanidyl residues in protein structure are responsible for release of pancreatic secretagogues from isolated intestinal mucosal cells. Finally, the increment of amylase release from pancreatic acini in response to the perifused effluent with HGC was eliminated in acini treated with a potent CCK antagonist, FK480. We conclude that in rats with BPJ diversion, HGC stimulates CCK release from the intestine by direct action on intestinal mucosa.

Ammonia-hyperproducing bacteria from New Zealand ruminants.

Pasture-grazed dairy cows, deer, and sheep were tested for the presence of ammonia-hyperproducing (HAP) bacteria in roll tubes containing a medium in which tryptone and Casamino Acids were the sole nitrogen and energy sources. Colonies able to grow on this medium represented 5.2, 1.3, and 11.6% of the total bacterial counts of dairy cows, deer, and sheep, respectively. A total of 14 morphologically distinct colonies were purified and studied further. Restriction fragment length polymorphisms of 16S rRNA genes indicated that all isolates differed from the previously described HAP bacteria, Clostridium aminophilum, Clostridium sticklandii, and Peptostreptococcus anaerobius. Carbon source utilization experiments showed that five isolates (C2, D1, D4, D5, and S1) were unable to use any, or very few, of the carbon sources tested. Biochemical tests and phylogenetic analyses of 16S ribosomal DNA sequences indicated that all isolates were monensin sensitive; that D1 and S1 belonged to the genus Peptostreptococcus, that D4 and D5 belonged to the family Bacteroidaceae, where D4 was similar to Fusobacterium necrophorum; and that C2 was most similar to an unidentified species from the genus Eubacterium. Growth on liquid medium containing tryptone and Casamino Acids as the sole nitrogen and energy source showed that D1, D4, and S1 grew rapidly (specific growth rates of 0.40, 0.35, and 0.29 h-1, respectively), while C2 and D5 were slow growers (0.25 and 0.10 h-1, respectively). Ammonia production rates were highest in D1 and D4, which produced 945.5 and 748.3 nmol/min per mg of protein, respectively. Tests of individual nitrogen sources indicated that D1 and D4 grew best on tryptone, S1 grew equally well on Casamino Acids or tryptone, and C2 and D5 grew poorly on all nitrogen sources. The intact proteins casein and gelatin did not support significant growth of any of the isolates. These isolates extend the diversity of known HAP rumen bacteria and indicate the presence of significant HAP bacterial populations in pasture-grazed New Zealand ruminants.

Selection of an Intact Casein or Casein Hydrolysate Diet by Rats Submitted to Protein Deprivation and Bowel Resection

Ribiero, R. P. P., L. M. De Oliveira and J. E. Dos Santos.Selection of an intact casein or casein hydrolysate diet by rats submitted to protein deprivation and bowel resection.PHYSIOL BEHAV 63(2) 185–189, 1998.—The feeding preference of normal rats (n = 14), malnourished rats (n = 14), and enterectomized rats (n = 16) was determined in a situation of free choice of three complete solid diets which only differed in extent of protein polymerization: intact casein, casein hydrolysate, and an amino acid mixture with a composition similar to that of casein. The animals were housed in metabolic cages for 30 days and allowed to freely choose among the three diets presented simultaneously. All three groups showed an initial preference for the intact casein diet. The control group maintained this preference, whereas the malnourished and enterectomized groups reduced the ingestion of the intact casein diet and increased the ingestion of the amino acid diet. The nitrogen balance, which was always positive in all three groups, was constant in the control group (1.51 ± 0.26 g), initially higher (1.77 ± 0.19 g) in the malnourished group, with a subsequent fall (1.13 ± 0.24 g), and lower in the enterectomized group (0.83 ± 0.32 g). Although total intake was similar for the control and malnourished groups, the malnourished group presented a higher weight recovery (130.2%). In contrast, the food intake of the enterectomized group was much lower, with a small weight gain. After treatment that impairs the digestive tract, intact casein was the initially preferred nitrogen source, which later tended to be replaced with free amino acids. This change was accompanied by an improvement in nitrogen balance and body weight, especially after protein malnutrition. These data may suggest that, in clinical practice, the use of enteral diets containing fully hydrolyzed protein may be of benefit in terms of the recovery of malnourished patients and of patients with short bowel syndrome.

Effect of Sodium Chloride on the Serum Phase of Mozzarella Cheese

The objective was to compare changes in the serum phase of brine-salted and unsalted Mozzarella cheeses during aging to elucidate the impact of NaCl on the serum phase and physico-chemical changes. Brine-salted and unsalted blocks of low moisture, part-skim Mozzarella cheese that had been commercially produced were analyzed for expressible serum after 2, 4, 6, 8, and 10 d of storage at 4°C. The amount of expressible serum decreased during aging of both brine-salted and unsalted cheeses; however, amounts were higher for unsalted cheese and decreased more slowly than for brine-salted cheese. The amounts of protein that were insoluble at pH 4.6 in the expressible serum increased dramatically during aging of brine-salted cheeses, but increased only slightly during aging of unsalted cheeses. Urea-PAGE and SDS-PAGE confirmed that intact αs1 -casein (CN), αs2 -CN, β-CN, and para-κ-CN were present at higher concentrations and increased to a greater extent in the expressible serum of brine-salted than in unsalted Mozzarella cheeses. The data are consistent with the hypothesis that NaCl in the serum phase of Mozzarella cheese promotes microstructural swelling, a concomitant increase in water-holding capacity, and the solubilization of intact caseins from the paracasein matrix.

Elemental diets in the repair of small intestinal damage

We have investigated the possible benefits of elemental diets, especially a diet supplemented with l-glutamine, on maintenance of intestinal absorptive function in rat small intestine damaged by 5-fluorouracil. Although a standard rat diet sustained better body growth in control rats, each of the elemental diets and the diet containing intact casein in place of hydrolyzed casein was beneficial in promoting less body weight loss during the 3 d after 5-fluorouracil injection. The same significant benefit was seen in absorptive activity measured in small intestine in vitro 3 d after the cytotoxic injury. Glutamine supplementation, however, did not confer any significant advantages, although it did cause significant elevation of muscle glutamine pools. This elevation was substantially less than the corresponding increase in muscle glycine content after dietary supplementation with glycine.

A zinc-dependent proteinase from the cell wall of Lactobacillus delbrueckii subsp. bulgaricus.

A zinc-dependent proteinase was extracted from the cell wall of Lactobacillus delbrueckii subsp. bulgaricus and partially purified despite a marked unstability. The caseinolytic activity was associated with a polypeptide chain of 65 kDa that belonged to the M1 family of zinc-dependent proteases. This zinc-dependent proteinase could degrade intact caseins, with a significant preference for beta-casein. The pH-profile of its activity indicated that its relative contribution to the caseinolytic activity increased at acidic pH, suggesting that this zinc proteinase could be involved in the late stages of milk fermentation.