Extraction, partial purification and characterization of proteases from the red seaweed Gracilaria edulis with similar cleavage sites on κ-casein as calf rennet.

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60 °C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108 kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.

Protease activity of enzyme extracts from tamarillo fruit and their specific hydrolysis of bovine caseins.

The characterisation of a serine protease isolated from tamarillo (Solanum betaceum) fruit and its milk casein hydrolysis activity were investigated. Compared with calf rennet, a crude extract from tamarillo exhibited wider caseinolytic activity on sodium caseinate. The purified protease was named "tamarillin" and revealed proteolytic activity toward purified α-, ß- and κ-casein. Similar to calf rennet, tamarillin preferably hydrolysed κ-casein, but, unlike calf rennet, it also displayed high proteolytic activity toward both α- and ß-casein. The major peptide generated from κ-casein by tamarillin was analysed by gel electrophoresis and liquid chromatography mass spectrometry to confirm its molecular mass as 14,290 Da. The cleavage site was confirmed by in-gel tryptic digestion and time-of-flight mass spectrometry analysis to be at Asn123-Thr124. This was in contrast to the Phe105-Met106 cleavage site of rennet hydrolysis.

Effects of heating and calcium and phosphate mineral supplementation on the physical properties of rennet-induced coagulation of camel and cow milk gels.

The physical properties of rennet-induced coagulation of preheated camel and cow milk gels (50 and 70 °C for 10 min) enriched with calcium chloride (CaCl2) and hydrogen phosphate dihydrate (Na2HPO42H2O) were evaluated using the dynamic low amplitude oscillatory shear analysis. The storage modulus (G') and loss modulus (G") of camel milk gels showed significant (P < 0·05) lower values than those of cow milk gels. The preheating of camel milk at 50 °C affected negatively the gelation properties, while the preheating at 70 °C prevented the formation of rennet-induced milk gels. No effect was observed on the gelation properties of cow milk gels. The CaCl2 added at 10 and 20 mM to preheated camel and cow milk reduced significantly (P < 0·05) the gelation time and increased the gel firmness. In contrast, Na2HPO42H2O added at 10 and 20 mM induced the formation of weak gels for preheated camel and cow milk at 50 °C, and even no gelation for preheated camel milk at 70 °C.

Low-sodium Cheddar cheese: Effect of fortification of cheese milk with ultrafiltration retentate and high-hydrostatic pressure treatment of cheese.

Low-sodium cheeses often exhibit an acidic flavor due to excessive acid production during the manufacturing and the initial stage of ripening, which is caused by ongoing starter culture activity facilitated by the low salt-in-moisture levels. We proposed that this excessive starter-induced acidity could be prevented by the fortification of cheese milk with ultrafiltration (UF) retentates (to increase curd buffering), and by decreasing microbial activity using the application of high-hydrostatic pressure (HHP) treatment (that is, to reduce residual starter numbers). Camel chymosin was also used as a coagulant to help reduce bitterness development (a common defect in low-sodium cheeses). Three types of low-Na (0.8% NaCl) Cheddar cheeses were manufactured: non-UF fortified, no HHP applied (L-Na); UF-fortified (cheese milk total solids = 17.2 ± 0.6%), no HHP applied (L-Na-UF); and UF-fortified, HHP-treated (L-Na-UF-HHP; 500 MPa for 3 min applied at 1 d post-cheese manufacture). Regular salt (2% NaCl) non-UF fortified, non-HHP treated (R-Na) cheese was also manufactured for comparison purposes. Analysis was performed at 4 d, 2 wk, and 1, 3, and 6 mo after cheese manufacture. Cheese functionality during ripening was assessed using texture profile analysis and dynamic low-amplitude oscillatory rheology. Sensory Spectrum and quantitative descriptive analysis was conducted with 9 trained panelists to evaluate texture and flavor attributes using a 15-point scale. At 4 d and 2 wk of ripening, L-Na-UF-HHP cheese had ~2 and ~4.5 log lower starter culture numbers, respectively, than all other cheeses. Retentate fortification of cheese milk and HHP treatment resulted in low-Na cheeses having similar insoluble calcium concentrations and pH values compared with R-Na cheese during ripening. The L-Na-UF cheese exhibited significantly higher hardness values (measured by texture profile analysis) compared with L-Na cheese until 1 mo of ripening; however, after 1 mo, all low-Na cheeses exhibited similar hardness values, which were significantly lower than R-Na cheese. Pressure treatment significantly increased maximum loss tangent (meltability) from rheology testing and decreased melt temperature. Sensory results indicated only very slight bitterness (<2.5 out of 15-point scale) was detected in all cheeses during the 6 mo of ripening. The L-Na-UF-HHP cheese did not significantly differ in bitterness and acidity from R-Na cheese during ripening. Pressures treatment of cheese at 500 MPa and cheese milk retentate fortification could be used to improve the quality of low-Na cheese.

The influence of protein fractions from bovine colostrum digested in vivo and in vitro on human intestinal epithelial cell proliferation.

Colostrum consists of a number of biologically active proteins and peptides that influence physiological function and development of a neonate. The present study investigated the biological activity of peptides released from first day bovine colostrum through in vitro and in vivo enzymatic digestion. This was assessed for proliferative activity using a human intestinal epithelial cell line, T84. Digestion of the protein fraction of bovine colostrum in vitro was conducted with the enzymes pepsin, chymosin and trypsin. Pepsin and chymosin digests yielded protein fractions with proliferative activity similar to that observed with undigested colostrum and the positive control foetal calf serum (FCS). In contrast trypsin digestion significantly (P<0·05) decreased colostral proliferative activity when co-cultured with cells when compared with undigested colostrum. The proliferative activity of undigested colostrum protein and abomasal whey protein digesta significantly increased (P<0·05) epithelial cell proliferation in comparison to a synthetic peptide mix. Bovine colostrum protein digested in vivo was collected from different regions of the gastrointestinal tract (GIT) in newborn calves fed either once (n=3 calves) or three times at 12-h intervals (n=3 calves). Digesta collected from the distal duodenum, jejunum and colon of calves fed once, significantly (P<0·05) stimulated cell proliferation in comparison with comparable samples collected from calves fed multiple times. These peptide enriched fractions are likely to yield candidate peptides with potential application for gastrointestinal repair in mammalian species.

Influence of micellar calcium and phosphorus on rennet coagulation properties of cows milk.

The main requirement for milk processed in most cheese typologies is its rennet coagulation ability. Despite the increasing number of studies, the causes for abnormal coagulation of milk are not fully understood. The aim of this study was to ascertain relationships between milk characteristics and its rennet coagulation ability, focusing on the influence of calcium (Ca) and phosphorus (P). Ca and P are essential constituents of the micelles. Micellar P can be present as part of colloidal calcium phosphate (inorganic-P) or covalently bound to caseins as phosphate groups (casein-P). Eighty one herd milk samples (SCC<400 000 cell/ml) were classified as Optimal (8), Suboptimal (39) Poor (29) and Non-coagulating milk (5), according to their rennet coagulation parameters as assessed by lactodynamographic test. Samples were analysed for their chemical composition (basic composition, protein fractions, minerals and salt equilibria), physicochemical parameters (pH and titratable acidity) and rheological properties. Optimal milk was characterised by the highest contents of major constituents, protein fractions and minerals, lowest content of chloride and highest values of titratable acidity. Non-coagulating milk was characterised by the highest values of pH and the lowest of titratable acidity. At micellar level, Optimal milk showed the highest values of colloidal Ca, casein-P and colloidal Mg (g/100 g casein), while Non-coagulating milk showed the lowest values. Interestingly, there was no statistical difference regarding the content of colloidal inorganic-P (g/100 g casein) between Optimal and Non-coagulating milks. Overall, high mineralisation of the micelle (expressed as g inorganic-P/100 g casein) positively affect its rennetability. However, excessive mineralisation could lead to a reduction of the phosphate groups (g casein-P/100 g casein) available for curd formation.

Effect of feeding buckwheat and chicory silages on fatty acid profile and cheese-making properties of milk from dairy cows.

Fresh buckwheat (Fagopyrum esculentum) and chicory (Cichorium intybus) had been shown to have the potential to improve certain milk quality traits when fed as forages to dairy cows. However, the process of ensiling might alter these properties. In the present study, two silages, prepared from mixtures of buckwheat or chicory and ryegrass, were compared with pure ryegrass silage (Lolium multiflorum) by feeding to 3 × 6 late-lactating cows. The dietary dry matter proportions realised for buckwheat and chicory were 0.46 and 0.34 accounting also for 2 kg/d of concentrate. Data and samples were collected from days 10 to 15 of treatment feeding. Buckwheat silage was richest in condensed tannins. Proportions of polyunsaturated fatty acids (PUFA) and α-linoleic acid in total fatty acids (FA) were highest in the ryegrass silage. Feed intake, milk yield and milk gross composition did not differ among the groups. Feeding buckwheat resulted in the highest milk fat concentrations (g/kg) of linoleic acid (15.7) and total PUFA (40.5; both P < 0.05 compared with ryegrass). The concentration of α-linolenic acid in milk fat was similar across treatments, but its apparent recovery in milk relative to the amounts ingested was highest with buckwheat. The same was true for the occurrence of FA biohydrogenation products in milk relative to α-linolenic acid intake. Recovery of dietary linoleic acid in milk remained unaffected. Feeding buckwheat silage shortened rennet coagulation time by 26% and tended (P < 0.1) to increase curd firmness by 29%. In conclusion, particularly buckwheat silage seems to have a certain potential to modify the transfer of FA from feed to milk and to contribute to improved cheese-making properties.

Seasonal changes in the technological and compositional quality of ewe's raw milks from commercial flocks under part-time grazing.

Rennet coagulation parameters, curd texture and gross compositional variables were studied in ewes' raw milk samples from nine commercial flocks using different concentrate:forage ratios and grazing times. From early lactation to March flocks were fed concentrate pellets and hay whereas from April to the end of lactation flocks were allowed to graze from 6 to 19 h/day receiving concentrate supplementation in the morning and evening. Milk from late-lactation flocks, when allowed to graze, showed higher content of fat, dry matter, protein, casein, soluble protein, total calcium, curd firmness and curd resistance to compression than the milk from early lactation flocks. Higher total calcium content and lower fat content were found when the early lactation flocks were fed high concentrate:forage ratio than when the flocks were fed low ratio. Curd firmness were lower for milk from flocks fed high concentrate:forage ratio, and the curd resistance to compression was greater, than for milk from flocks fed low ratio. At late lactation, when flocks grazed for a long time per day the total calcium content was higher than when the flocks grazed for a short time per day. Principal component analysis showed that protein and fat content were highly correlated with coagulum and curd firmness, whereas total calcium content was highly correlated with curd resistance to compression, and milk pH with rennet coagulation time.

Rennet paste from lambs fed a milk substitute supplemented with Lactobacillus acidophilus: effects on lipolysis in ovine cheese.

The present work was undertaken to evaluate the effects of Lactobacillus acidophilus supplementation of a milk substitute on the features of lamb rennet paste used for cheese making. Lipolysis in cheese manufactured with rennet paste from lambs receiving supplemented milk was also evaluated. Lambs were subjected to 3 different feeding regimens (mother suckling, MS; artificial rearing, AR; and artificial rearing with 7 log10 cfu/mL of Lb. acidophilus supplementation of the milk substitute, ARLb) and slaughtered at 20 and 40 d of age for each feeding treatment. Abomasa of the lambs were processed to rennet paste. Microbial loads, enzymatic activities (chymosin, pepsin, and lipases), and renneting characteristics of the lamb rennet paste were determined. Free fatty acids and conjugated linoleic acids were detected in cheese at 60 d of ripening. Addition of 7 log10 cfu/mL of Lb. acidophilus to the milk substitute was carried out successfully. Total recovery of viable cells was recorded in milk supplied daily to the lambs in the ARLb group. The ARLb rennet had greater amounts of lactobacilli than did the MS or AR rennet, irrespective of the slaughter age of the lambs, and the ARLb rennet had higher concentrations of lactococci when lambs were slaughtered at 40 d of age. Chymosin and lipase activities were also higher in ARLb rennet than in MS or AR rennet from lambs slaughtered at an older age. Milk supplementation of ARLb lambs resulted in improved coagulating ability of the rennet and enhanced cheese lipolysis after 60 d of ripening. A reduction of all free fatty acids was observed in all cheeses when passing from 20 to 40 d of slaughter of the lambs. Conjugated linoleic acids were more abundant in ARLb cheeses at both 20 and 40 d. Therefore, supplementation of the milk substitute with Lb. acidophilus improved the enzymatic features of rennet and the healthful and nutritional characteristics of it the ovine cheese. Moreover, the addition of lactobacilli to the milk substitute made it possible to increase the slaughter age of lambs without detrimental effects on rennet characteristics.

Production of ingredient-type cheddar cheese with accelerated flavor development by addition of enzyme-modified cheese powder.

Fast-ripened Cheddar cheeses for ingredient purposes were produced by addition of a dried enzyme-modified cheese (EMC; 0.25 and 1 g/100 g of milled curd) at the salting stage during a standard Cheddar cheese-making procedure. Populations of starter and nonstarter lactic acid bacteria (NSLAB), levels of proteolysis and lipolysis, volatile analysis, and flavor development (by quantitative descriptive sensory analysis) were monitored over a 6-mo ripening period. Levels of free AA and free fatty acids were elevated in the experimental cheeses on d 1 because of inclusion of the EMC. Counts of NSLAB were also elevated in the experimental cheeses compared with the control cheese from the start of ripening. Levels of free AA were slightly elevated in the experimental cheeses at 1, 2, and 4 mo, but significantly greater accumulations were detected by 6 mo of ripening, with His, Leu, and glutamate reflecting the greatest increases. Levels of long-chain free fatty acids increased up to 2 mo, indicating an initial stimulation of lipolysis, but had decreased by 6 mo, indicating greater catabolism, probably caused by NSLAB and increased starter lysis. Principal component analysis of the volatile compounds showed few differences in the aroma profiles among the cheeses up to 4 mo of ripening, but a large separation of the cheeses supplemented with EMC relative to the control was observed by 6 mo. Sensory analysis of the cheeses with added EMC showed an acceleration of 2 mo in flavor development compared with the control cheese with the addition of 1 g/100 g of EMC developing a flavor profile at 4 mo similar to the control cheese at 6 mo of ripening. However, atypical Cheddar flavors developed on prolonged storage. This study shows the potential of adding EMC during Cheddar production to produce a fast-ripened ingredient-type Cheddar cheese.

Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: proteolysis during ripening.

Proteolysis in cheese is influenced by the state of proteins (protein-calcium-phosphate interactions), level of indigenous milk enzymes (plasmin), externally added milk-clotting enzymes (chymosin), and endogenous and exogenous enzymes from starter and non-starter lactic acid bacteria (NSLAB). The objective of this study was to determine how different levels of calcium (Ca) and phosphorus (P), residual lactose, and salt-to-moisture ratio (S/M) in cheese influence proteolysis during ripening. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), 2 levels of lactose at pressing (2.4 vs. 0.78%), and 2 levels of S/M (6.4 vs. 4.8%) were manufactured. The cheeses were analyzed for changes in pH 4.6-soluble N, and starter and NSLAB counts during 48 wk of ripening. Cheeses at d 1 were also analyzed for residual chymosin, plasmin, and plasminogen activity. A significant increase in soluble N was observed during ripening for all the treatments. Cheeses with low Ca and P, low lactose, and low S/M treatments exhibited higher levels of proteolysis as compared to their corresponding high treatments. Differences in the rate of proteolysis for cheeses with different levels of Ca and P might be due to changes in protein conformation and differences in residual chymosin in the cheeses. Cheeses with low Ca and P were manufactured by lowering the pH at set and drain, which led to higher chymosin retention in cheeses with low Ca and P compared with high Ca and P. Differences in proteolysis between treatments with different levels of lactose were also partly attributed to residual chymosin activity. In all treatments, a major fraction of plasmin existed as plasminogen, indicating minimal contribution of plasmin to proteolysis in Cheddar cheeses. The number of starter bacteria, in all treatments, decreased significantly during ripening. However, the decrease was larger in the case of high S/M treatments compared with low S/M treatments. In contrast, the number of NSLAB increased during ripening, and low S/M cheeses had higher counts compared with high S/M cheeses. The differences in proteolysis due to S/M were partially attributed to changes in protein conformation or bacterial proteolytic activity.

Isolation and partial characterization of rennet-like proteases from Australian cardoon (Cynara cardunculus L.).

The yield, protein content, proteolytic activity, and substrate specificity of crude and partially purified extracts from dried and fresh Australian cardoon (Cynara cardunculus L.) flowers were determined. Crude water extracts had high yield but low protein content and proteolytic activity, whereas citric acid extracts had low yield but high protein content and proteolytic activity. Fresh flower extracts gave higher yield and proteolytic activity but lower protein content in comparison with dried flower extracts. Purification with ammonium sulfate resulted in significantly increased proteolytic activity for water extracts from both fresh and dried cardoon flowers, whereas the proteolytic activity of citric acid extracts did not change significantly after purification. Irrespective of extraction method, all extracts had higher proteolytic activity against ovine whole and kappa-caseins compared to their bovine counterparts, showing optimal activity at 37 degrees C and pH 6.0. Separation of purified extracts by ion-exchange liquid chromatography yielded three active fractions, each of which when assayed with sodium dodecyl sulfate capillary electrophoresis revealed two subunits with molecular masses of 15.5 and 33.1 kDa, respectively.

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.

Cloning of a marsupial kappa-casein cDNA from the brushtail possum (Trichosurus vulpecula).

The main role of kappa-casein in milk is to stabilize the formation of casein micelles. Although marsupial milk contains casein micelles, kappa-casein had not been identified in any species. In these experiments, the first marsupial kappa-casein has been prepared as enriched casein fractions and the cDNA cloned from the brushtail possum (Trichosurus vulpecula). Possum kappa-casein is a 158 amino acid peptide that shares low amino acid sequence identity (20-30%) with that of eutherian kappa-caseins. In the gut of suckling young, casein micelles clot when kappa-casein is cleaved by chymosin at a specific site. Eutherian kappa-casein sequences are classified according to the sequence of the chymosin cleavage site: Phe-Met, Phe-Ile or Phe-Leu. Possum kappa-casein appears to form a separate class, with a putative chymosin cleavage site of Phe-Ala, which is different from that found in eutherian mammals. Other features of kappa-caseins, such as the location of the N-terminal cysteine, solubility in the presence of calcium, and the O-glycosylation sites on threonine residues in the C-terminus of the molecule, are conserved in the possum sequence. The kappa-casein gene was expressed throughout lactation in the mammary gland, and although mRNA levels of kappa-, alpha- and beta-casein varied between animals there appeared to be a correlation in the expression of these genes within an individual animal. This suggests that a common transcription regulatory region may be controlling expression of all three genes in the possum.

Effect of the levels of N fertilizer, grass and supplementary feeds on nitrogen composition and renneting properties of milk from cows at pasture.

In a 2 x 2 x 3 factorial design grazing experiment we investigated the effect of fertilizer (none or 240 kg N/ha), amounts of clover grass available (low or high) and type and level of daily supplementary feed for each cow (3.5 kg barley, 3.5 kg concentrate mixture rich in protein and fat, or both, 7 kg) on the protein composition and renneting properties of their milk. The experiment was carried out in two successive grazing seasons (years) and included a total of 79 Danish Holstein cows. The effect on milk protein composition was determined in both years whereas the effect on renneting properties was determined only in the second year. Fertilization of the clover grass significantly decreased total milk protein concentration (-1.4 g/kg; P < 0.01) and tended also to decrease the relative proportion of whey protein N. Fertilization had no effect on renneting properties. Increased availability of clover grass significantly increased milk protein concentration (1 g/kg; P < 0.05) and resulted in significantly poorer renneting properties, that is increased clotting time (P < 0.01) and decreased coagulum development. These effects seemed to be mediated through an effect on the pH of the milk (+0.05; P < 0.05) as the effect was markedly reduced when statistical correction was made for the actual pH. Use of the protein- and fat-rich concentrate mix (3.5 kg) significantly reduced the total protein content of the milk (P < 0.05) and increased the proportion of non-protein N (NPN) in total N compared with use of the other supplementary feeds (P < 0.05). We found no effect on renneting properties of the different supplementary feeds. Throughout the grazing season and independent of the main treatments, the NPN proportion of milk N increased at the expense of casein N. At the same time, renneting properties became poorer, especially with high clover grass availability.