Residual Chymosin Activity Research Articles

Effect of chymosin‐induced hydrolysis of αS1‐casein on the tribological behaviour of brine‐salted semihard cheeses

This study investigated the effect of chymosin‐induced hydrolysis of αS1‐casein on the tribological behaviour of cheese. Residual chymosin activity was inhibited by adding a chymosin inhibitor (i.e. pepstatin A) to the curd–whey mixture during cheese manufacture. A significant difference was observed in the static and kinetic regimes of the extended Stribeck curves of cheese samples, suggesting that the chymosin‐induced hydrolysis of αS1‐casein affects the friction and lubrication properties of cheese, which may be relevant to the mouthfeel‐related sensory attributes of the cheese. Overall, the modulation of hydrolysis of αS1‐casein could be an approach to alter the friction‐related sensory attributes of cheese.

Effect of phenolic compounds on the activity of proteolytic enzymes during rennet induced coagulation of milk and ripening of miniature cheese

The effect of several purified phenolic compounds and green tea extract (GTE) on chymosin activity in model buffer solutions were found to be insignificant. The conserved secondary structure of chymosin upon interaction with phenolics, revealed by circular dichroism (CD) spectroscopy measurements, was considered to be the reason for unchanged enzyme activity. The coagulation properties of milk, however, was affected negatively by GTE addition. The RP-HPLC analysis and particle size measurements revealed that the caseinomacropeptide formation and aggregation of micelles was retarded in the presence of GTE during enzymatic coagulation of milk. The plasmin activity values during the ripening of miniature cheese samples were not affected by GTE addition while residual chymosin activity was decreased for cheeses fortified with GTE. Decreased levels of proteolysis of GTE-added cheese samples were observed with a 13–24% reduction in pH 4.6 SN/TN values, indicating a hindered primary proteolysis which was also confirmed by urea-PAGE and peptide profile analysis. The results of this study showed the importance of the interactions between phenolics and enzymes, as minor constituents of milk, for functional dairy product manufacturing.

Dynamic in situ imaging of semi-hard cheese microstructure under large-strain tensile deformation: Understanding structure-fracture relationships

Changes in the microstructure of semi-hard cheeses were observed in situ under tensile deformation by placing a microtensile stage directly under a confocal scanning laser microscope, and recording force/displacement data simultaneously. On tensile deformation, detachment of fat globules and their subsequent release from the cheese matrix were observed, suggesting that they are weakly bonded to or entrapped within the cheese matrix. Moreover, an inherent micro-defect was observed at a curd granule junction within the cheese matrix, which fractured along the curd granule junction under tensile deformation, suggesting that such micro-defects could be a key to the formation of undesirable slits or cracks. Furthermore, the fracture behaviour of semi-hard cheese varied with ripening temperature, coagulant type, and inhibition of residual chymosin activity. Overall, this study demonstrated the potential of dynamic in situ imaging of cheese microstructure for developing a greater understanding of the breakdown behaviour of cheese matrices.

Para-κ-casein during the ripening and storage of low-pH, high-moisture Feta cheese.

The hypothesis of this research paper was that the physicochemical conditions in a low-pH, high-moisture white brined cheese such as Feta would make para-κ-casein vulnerable to residual chymosin activity during ripening and storage. It was important to address this hypothesis, since cheese para-κ-casein could theoretically be used for the assessment of the origin of cheese milk by means of various analytical methods. Feta cheese was manufactured from sheep milk and from four different mixtures of sheep and goat milk in triplicate. The para-κ-casein of Feta samples taken during 120 d of ripening and storage was estimated by means of cation-exchange HPLC and proteolysis was determined in terms of free amino groups. Despite their similarity, sheep and goat para-κ-casein were efficiently separated and the changes of their chromatographic areas indicated that hydrolysis took place during the first stage of ripening. In accordance to the evolution of free amino groups, para-κ-caseins remained stable thereafter. The hydrolysis pattern was not affected by the composition of the cheese milk mixture and after 120 d at least two thirds of the initial quantity remained intact. Considering the efficient separation of sheep and goat para-κ-caseins and their stability during Feta storage, the same method was used for the evaluation of the percentage of goat milk in the cheese milk. The actual and the estimated percentage of goat milk within the range 0-40 were strongly correlated (R = 0·997, n = 60) and the standard error of estimation was 0·914.

Effect of high-pressure-treated starter on ripening of Feta cheese

High-pressure (HP) treatment of cheese or of bacterial starters used in cheese manufacture has the potential to modify the cheese ripening process. The objective of the present study was to investigate the effect of a DVI starter mixture HP-treated at 200 MPa, at 20 °C for 15 min, on the ripening of Feta cheese. Microbiological and biochemical characteristics, including enzymatic activities, in cheese were studied throughout ripening. Mesophilic, thermophilic, and non-starter bacteria counts were not affected significantly (P > 0.05) by the starter HP treatment and the same was also true for lactate dehydrogenase activity, indicating that there was no enhancement of autolysis in the cheese matrix due to starter treatment. However, the use of HP-treated starter increased secondary proteolysis in cheese, as shown by levels of nitrogen soluble in 12% trichloroacetic acid and in 5% phosphotungstic acid. These findings were consistent with the significant (P < 0.05) increase of the area of small hydrophilic peptides in the HPLC profiles of Feta made with treated starter. Since residual chymosin activity and aminopeptidase activity on Leu-p-NA did not differ significantly compared to those of control cheese throughout ripening, an HP-treatment-induced modification of other proteolytic enzymes was possible. These findings, together with the slow decrease in pH during the first days of ripening, suggest that HP-treated starter can be used as an adjunct starter in Feta manufacture rather than as a regular starter.

Proteolysis and related enzymatic activities in ten Greek cheese varieties

The objective of this study was to assess indices of proteolysis and related enzymatic activities of various cheese varieties produced in Greece. Physicochemical composition, extent of proteolysis (nitrogen fraction and reversed-phase high-performance liquid chromatography (RP-HPLC) profiles of cheese soluble fraction) and residual chymosin and plasmin activities were analyzed in 57 commercial samples, grouped according to cheesemaking technologies. The mean values of soluble nitrogen fraction on total nitrogen and trichloroacetic acid-soluble nitrogen fraction on total nitrogen did not differ significantly between the different cheese varieties, with mean values of 16.03% and 9.97%, respectively. Brined cheeses had the highest mean residual chymosin activity 0.166 International Milk Clotting units (IMCU).g−1 of cheese dry matter and the lowest mean plasmin plus plasminogen-derived activity 3.88 U.g−1 of cheese. The respective values for Gruyere and hard type cheeses were 0.029 and 0.047 IMCU.g−1 of cheese dry matter for chymosin and 6.22 and 6.94 U.g−1 of cheese for plasmin. It was interesting that both enzymatic activities were high in pasta filata type cheeses, i.e., respective mean values of 0.086 IMCU.g−1 of cheese dry matter and 7.42 U.g−1 of cheese. Intermediate regions on the RP-HPLC profiles were positively correlated with residual chymosin activity and negatively correlated with plasmin activity. It was concluded that cooking at pH close to cheesemilk pH and pressing are the most important technological factors both for proteolysis and activity of major proteolytic enzymes in various cheese varieties.

Inhibition of rennet activity in cheese using equine blood serum

The objective of this study was to inactivate chymosin in Cheddar-type cheese curd using equine blood serum. This blood serum was added (0.1–2%, v/v) to cheesemilk at 15 °C after completion of the first phase of renneting and before aggregation of the rennet-altered casein micelles, to inhibit the residual coagulant in Cheddar-type cheeses. Throughout ripening the level of pH 4.6-soluble nitrogen expressed as a percentage of total nitrogen was significantly higher in the control cheeses than in experimental cheeses and was about twice that of the experimental cheeses after ripening for 180 days. During ripening, there was almost no hydrolysis of αs1-casein in the cheeses made from milk containing 0.25–2% blood serum. Throughout ripening there were large quantitative differences between the peptide profiles of control and experimental cheeses. The results of this study suggest that the addition of equine blood serum to cheesemilk (0.25–2%) was very effective at inhibiting the residual chymosin activity in Cheddar-type cheeses during ripening; the activity of plasmin remained unaffected by the added blood serum. This study describes an easy and effective method for producing cheese curd free from residual coagulant activity, which will help to study the separate roles of the coagulant and other proteolytic enzymes in cheese ripening.

Impact of Chymosin- and Plasmin-Mediated Primary Proteolysis on the Growth and Biochemical Activities of Lactobacilli in Miniature Cheddar-Type Cheeses

Strongly proteolytic starters seem to improve the growth of nonstarter lactobacilli during cheese ripening, but no information is available on the impact of the nonmicrobial proteases usually active in cheese on their development. In the current study, the influence of chymosin- and plasmin-mediated proteolysis on the growth and biochemical activities of lactobacilli during ripening of miniature Cheddar-type cheeses, manufactured under controlled microbiological conditions, was studied. Two experiments were performed; in the first, residual chymosin activity was inhibited by the addition of pepstatin, and in the second, plasmin activity was increased by adding more enzyme, obtained in vitro through the activation of plasminogen induced by urokinase. Cheeses with or without a Lactobacillus plantarum I91 adjunct culture and with or without added pepstatin or plasmin solution were manufactured and ripened for 60 d. The addition of the adjunct culture resulted in enhancement of secondary proteolysis, evidenced by an increase in the total content of free amino acids (FAA) and modifications of the individual FAA profiles. Reduction in residual chymosin activity caused a decrease in primary and secondary proteolysis, characterized by the absence of αs1-casein hydrolysis and reduced production of peptides and FAA, respectively. The increase in plasmin activity accelerated primary proteolysis but no enhancement of secondary proteolysis was observed. Chymosin- and plasmin-mediated proteolysis did not influence the growth and biochemical activities of adventitious or adjunct lactobacilli, indicating that it is not a limiting factor for the development and proteolytic-peptidolytic activities of lactobacilli in the cheese model studied.

Characteristics of reduced fat Cheddar cheese made from ultrafiltered milk with an exopolysaccharide-producing culture

In a previous study, ultrafiltration (UF) at 1.2x reduced residual chymosin activity and bitterness in exopolysaccharide (EPS)-positive reduced fat Cheddar cheese. The objective of this research was to study the effect of this level of concentration on the textural and functional characteristics of the reduced fat cheese. Ultrafiltration (1.2x) did not affect the hardness, cohesiveness, adhesiveness, chewiness, and gumminess of EPS-positive cheese. The 6-month old UF cheeses were springier than non-UF cheeses. However, the springiness of the EPS-positive cheese made from UF milk was much lower than that of the EPS-negative cheeses. Texture of the EPS-negative cheese was more affected by UF than that of the EPS-positive cheese. Differences were seen in the extent of flow between UF and non-UF cheeses at 1 and 3-months but not after 6 months ripening. Ultrafiltration increased the elastic modulus in the 6-month old EPS-positive cheeses. Higher body and texture scores were given to EPS-positive cheeses than the EPS-negative ones. Sensory panelists found the body of the UF and non-UF cheeses to be similar.

Ultrafiltered Milk Reduces Bitterness in Reduced-Fat Cheddar Cheese Made with an Exopolysaccharide-Producing Culture

The objectives were to reduce bitterness in reduced-fat Cheddar cheese made with an exopolysaccharide (EPS)-producing culture and study relationships among ultra-filtration (UF), residual chymosin activity (RCA), and cheese bitterness. In previous studies, EPS-producing cultures improved the textural, melting, and viscoelastic properties of reduced-fat Cheddar cheese. However, the EPS-positive cheese developed bitterness after 2 to 3 mo of ripening due to increased RCA. We hypothesized that the reduced amount of chymosin needed to coagulate UF milk might result in reduced RCA and bitterness in cheese. Reduced-fat Cheddar cheeses were manufactured with EPS-producing and nonproducing cultures using skim milk or UF milk (1.2×) adjusted to a casein:fat ratio of 1.35. The EPS-producing culture increased moisture and RCA in reduced-fat Cheddar cheese. Lower RCA was found in cheese made from UF milk compared with that in cheese made from control milk. Ultrafiltration at a low concentration rate (1.2×) produced EPS-positive, reduced-fat cheese with similar RCA to that in the EPS-negative cheese. Slower proteolysis was observed in UF cheeses compared with non-UF cheeses. Panelists reported that UF EPS-positive cheese was less bitter than EPS-positive cheese made from control milk. This study showed that UF at a low concentration factor (1.2×) could successfully reduce bitterness in cheese containing a high moisture level. Because this technology reduced the RCA level (per g of protein) to a level similar to that in the control cheeses, the contribution of chymosin to cheese proteolysis would be similar in both cheeses.

Effect of cook temperature on primary proteolysis and predicted residual chymosin activity of a semi-hard cheese manufactured using thermophilic cultures

Novel semi-hard cheeses were manufactured using Streptococcus thermophilus and Lactobacillus helveticus as starter cultures and with cook temperatures of 47, 50 or 53 °C. There was a progressive and significant degradation of both α s1- and β-caseins during ripening of all cheeses. Increasing cook temperature significantly reduced degradation of α s1-casein during ripening, in the order 53<50<47 °C, as measured by densitometric analysis of urea-polyacrylamide gel electrophoresis (urea-PAGE) electrophoretograms. Mean levels of primary proteolysis, as measured by amounts of pH 4.6-soluble N, were also significantly reduced. A mathematical model, incorporating changes in pH and temperature during manufacture of the cheeses, predicted near to total inactivation of residual chymosin as a result of the cooking profiles used in cheesemaking. Increasing cook temperature did not completely inhibit primary proteolysis or hydrolysis of α s1-casein to α s1-casein (f24–199) during ripening, although these reactions were slowed.

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.

Thermal inactivation of chymosin during cheese manufacture.

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100 +/- 21 min and a z-value of 5.9 +/- 0.3 degrees C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.

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.