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.     

Effects of blends of camel and calf chymosin on proteolysis,residual coagulant activity,microstructure, and sensory characteristics of Beyaz peynir

Beyaz peynir, a white brined cheese, was manufactured using different blends of camel chymosin (100, 75, 50, 25, and 0%) with calf chymosin and ripened for 90 d. The purpose of this study was to determine the best mixture of coagulant for Beyaz peynir, in terms of proteolysis, texture, and melting characteristics. The cheeses were evaluated in terms of chemical composition, levels of proteolysis, total free amino acids, texture, meltability, residual coagulant activity, microstructure, and sensory properties during 90 d of ripening. Differences in the gross chemical composition were statistically significant for all types of cheeses. Levels of proteolysis were highly dependent on the blends of the coagulants. Higher proteolysis was observed in cheeses that used a higher ratio of calf chymosin. Differences in urea-PAGE and peptide profiles of each cheese were observed as well. Meltability values proportionally increased with the higher increasing levels of calf chymosin in the blend formula. These coagulants had a slight effect on the microstructure of cheeses. The cheese made with camel chymosin had a harder texture than calf chymosin cheese, and hardness values of all cheese samples decreased during ripening. The cheeses with a high ratio of calf chymosin had higher residual enzyme activity than those made with camel chymosin. No significant difference in sensory properties was observed among the cheeses. In conclusion, cheeses made with a high level of calf chymosin had a higher level of proteolysis, residual coagulant activity, and meltability. The cheeses also had a softer texture than cheeses made with a high content of camel chymosin. Camel chymosin may be used as a coagulant alone if low or limited levels of proteolysis are desired in cheese.     

Proteolysis of alphas-casein as a marker of Grana Padano cheese ripening

Since casein proteolysis has a critical role in defining the typical characteristics of Grana Padano cheese, we evaluated the hydrolysis of alphas-casein during the ripening process. Thanks to the high specificity of the anti-alphas((alphas1 + alphas2)-casein monoclonal antibody and amino acid sequence determination, it was possible to identify three main alphas-casein-derived polypeptides in cheese: alphaa, alphab, and alphac. Their production by the three enzymes most involved in cheese proteolysis (pepsin, chymosin, and plasmin) was evaluated by performing in vitro digestions. Data showed that alphaa was released in cheese mainly by the chymosin attack, while alphab and alphac were due to the action of plasmin. A significant correlation between the abundance of some polypeptides and ripening process was shown.     

Ripening of extra-hard cheese made with mesophilic DL-starter

Extra hard cheese is commonly made with thermophilic starters using high temperatures to stimulate expulsion of whey. In this work, microflora, proteolysis and volatiles were investigated in an extra-hard cheese made with mesophilic DL-starter, produced using challenging cooking temperatures for the starter bacteria over several hours. Cheese from six commercially produced vats was investigated over 56 weeks. The number of starter bacteria decreased after three weeks of ripening. Casein breakdown was characterised by chymosin and plasmin activity on α_s1- and β-caseins, respectively. Peptide profiles showed accumulation of Lactococcus derived peptides from α_s1-CN f1–23, and the peptide β-CN 29–93 as a result of joint plasmin and chymosin activity and absence of highly proteolytic thermophilic Lactobacillus, commonly present in extra-hard cheese. The composition of amino acids depended mainly on starter during the first 26 weeks of ripening. The content of volatiles depended both on ripening time and the starter used.     

Chymosin-mediated proteolysis, calcium solubilization, and texture development during the ripening of cheddar cheese

Full fat, milled-curd Cheddar cheeses (2 kg) were manufactured with 0.0 (control), 0.1, 1.0, or 10.0 μmol of pepstatin (a potent competitive inhibitor of chymosin) added per liter of curds/whey mixture at the start of cooking to obtain residual chymosin levels that were 100, 89, 55, and 16% of the activity in the control cheese, respectively. The cheeses were ripened at 8°C for 180 d. There were no significant differences in the pH values of the cheeses; however, the moisture content of the cheeses decreased with increasing level of pepstatin addition. The levels of pH 4.6-soluble nitrogen in the 3 cheeses with added pepstatin were significantly lower than that of the control cheese at 1 d and throughout ripening. Densitometric analysis of urea-PAGE electro-phoretograms of the pH 4.6-insoluble fractions of the cheese made with 10.0 μmol/L of pepstatin showed complete inhibition of hydrolysis of α_S1-casein (CN) at Phe₂₃-Phe₂₄ at all stages of ripening. The level of insoluble calcium in each of 4 cheeses decreased significantly during the first 21 d of ripening, irrespective of the level of pepstatin addition. Concurrently, there was a significant reduction in hardness in each of the 4 cheeses during the first 21 d of ripening. The softening of texture was more highly correlated with the level of insoluble calcium than with the level of intact α_S1-CN in each of the 4 cheeses early in ripening. It is concluded that hydrolysis of α_S1-CN at Phe₂₃-Phe₂₄ is not a prerequisite for softening of Cheddar cheese during the early stages of ripening. We propose that this softening of texture is principally due to the partial solubilization of colloidal calcium phosphate associated with the para-CN matrix of the curd.     

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.     

Use of high pressure treatment to attenuate starter bacteria for use as adjuncts for Cheddar cheese manufacture

Attenuated starter bacteria cannot produce acid during cheese manufacture, but contain enzymes that contribute to cheese ripening. The aim of this study was to investigate attenuation of starter bacteria using high pressure treatment, for use in combination with a primary starter for Cheddar cheese manufacture, and to determine the effect of such adjunct cultures on secondary proteolysis during ripening. Lactococcus lactis ssp. cremoris HP and L. lactis ssp. cremoris 303 were attenuated by pressure treatment at 200 MPa for 20 min at 20 °C. Cheddar cheese was manufactured using untreated cultures of both these starter strains, either alone or in combination with their high pressure-treated equivalents. High pressure-treated starters did not produce acid during cheese manufacture and starter counts in cheeses manufactured using high pressure-treated starter did not differ from those of the controls. Higher levels of cell lysis were apparent in cheese manufactured using high pressure-treated strains than in the controls after 26 d of ripening. Small differences were observed in the peptide profiles of cheeses, analysed by reversed-phase HPLC; cheeses manufactured using high pressure-treated starters also had slightly higher levels of amino acids than the relevant controls. Overall, addition of high pressure-treated starter bacteria as a secondary starter culture accelerated secondary proteolysis in Cheddar cheese.

Industrial relevance

Attenuated starters provide extra pool of enzymes, which can influence cheese ripening, without affecting the cheese making schedule. This paper presents an alternative method for attenuation of starter bacteria using high pressure treatment and their subsequent use to accelerate secondary proteolysis in Cheddar cheese during ripening.     

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.     

Accelerated ripening of Caciocavallo Pugliese cheese with attenuated adjuncts of selected nonstarter lactobacilli

The nonstarter lactic acid bacteria Lactobacillus plantarum CC3M8, Lactobacillus paracasei CC3M35, and Lactobacillus casei LC01, previously isolated from aged Caciocavallo Pugliese cheese or used in cheesemaking, were used as adjunct cultures (AC) or attenuated (by sonication treatment) adjunct cultures (AAC) for the manufacture of Caciocavallo Pugliese cheese on an industrial scale. Preliminary studies on the kinetics of growth and acidification and activities of several enzymes of AAC were characterized in vitro. As shown by the fluorescence determination of live versus dead or damaged cells and other phenotype features, attenuation resulted in a portion of the cells being damaged and a portion of the cells being capable of growing with time. Compared with the control cheese (without adjunct cultures) and the cheese with AAC, the addition of AC resulted in a lower pH after manufacture, which altered the gross composition of the cheese. As shown by plate count and confirmed by random amplification of polymorphic DNA-PCR, the 3 species of nonstarter lactobacilli persisted during ripening but the number of cultivable cells varied between AC and AAC. Slight differences were found between cheeses regarding primary proteolysis. The major differences between cheeses were the accumulation of free amino acids and the activity levels of several enzymes, which were highest in the Caciocavallo Pugliese cheeses made with the addition of AAC. As shown by triangle test, the sensory properties of the cheese made with AAC at 45d did not differ from those of the control Caciocavallo Pugliese cheese at 60d of ripening. In contrast, the cheese made with AC at 45d differed from both the Caciocavallo Pugliese cheese without adjuncts and the cheese made with AAC. Attenuated adjunct cultures are suitable for accelerating the ripening of Caciocavallo Pugliese cheese without modifying the main features of the traditional cheese.     

Effect of chymosin and salt reduction on the quality of ultrafiltrated white-salted cheese

This study demonstrated that both chymosin and salt-in-moisture (SM) were important factors for proteolysis in the manufacture of ultrafiltrated white-salted cheese, with significant effects on water-soluble nitrogen and nitrogen soluble in trichloroacetic acid. In contrast, the levels of free amino acids were not significantly affected by chymosin and salt treatments. The cheeses made, using high levels of chymosin with low SM had lower levels of residual alpha(S1)- and beta-casein at the end of ripening. On texture profile analysis, the hardness and fracturability of the cheeses significantly increased with SM and decreased during ripening. Increases in chymosin significantly contributed to the overall weakening of the structure throughout ripening. Bitter flavour was detected after 12 weeks in the cheese made with the higher chymosin level and lower SM, which could be the result of accumulation of gamma-casein fractions. The sensory data indicated that the hedonic responses for low chymosin with low SM cheeses were good and acceptable in flavour, which may be due to the moderate levels of proteolysis products.     

Microbiological,biochemical and compositional changes during ripening of São Jorge – a raw milk cheese from the Azores (Portugal)

The microbial, compositional and biochemical profiles of São Jorge cheese (PDO) obtained from three distinct cheese plants, throughout the ripening period were determined. Fully ripened cheeses (i.e. by 130 days) contained a total of 3.1 × 10⁷ CFU g⁻¹ mesophilic bacteria, and a decrease in moisture content, concomitantly with an increase in salt content, was observed throughout the same time frame. The pH decreased until 30 days of ripening; thereafter, a slight increase was reported, up to 5.6 by the end of ripening. Urea-PAGE results showed extensive primary proteolysis, of both β-casein and α_s1-casein − degraded at essentially similar rates; plasmin and chymosin accordingly appear to be active in the cheese curd. RP-HPLC profiles of water-soluble fractions showed minor differences between 1 and 130 day old cheeses, whereas equivalent profiles of 7% (v/v) ethanol-soluble fractions contained several peaks, indicative of a heterogeneous mixture of products of proteolysis, that evolved with time.     

Texture Development During Cheese Ripening

The texture of a cheese is determined primarily by its pH and the ratio of intact casein to moisture. The texture generally changes markedly in the first 1 to 2 wk of ripening as the hydrolysis of a small fraction of α_s1-casein by the rennet to the peptide α_s1-I results in a general weakening of the casein network. The relatively slow change in texture thereafter is determined mainly by the rate of proteolysis, which in turn is controlled largely by the proportion of residual rennet and plasmin in the cheese, salt to moisture ratio, and storage temperature. The rise in pH that occurs during ripening is also important in many cheese varieties. Cheese texture may be significantly changed by the use of coagulants other than chymosin, addition of neutral proteases, and incorporation of whey proteins. The stretching characteristics of natural cheese curd depend upon both its pH and the proportion of colloidal calcium phosphate that has been removed. Cheese containing residual coagulant loses stretchability very rapidly with age. Eye formation in Swiss-type cheese is dependent upon the cheese pH at the time of transfer to the hot room.     

Ripening of cheese made from full concentrated milk retentate with and without peptidase addition

The aim of this study was to determine ripening of cheese made from full concentrated (FC) milk retentate with and without peptidase addition. No free amino acids (FAAs) were found in FC cheese at the end of ripening. However, added peptidase increased FAA formation. Protein and peptide profile analysis showed that FAA and small peptides increased during ripening and therefore some secondary proteolysis occurred. Added peptidase increased D‐lactic acid formation during ripening of cheeses. This kind of changes in lactose fermentation should be considered during developing the making cheese with different enzyme addition.     

Ultrafiltration of cheese milk: Effect on plasmin activity and proteolysis during cheese ripening

Havarti 45+ cheese was manufactured from milk concentrated 1.8–4.6-fold by ultrafiltration (UF) and from normal milk, and the effect of concentration factor on plasmin activity and subsequent proteolysis in cheese during ripening was examined. There was decreased plasmin activity and a reduced rate of proteolysis of α_S2-casein and β-casein in the UF-cheeses, compared with the corresponding controls, independent of concentration factor. The decreased plasmin activity and slower breakdown of α_S2-casein and β-casein in UF-cheeses compared with traditional cheeses can be partly explained by the inclusion of an increased amount of plasmin inhibitors into the UF-cheeses. However, it is suggested that the differences in plasmin activity and proteolysis arise mainly as a result of inactivation of the plasminogen activation system during UF-concentration, due to a combination of time, temperature and the presence of air in the UF-equipment. The effect of milk treatment on the plasminogen activation system should be further investigated.     

Biochemistry of cheese ripening

Rennet-coagulated cheeses are ripened for periods ranging from about two weeks to two or more years depending on variety. During ripening, microbiological and biochemical changes occur that result in the development of the flavour and texture characteristic of the variety. Biochemical changes in cheese during ripening may be grouped into primary (lipolysis, proteolysis and metabolism of residual lactose and of lactate and citrate) or secondary (metabolism of fatty acids and of amino acids) events. Residual lactose is metabolized rapidly to lactate during the early stages of ripening. Lactate is an important precursor for a series of reactions including racemization, oxidation or microbial metabolism. Citrate metabolism is of great importance in certain varieties. Lipolysis in cheese is catalysed by lipases from various source, particularly the milk and cheese microflora, and, in varieties where this coagulant is used, by enzymes from rennet paste. Proteolysis is the most complex biochemical event that occurs during ripening and is catalysed by enzymes from residual coagulant, the milk (particularly plasmin) and proteinases and peptidases from lactic acid bacteria and, in certain varieties, other microorganisms that are encouraged to grow in or on the cheese. Secondary reactions lead to the production of volatile flavour compounds and pathways for the production of flavour compounds from fatty acids and amino acids are also reviewed.     

Effects of Penicillium roqueforti and whey cheese on gross composition,microbiology and proteolysis of mould‐ripened Civil cheese during ripening

Four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as the secondary starter with and without addition of the whey cheese (Lor); in parallel, secondary starter‐free counterparts were manufactured. Chemical composition, microbiology and proteolysis were studied during the ripening. The incorporation of whey cheese in the manufacture of mould‐ripened Civil cheese altered the gross composition and adversely affected proteolysis in the cheeses. The inoculated P. roqueforti moulds appeared to grow slowly on those cheeses, and little proteolysis was evident in all cheese treatments during the first 90 days of ripening. However, sharp increases in the soluble nitrogen fractions were observed in all cheeses after 90 days. Microbiological analysis showed that the microbial counts in the cheeses were at high levels at the beginning of ripening, while their counts decreased approximately 1–2 log cfu/g towards the end of ripening.     

The effect of refrigerated storage of raw milk on the physicochemical and microbiological quality of Tunisian semihard Gouda‐type cheese during ripening

A Tunisian semihard Gouda‐type cheese made from milk kept at 4 °C for 24, 48, 72 and 96 h was monitored during 45 days of ripening. The effect of milk refrigeration on the evolution of physicochemical parameters in relation to the quantitative variation of the microbial population during ripening of Gouda‐type cheese was investigated. Microbiological and physicochemical analyses were performed on raw milk and cheese samples after curding, 2, 9, 16, 23, 30, 37 and 45 days of ripening time. The raw milk kept under refrigeration at 4 °C for 96 h showed the highest microbial count and proteolysis level. The duration of storage significantly reduced the cheese yield as a result of important solubilisation casein in proteoses‐peptones. Results of different nitrogenous fractions by Kjeldahl method showed enzymatic hydrolysis products of casein whose intensity depended on the maturing stage as well as the refrigeration time. Besides the evident action of the plasmin, original milk protease, on the hydrolysis of casein in soluble fractions, the proteolysis of cheese caseins is also initiated by proteolytic action of the chymosin and extracellular heat‐resistant proteases notably produced by the same psychrotrophic microflora. Lactic acid bacteria starters that constitute the dominant microflora of this type of cheese are also considered as aroma precursors.     

Quality of milk and of Canestrato Pugliese cheese from ewes exposed to different ventilation regimens

Effects of ventilation regimen on the quality of ewes' milk and on proteolysis in Canestrato Pugliese cheese during ripening were studied. Cheeses were manufactured from the bulk milk of Comisana ewes subjected to three different ventilation regimens, which were designated low (LOV, 23 m3/h per ewe), moderate (MOV, 47 m3/h per ewe) and programmed ventilation regimen (PROV, 73 m3/h per ewe; fan set to maintain 70% relative humidity). Bulk milk was analysed for chemical and microbial composition, renneting parameters and plasmin-plasminogen activities. At 1, 15, 30 and 45 d of ripening, the cheeses were analysed for gross chemical composition, nitrogen fractions, and plasmin and plasminogen activities. The pH 4.6-insoluble nitrogen fractions were analysed by urea-PAGE. Free amino acid content was determined at the end of ripening. Lower concentrations of bulk milk somatic cell count (BMSCC) and of mesophilic bacteria were found in the MOV group than in the LOV and the PROV groups. A lower plasminogen (PG) to plasmin (PL) ratio (PG/PL) was observed in the MOV and PROV than in the LOV cheeses. Irrespective of treatment, PL activity in cheeses was higher at 15d of ripening, while a sudden decrease of PL and PG activities was observed at 30 d, which was associated with a marked increase in non-protein nitrogen. The peptide profile characterized in the urea-PAGE showed a greater intensity of alpha- and beta-CN hydrolysis in the MOV than in the PROV and LOV cheeses. The results provide evidence that a proper ventilation regimen is critical for optimizing the hygienic quality of milk and the proteolysis of Canestrato Pugliese cheese during ripening.     

Size-Exclusion HPLC Separation of Bitter and Astringent Fractions from Cheddar Cheese Made with Added Lactobacillus Strains to Accelerate Ripening

Lactobacillus strains were added as an adjunct to the regular lactic starter in Cheddar cheese manufacture in order to accelerate ripening. Microbial cheese proteolysis resulted in the release of free amino acids which were extracted with the astringent and bitter fractions and separated by size-exclusion and reversed-phase HPLC chromatography. Lactobacillus strains generally increased the degree of proteolysis. L. plantarum and L. brevis produced off-flavors possibly due to an accumulation of medium-size peptides. The control cheese (without lactobacilli) had the most peptides with a mean molecular- weight of < 1000 daltons and had a flavor described as slightly bitter. Addition of L. casei-casei L2A accelerated ripening and yielded a well-aged Cheddar cheese without any bitterness even after 7 months at 6°C.