Inhibition of Residual Coagulant in Cheese using Pepstatin

Pepstatin A, an inhibitor of acid proteases, was added (7.5, 15 or 30 μmol L^-1) to the curds/whey mixture at the start of cooking to inhibit residual coagulant in miniature (20 g) Cheddar-type cheeses. No degradation of _s1-casein was observed by urea–polyacryl amide gel electrophoresis (PAGE) in the pepstatin-treated cheeses, indicating that all the concentrations of pepstatin used in this study effectively inhibited residual coagulant throughout ripening. The level of water-soluble N (WSN) as % of total N increased very slowly in the pepstatin-treated cheeses, while there was a steady increase in WSN in the control cheeses; after 4 months of ripening, the level of WSN in the control cheese was nearly three times as high as in the cheese treated with 30 μmol L^-1 pepstatin. Urea–PAGE of water-soluble fractions (WSF) showed marked differences between pepstatin-treated cheeses and their respective controls throughout ripening. Reverse-phase HPLC of the WSF of the cheeses showed that the peptides _s1-CN f1-9/13, which are formed from the chymosin-produced peptide, _s1-CN f1-23, by the action of the cell envelope-associated proteinase of Lactococcus, were not present in pepstatin-treated cheeses. Levels of total free amino acids (as determined by the Cd–ninhydrin method) were higher in controls than in pepstatin-treated cheeses throughout ripening. The results of this study demonstrated that pepstatin is a very effective inhibitor of residual coagulant in cheese.     

Possible implications of milk pasteurization on the manufacture and sensory quality of ripened cheese

Cheese made from raw milk represents an important proportion of the traditional cheeses, particularly in South European countries. Besides destruction of pathogenic bacteria, the most significant changes in milk relevant to cheesemaking, which are induced by pasteurization are:

• a partial elimination of the milk microorganisms which may grow in cheese during ripening,

• a partial or total activation or inhibition of the plasmin/plasminogen complex, cathepsin D, lipoprotein lipase and alkaline phosphatase. Enzymes from psychrotrophic bacteria, acid phosphatase and xanthine oxidase, which may be active during ripening, withstand pasteurization.,

• a slight (7%) denaturation of serum proteins and little or no modification of the cheesemaking properties (coagulation, acidification by lactic acid bacteria).

From experimental work carried out on several cheese varieties, comparing pasteurized or microfiltered milk and raw milk cheeses, it was found that facultatively heterofermentative lactobacilli, Micrococcaceae, enterococci, and propionibacteria in Swiss-type cheese, are found at higher levels in raw milk cheese. The main biochemical modification of cheese during ripening concerns the nature and extent of proteolysis. Although there is no clear trend in the breakdown of _s1- and β-caseins, milk pasteurization leads to a significant decrease of the amount of small peptides and free amino acids and to different HPLC profiles. Experiments carried out with sensory analysis show that, in all cases, pasteurized or microfiltered milk cheeses have received lower flavour intensity scores than raw milk cheeses. From this review, it is concluded that the indigenous milk microflora, with its diversity of species and strains, appears to be mainly responsible of the specific sensory properties of raw milk cheeses.     

Lipolysis and proteolysis profiles of fresh artisanal goat cheese made with raw milk with 3 different fat contents

The objective of this study was to describe the proteolysis and lipolysis profiles in goat cheese made in the Canary Islands (Spain) using raw milk with 3 different fat contents (0.5, 1.5, and 5%) and ripened for 1, 7, 14, and 28 d. β-Casein was the most abundant protein in all cheeses and at all ripening times. Quantitative analysis showed a general decrease in caseins as ripening progressed, and degradation rates were higher for α_S1-casein than for β-casein and α_S2-casein. Furthermore, the degradation rate during the experimental time decreased with lower fat contents. The α_S2-casein and α_S1-casein levels that remained in full-fat and reduced-fat cheeses were less than those in low-fat cheese. In contrast, β-casein also showed degradation along with ripening, but differences in degradation among the 3 cheese types were not significant at 28 d. The degradation products increased with the ripening time in all cheeses, but they were higher in full-fat cheese than in reduced-fat and low-fat cheeses. The free fatty acid concentration per 100 g of cheese was higher in full-fat cheese than in reduced- and low-fat cheese; however, when the results were expressed as milligrams of free fatty acids per gram of fat in cheese, then lipolysis occurred more rapidly in low-fat cheese than in reduced- and full-fat cheeses. These results may explain the atypical texture and off-flavors found in low-fat goat cheeses, likely the main causes of non-acceptance.     

Proteolysis texture and microstructure of low‐fat Tulum cheese affected by exopolysaccharide‐producing cultures during ripening

The objective of the study was to determine the effects of exopolysaccharide (EPS)‐producing or non‐EPS‐producing starters on proteolysis, physical and microstructural characteristics of full‐fat or low‐fat Tulum cheeses during ripening. For this purpose, Tulum cheese was manufactured using full‐ or low‐fat milk with EPS‐producing and non‐EPS‐producing starter cultures. Chemical composition, proteolysis, texture profiles and microstructure of the cheeses were studied during 90 days of ripening. Urea‐PAGE of water‐insoluble and RP‐HPLC peptide profiles of water‐soluble fractions of the cheeses showed that the use of starters resulted in different degradation patterns in all cheeses during ripening. Although β‐casein exhibited similar degradation patterns in all cheeses, small differences are present in α_s1‐casein degradation during ripening. Reducing fat in Tulum cheese changed the RP‐HPLC peptide profile of the cheeses. The use of EPS‐producing cultures improved the textural characteristics and changed the microstructure and proteolysis of low‐fat Tulum cheese.     

Proteolytic and lipolytic changes during the ripening of León raw cow's milk cheese, a Spanish traditional variety

Proteolytic and lipolytic changes were studied throughout ripening of five batches of León cow's milk cheese, a traditional variety made in the north of Spain. Total soluble nitrogen, non-protein nitrogen, oligopeptides nitrogen, amino nitrogen and ammonia nitrogen fractions increased slightly during the ripening process. The final values of these nitrogen fractions indicate that this cheese undergoes a very slight proteolysis as much in extent as in depth. This weak protein degradation is corroborated when the caseins and their degradation products were quantified by electrophoresis. β-Casein stayed practically intact throughout the ripening process and only 10% of α_s-casein became degraded. The content of total free amino acids increased progressively but in a slightly increased way during ripening, reaching final average values of 592 mg (100 g)⁻¹ of total solids. The most abundant free amino acid at the end of ripening was lysine, followed by leucine, glutamic acid, tryptophan, valine and phenylalanine. The acidity index of the fat values increased during ripening by a factor of 4.39. The final values of this parameter are in the range of those observed in other cow's milk cheeses ripened by bacteria. The content in total free fatty acids underwent an increase throughout ripening reaching final average values of 6669 ppm. The most abundant free fatty acid at the end of ripening was oleic acid followed by butyric and palmitic acids. The high content of short-chain fatty acids is outstanding, specially that of butyric acid.     

Influence of rennet milk-clotting activity on the proteolytic and sensory characteristics of an ovine cheese

Roncal cheese (regulated by an Apellation of Origin) is a traditional hard cheese manufactured from raw ewe's milk in the region of Navarre in Spain. Roncal cheeses, manufactured using two lamb rennets with different milk-clotting activity levels, were evaluated to compare their chemical, proteolytic, and sensory characteristics. A preliminary study of samples of lamb rennets indicated that a large proportion of such rennets did not fulfil current microbiological requirements and likewise revealed considerable variation in the milk-clotting activity of the samples examined. Trends in the overall physicochemical parameter values (pH, dry matter, fat, and protein) were similar in both cheese batches. Proteolysis of the nitrogen fractions was observed to take place at a faster rate in the cheeses made using the rennet with the higher milk-clotting activity (soluble nitrogen, non-protein nitrogen, and amino acid nitrogen values around 13–20% higher than in the cheeses made using the rennet with the lower milk-clotting activity after 180 days of ripening). Urea-PAGE electrophoretic analysis of the caseins from the cheeses manufactured using both types of rennet showed that the β-caseins were less susceptible to proteolysis than the α_s-caseins. The effect of the different milk-clotting activity levels was most pronounced on the α_s-caseins, in which the rennet with the higher milk-clotting activity gave higher breakdown. Nevertheless, the differences in the proteolysis rates did not yield any appreciable sensory differences.     

Contribution of Geotrichum candidum to the proteolysis of soft cheese

To determine the action of the yeast Geotrichum candidum on the proteolysis of soft cheese, Camembert-type cheeses were manufactured with and without this surface flora. Casein degradation and the release of peptides and amino acids at the cheese surface were studied to assess overall proteolysis. The results showed extensive proteolytic activity at the surface of cheese with G. candidum, and suggested that G. candidum is able to contribute to both primary and secondary proteolysis. α_s1- and βA²-caseins were preferentially hydrolysed at the surface of cheese with G. candidum from the first week of ripening. This proteolytic activity led to the production of numerous peptides that were subsequently hydrolysed, as indicated by the large increase in the concentration of free amino acids from the second week to the end of ripening.     

Proteolysis in ultra-filtered and conventional Iranian white cheese during ripening

Conventional and ultra-filtered (UF) Iranian white cheeses were made with almost identical gross chemical composition and the extent and characteristics of proteolysis were studied during ripening. UF cheeses exhibited a lower rate of development of pH 4.6-soluble nitrogen than conventional cheeses. The rates of degradation of α_s1-casein and particularly β-casein were lower in UF cheeses than in conventional cheeses. Plasmin activity was lower in UF cheeses than that in conventional cheese, whereas coagulant activity was higher in the former. Noticeable qualitative and quantitative differences were observed in reverse-phase high performance liquid chromatography (RP-HPLC) peptide profiles between UF and conventional white cheeses and chemometric analysis of peak height data distributed the cheeses into two separate groups. The levels of free amino acids in UF cheeses were lower than in conventional cheeses. Lower peptide degradation and production of amino acids suggested slower ripening, which may have been associated with the weak aroma development characteristic of UF cheeses.     

Blue like cheese from dried milk

A trial was made to produce Blue like cheese from both whole dry milk and non fat one. The resultant cheese was kept for ripening at 5°C for two months. Cheese made from reconstituted whole dried milks were characterized with higher moisture, salt, and protein contents and acidity than the control. Protein degradation and fat hydrolysis were found to be lower in these cheeses than the control. Organoleptically, cheese made from cow's milk was found to be superior to cheeses produced from reconstituted either non fat or whole dried milk, as regards flavour, body and texture and the distribution of P. requeforti.     

CHANGES IN EQUILIBRIUM MODULUS AND αsl-CASEIN BREAKDOWN DURING THE RIPENING OF PORT SALUT ARGENTINO CHEESE AS AFFECTED BY FROZEN STORAGE

The effect of the freezing, frozen storage and thawing on textural parameters and α_sl-casein breakdown during the ripening of Port Salut Argentino cheese was studied. Moisture content, salt concentration, casein profiles and asymptotic equilibrium modulus were monitored in control cheeses ripened at 5C and in cheeses, stored at -22C for 30 days, thawed and ripened at 5C, for different ripening times (1, 6, 13, 27 and 56 days) and two sampling zones (central and external). The freezing process significantly increased the rate of α_sl-casein and α_sl-I-casein hydrolysis. This process may affect the susceptibility of α_sl-casein to chymosin attack and also the availability of hydrolytic enzymes released by damaged microorganisms, which may contribute to the faster hydrolysis of α_sl-I-casein. The freezing process did not significantly affect the decay rates of asymptotic equilibrium modulus. First order kinetics constants for decay of the asymptotic equilibrium modulus were 3.71 10^-2day^-1 (control cheeses, central zone), 8.48 10^-2 day^-1 (control cheeses, external zone), 4.52 10^-2 day^-1 (frozen cheeses, central zone), and 11.43 10^-2 day^-1 (frozen cheeses, external zone). Significant differences in the decay rates of asymptotic equilibrium modulus were found between central and external zones in control and frozen cheeses primarily due to differences in moisture contents of the sampling zones.     

Effects of coagulant type on the physicochemical and organoleptic properties of Kashar cheese

Kashar cheeses were manufactured using different coagulants (calf rennet, chymosin derived by fermentation and proteases from Rhizomucor miehei and Cryphonectria parasitica) and ripened for 90 days. Use of different coagulants did not influence the dry matter, fat, protein, salt, pH, titratable acidity, total free fatty acids and texture profile analyses. The levels of water‐soluble nitrogen, 12% trichloroacetic acid‐soluble nitrogen, and for 5% phosphotungstic acid‐soluble nitrogen, the sensory properties were significantly influenced by the use of different coagulants. β‐casein was more hydrolysed in the cheese manufactured using protease from Cryphonectria parasitica than the other cheeses during 90 d of ripening.     

Proteolysis and microstructure of Piacentinu Ennese cheese made using different farm technologies

The aim of this study was to provide the biochemical and structural characterization of Piacentinu Ennese cheese and to evaluate the impact of different farm technologies on cheese proteolysis and microstructure. Fifteen cheeses were manufactured according to traditional technology, i.e., from raw milk and farmhouse rennet in the absence of starter culture. Pasteurized milk, commercial rennet, and starter were used for production of 20 nontraditional cheeses. Proteolysis in Piacentinu Ennese cheese was monitored during a 2- to 10-mo ripening time. Low rates of overall proteolysis were observed in cheese, as percentages of total N soluble at pH 4.6 and in 12% trichloroacetic acid were about 11.40 and 8.10%, respectively, after 10 mo of age. Patterns of primary proteolysis by urea-PAGE showed that alpha(s)-caseins were degraded to a larger extent than were beta-caseins, although a considerable amount of both caseins was still intact after 10 mo. Reversed phase-HPLC analysis of the cheese peptide fractions showed a slow decrease in the levels of hydrophobic peptides coupled to increasing levels of hydrophilic compounds as the cheese aged. The structural characteristics of Piacentinu Ennese cheese were evaluated by scanning electron microscopy after 2, 4, and 6 mo of age. The micrographs showed a sponge-like structural network with a well-distributed system of empty spaces, originally occupied by whey and fat. The microstructure changed during cheese ripening to become more compact with cavities of smaller size. Farm technology significantly affected cheese proteolysis and microstructure. Nontraditional cheeses had higher levels of pH 4.6-soluble N and showed a larger hydrolysis of alpha(s)-casein fractions by urea-PAGE analysis than did traditional cheeses. Large differences between cheese-types also concerned the patterns of secondary proteolysis. Nontraditional cheeses had higher levels of 12% trichloroacetic acid-soluble N and showed larger proportions of free amino acids and hydrophilic peptides in the HPLC profiles of the corresponding 70% ethanol-soluble N fraction than traditional cheeses. Nontraditional cheeses also had a more open structure with a coarser and less continuous appearance than did traditional cheeses. A large amount of variability in cheese proteolysis and structure within nontraditional treatment reflected farm-dependent changes in manufacturing conditions related to the use of various types of rennet and starter.     

Accelerated ripening of low fat Ras cheese by attenuated lactobacilli cells

Thirteen Ras cheese were made from 4% fat raw milk; 3% raw and heat treated; 2% raw and heat treated milks in order to study the effect of freeze-shocked or heat-shocked L. casei NIH 334 or L. helveticus CNRZ 53 on the quality of the resultant cheeses. The soluble nitrogen, soluble tyrosine, soluble tryptophan, total volatile fatty acids, titratable acidity and organoleptic evaluation scores increased as ripening period progressed, while moisture decreased. Neither strain nor the heated lactobacilli had significant effects on moisture content of cheeses, while increasing their acidity. Cheeses with freeze-shocked L. casei or L. helveticus had higher titratable acidity than cheeses in which heat-shocked cells were added. However, cheeses added L. helveticus had higher acidity than those with L. casei. Ripening indices (soluble nitrogen, soluble tyrosine, soluble tryptophan and total volatile fatty acids) and organoleptic evaluation scores had similar trends. Cheeses with attenuated lactobacilli had higher ripening indices and cheese scores than cheeses without lactobacilli. Addition of either freeze-shocked L. casei or L. helveticus yielded cheeses having higher ripening indices and organoleptic scores than cheeses made with heat-shocked lactobacilli. The best cheeses were made from 3% fat milk heated to 70 °C, and containing freeze-shocked L. helveticus followed by cheeses made from 2% fat milk heated to 75 °C and containing freeze-shocked L. helveticus.     

Effects of pectic fat mimetics and transglutaminase on the regularity of protein and fat degradation and flavour compounds in Cheddar cheese during ripening

The effect of simultaneous transglutaminase (TGase) treatment with pectic fat mimetics (PFM) addition on regularity of protein and fat degradation and flavour compound variations in Cheddar cheese during ripening was investigated. In the early stages of fermentation, the cheese with 20% PFM and 15 U/L TGase had a higher amino acid content. In the middle stage of fermentation, cheese with high concentration of TGase hydrolysed more proteins. Cheese supplemented with PFM and TGase showed increased content of long-chain polyunsaturated fatty acids, especially linoleic acid (C18:2). In addition, PFM and TGase compensated the flavour deficiencies of low-fat cheeses and had positive effects on volatile compounds such as alcohols, acids and methyl ketones. Overall, the characteristics and flavours of Cheddar cheese with the addition of PFM and TGase were superior to the control group, which could provide a theoretical basis for the application of PFM and TGase in cheese production.     

The effect of fat content on the microbiology and proteolysis in cheddar cheese during ripening dairy foods

We investigated the effect of incremental reduction in fat content, in the range 33 to 6% (wt/wt), on changes in the microbiology and proteolysis of Cheddar cheese, over a 225-d ripening period at 7 degrees C. A reduction of fat content resulted in significant increases in contents of moisture and protein and a decrease in the concentration of moisture in nonfat substance. Reduced fat had little effect on the age-related changes in the population of starter cells. The populations of nonstarter lactic acid bacteria decreased with fat content, and counts in the low fat cheese (6% wt/wt) were significantly lower than those in the full fat cheese (33% wt/wt) at ripening times >1 and <180 d. Proteolysis as measured by the percentage of total N soluble at pH 4.6 or in 70% ethanol decreased significantly as the fat content decreased. However, the content of pH 4.6 soluble N per 100 g of cheese was not significantly influenced by fat content. At ripening times >60 d, the content of 70% ethanol soluble N per 100 g of full fat (33% wt/wt) cheese was significantly lower than that in either the half fat (17% wt/wt) or low fat (6% wt/wt) cheeses. The concentration of AA N, as a percentage of total N, was not significantly affected by fat content. However, when expressed as a percentage of total cheese, amino acid N increased significantly with decreasing fat content. Analysis of pH 4.6 soluble N extracts by reverse phase- and gel permeation HPLC revealed that fat content affected the pattern of proteolysis, as reflected by the differences in peptide profiles.     

The effect of varying casein/fat ratio on physicochemical and sensory qualities of Feta‐type cheese made using buffalo milk

The Feta‐type cheese was prepared with different casein/fat (C/F) ratios of buffalo milk using microbial rennet. The manufactured Feta cheeses were subjected to physicochemical and sensory quality at 15‐day interval up to 60 days of ripening. Sensory analysis discriminated the different level of C/F ratio of buffalo milk cheeses predominantly by age. There was no significant difference (P < 0.01) observed in cheese made from C/F ratio of 0.6–0.7 in terms of flavour. The titratable acidity (TA), soluble protein and free fatty acid appear to be age‐dependent and increased throughout the ripening in all experimental cheeses.     

Proteomic study of proteolysis during ripening of Cheddar cheese made from milk over a lactation cycle

Milk for cheese production in Ireland is predominantly produced by pasture-fed spring-calving herds. Consequently, there are marked seasonal changes in milk composition, which arise from the interactive lactational, dietary and environmental factors. In this study, Cheddar cheese was manufactured on a laboratory scale from milk taken from a spring calving herd, over a 9-month lactation cycle between early April and early December. Plasmin activity of 6-months-old Cheddar cheese samples generally decreased over ripening time. One-dimensional urea-polyacrylamide gel electrophoresis (PAGE) of cheese samples taken after 6 months of ripening showed an extensive hydrolysis of caseins, with the fastest hydrolysis of α(s1)-caseins in cheeses made in August. A proteomic comparison between cheeses produced from milk taken in April, August and December showed a reduction in levels of β-casein and appearance of additional products, corresponding to low molecular weight hydrolysis products of the caseins. This study has demonstrated that a seasonal milk supply causes compositional differences in Cheddar cheese, and that proteomic tools are helpful in understanding the impact of those differences.     

Accelerated ripening of Ras cheese using cell free extract,freeze- and heat-shocked Leuconostoc spp. cultures

Ras cheese was manufactured with cell free extract, freeze- and heat-shocked cultures of Leuconostoc spp. to enhance flavour development and accelerated ripening. Cheese analysis (pH, acidity, dry matter, fat, protein, soluble nitrogen, volatile acidity, free fatty acids) were carried out for 3 months. No significant differents were observed between the experiment cheeses and control, however, the cheese flavour was enhanced in cheeses with addition of heat treated Leuconostoc cells. Bitter taste was decreased in all experimental cheeses.     

Influence of Australian Native Herbs on the Maturation of Vacuum-packed Cheese

The composition, microbiology and biochemistry of semi-hard cheeses flavoured with native mint, lemon myrtle and bush tomato (BT) were compared with unflavoured (control) cheese during a 90-day period of maturation. Moisture, protein and salt levels of all cheeses were similar and did not change during maturation. However, the fat content of control cheese was significantly higher than that of the flavoured cheeses while the pH of cheese flavoured with BT was consistently lower throughout maturation. Total viable organisms, Lactobacillus and Lactococcus counts were between 10⁶ and 10⁷ colony forming units (cfu)/g cheese for all cheeses. Yeast and mould count was <10² cfu/g cheese throughout the maturation of all cheeses except in the cheese flavoured with BT which was >10³ cfu/g cheese. Biochemical indices of proteolysis and lipolysis increased with the extent of maturation in all cheeses but were most pronounced in the BT-flavoured cheese. The capillary electrophoretic profile of this cheese also indicated a more extensive hydrolysis of both α s- and β -caseins. The microbiological quality of BT appeared to have exerted a very significant influence on cheese properties.     

Biochemical changes in Picón Bejes-Tresviso cheese,a Spanish blue-veined variety,during ripening

The changes in the gross chemical composition, physico-chemical parameters, nitrogen fractions, caseins and their degradation products, and some fat characteristics were studied during the ripening process of 10 batches of Picón Bejes-Tresviso cheese, a traditional blue-veined variety made in the north of Spain. The values of the different compositional and physico-chemical parameters at the end of ripening did not differ very much from those found in other Spanish and European blue-veined cheeses. The total soluble nitrogen and the non-protein nitrogen increased by factors of 5.4 and 8, respectively, at the end of ripening compared to the values found in cheese curd after salting. The final values of all the nitrogen fractions showed that Picón Bejes-Tresviso cheese undergoes extensive and in depth proteolysis. The intense degradation of the caseins during ripening was confirmed when the caseins and their degradation products were quantified using PAGE techniques. The autooxidation of the fat does not seem very important during the ripening of this cheese. Nevertheless, lipolysis was very intense; the acidity index of the fat values (free fatty acid contents) increased by a factor of about 20 during ripening.