Specific effect of high-pressure treatment of milk on cheese proteolysis

The extent of primary and secondary proteolysis of cheeses made from raw (RA), pasteurized (PA, 72 degrees C, 15 s) or pressure-treated (PR, 500 MPa, 15 min, 20 degrees C) goats' milk was assessed. Modifications in cheese-making technology were introduced to obtain cheeses with the same moisture content, and thus studied per se the effect of milk treatment on cheese proteolysis.The PR milk cheese samples were differentiated from RA and PA milk cheeses by their elevated beta-lg content, and by the faster degradation of alphas1-, alphas2- and beta-CN throughout ripening. Non-significant differences were found in either pH 4.6 soluble-nitrogen or trichloracetic acid soluble-nitrogen contents of cheeses. However, the pasteurization of milk decreased the free amino acid production in cheese. The RA milk cheeses had the highest amount of proline and the lowest concentrations of serine, tyrosine, arginine and alpha-aminobutyric acid, whereas PR milk cheese showed higher levels of arginine.     

Changes in textural, microstructural, and colour characteristics during ripening of cheeses made from raw, pasteurized or high-pressure-treated goats’ milk

Goats’ milk cheeses were made from raw (RA), pasteurized (PA; 72°C, 15 s) or pressure-treated (PR; 500 MPa, 15 min, 20°C) milk to compare textural, microstructural, and colour characteristics in relation to ripening time. Texture, microstructure and colour were evaluated by uniaxial compression and stress relaxation tests, confocal laser scanning microscopy and Hunter colorimetry, respectively.Raw and PR cheeses were firmer and less fracturable than PA cheese, but differences became less notable toward the end of ripening. PA and PR cheeses were less cohesive than RA cheese. Although cheeses exhibited a loss of elastic characteristics with ageing, PR cheese showed the most elastic behaviour initially. Confocal laser scanning micrographs displayed PR cheese with a regular and compact protein matrix, with small and uniform fat globules resembling the structure of RA cheese. Finally, colour evaluation demonstrated significant differences between cheeses due to milk treatments and ripening time.     

Microbiological changes throughout ripening of goat cheese made from raw,pasteurized and high-pressure-treated milk

The bacteriological quality during ripening of raw (RA), pasteurized (PA; 72°C, 15 s) and pressure-treated (PR; 500 MPa, 20°C, 15 min) goat milk assessed by enumeration of total bacteria, psychrotrophic bacteria, Enterobacteriaceae, lactobacilli, enterococci, Micrococcaceae and lactococci was evaluated. The high pressure treatment applied was as efficient as pasteurization in reducing the bacterial population of milk. Experimental cheeses were made from RA, PA and PR milks to study the microbial population during ripening. Lactobacilli and lactococci were the predominant microbiota present during ripening in all the cheeses. There were no differences in numbers of starter bacteria during ripening. However, lactobacilli counts for RA milk cheese were significantly higher than for PA and PR cheeses in all the ripening stages studied. Micrococcaceae and enterococci remained at a secondary level, and no differences were observed between cheeses at the end of ripening. On the other hand, the number of Enterobacteriaceae decreased during ripening, but faster in PR milk cheese than in PA and RA milk cheeses. The results of this study suggest that goat cheese made from PR milk had similar microbiological characteristics to PA milk cheeses.     

Changes in organic acids during ripening of cheeses made from raw, pasteurized or high-pressure-treated goats’ milk

Organic acids of cheeses made from raw (RA), pasteurized (PA; 72°C, 15 s) or pressure-treated (PR; 500 MPa, 15 min, 20°C) goats’ milk were qualitatively and quantitatively assessed during ripening. Nine organic acids (citric, pyruvic, malic, lactic, formic, acetic, uric, propionic and butyric) were analysed in each sample by HPLC.Milk treatment did not affect the total organic acids content of 1-day-old cheeses, which increased steadily from day 1 to day 60. At the end of ripening, RA and PR milk cheeses both exhibited higher concentration of organic acids than in those made from PA milk.Lactic acid was found in higher concentration in PR milk cheese from 30 days of ripening. The RA milk cheese, that showed the highest nonstarter lactic acid bacteria counts, were characterized by an elevated amount of propionic and acetic acids. These cheeses also were negatively correlated with both pyruvic and citric acid contents. The PA milk cheese showed a high level of malic acid, and was clearly differentiate from RA and PR milk cheeses by its low level of butyric acid.     

Effects of genetic type,stage of lactation,and ripening time on Caciocavallo cheese proteolysis

The objective of this experiment was to evaluate the effects of genetic type, stage of lactation, and ripening time on proteolysis in Caciocavallo cheese. One hundred twenty Caciocavallo cheeses made from the milk of 2 breeds, Italian Brown and Italian Holstein and characterized by different stages of lactation were obtained and ripened for 1, 30, 60, 90, and 150 d. Cheese proteolysis was investigated by ripening index (ratio of water-soluble N at pH 4.6 to total protein, %) and by the study of degradation of the protein fractions (α_S1-, β-, and para-κ-casein), which was determined by densitometric analysis of isoelectric focusing results. The statistical analysis showed a significant effect of the studied factors. Ripening index was higher in Italian Brown Caciocavallo cheese and in cheeses made with early lactation milk, whereas casein solubilization was greater in the first 2 mo of ripening. Isoelectric focusing analysis of cheese samples during ripening showed extensive hydrolysis of caseins. In particular, the protein fraction that underwent major degradation by proteolytic enzymes was α_S1-casein, followed by β-casein, whereas para-κ-casein was less degraded. Italian Brown cheese showed a lower residual quantity of β- and para-κ-casein, whereas Italian Holstein cheese showed a lower residual quantity of α_S1-casein. In addition, significant interactions of both first and second order were found on both ripening index and degradation of protein fractions. This study demonstrated that the analyzed factors influenced proteolysis of Caciocavallo cheese, which forms the basis of new knowledge that could lead to the production of a pasta filata cheese with specific characteristics.     

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.     

Lipolysis in Urfa cheese produced from raw and pasteurized goats’ and cows’ milk with mesophilic or thermophilic cultures during ripening

Lipolysis was evaluated in Urfa cheese made from raw and pasteurized goats’ and cows’ milk with mesophilic or thermophilic cultures. The acid degree values (ADVs) of the cows’ milk cheeses were significantly (P < 0.05) higher until 60 d of storage than that of cheese made from goats’ milk. Total free fatty acid (FFA) contents of goats’ milk cheese were significantly (P < 0.001) lower than that of cows’ milk cheese throughout ripening, whereas goats’ milk cheese flavour was higher (P < 0.05) than cows’ milk cheese. Pasteurization of milk prior to cheese-making has a negative influence, not only on the level of lipolysis throughout ripening, but also on the relative amounts of short chain FFAs and sensory properties of the cheeses (P < 0.001). Cheese produced without starter bacteria underwent significantly (P < 0.05) higher lipolysis than cheeses produced with mesophilic or thermophilic starter bacteria, while cheese made with thermophilic starter culture had similar flavour to cheese made without starter culture.     

Study of the chemical composition,proteolysis, volatile compounds,and textural properties of industrial and traditional Beaten (Bieno sirenje) ewe milk cheese

The objective of this study was to determine the gross composition, proteolysis, and volatile and texture profiles during ripening of industrial (IND) and traditional (TRD) Beaten (Bieno sirenje) cheeses made by using ewe milk. In the course of the analyses, statistical differences were determined in some physicochemical parameters, nitrogen fractions, and total free amino acid levels between TRD and IND types of cheese. Higher levels of proteolysis were observed in IND cheeses than in TRD cheeses during ripening. Levels of residual β- and α_s-caseins were 72.2 and 48.7%, respectively, in 180-d-old TRD cheeses. However, the residual levels were 52.8% for β-casein and 18% for α_s-casein in IND cheeses. Similar differences were noted for the reversed-phase HPLC peptide profiles of 2 types of cheeses. Also, higher concentrations of peptides were eluted in IND cheeses than in TRD cheeses during ripening. A total of 73 volatile compounds, including alcohols (16), esters (17), acids (14), terpenes (7), ketones (5), aldehydes (4), and miscellaneous (10) were identified. The IND cheeses contained higher levels of carboxylic acids, esters, alcohols, and terpenes than the TRD cheeses; however, the same levels of methyl ketones were determined in the 2 types of cheeses at the end of ripening. These may be due to some differences (e.g., pasteurization and scalding temperature, among other factors) in the manufacture of the 2 types of Beaten cheeses. The textural profile of Beaten cheeses showed that TRD production method resulted in firmer, less fracturable, and stiffer cheeses than the IND production method. In conclusion, the results suggest that the use of industrial production method (pasteurization of cheese milk and curd scalding at 70°C) in the manufacture of Beaten ewe milk cheese enriched the volatile profile of the cheese.     

Effect of β-casein concentration in cheese milk on rennet coagulation properties,cheese composition and cheese ripening

Effects of the use of a β-casein powder to enrich cheese milk on rennet coagulation properties of milk, cheese composition and cheese ripening were investigated. Casein content of control milk was 2.5%, whereas that for the three enriched milks was adjusted with β-casein powder at 2.7%, 2.9% and 3.1%. The β-casein to α-casein ratio of these cheese milks was, respectively, 0.70, 0.79, 0.89 and 0.99. Rennet coagulation properties were related not only to casein concentration but also to the proportion of β-casein and α_s-casein presents in milks. Milk with higher concentration of β-casein had poorer coagulation properties. Cheeses could be produced by using a miniature cheese making process. Moisture, ash and calcium contents decreased, while protein content and β-casein increased in cheese as casein and β-casein concentration increased in milk. As a result, hardness was higher in enriched cheeses than in control cheese. During cheese ripening, α-casein was hydrolyzed, but the rate of degradation of α-casein decreased as protein and β-casein concentration increased in cheese. β-Casein seemed to be not hydrolyzed. The rate of decrease of hardness was also slower for enriched cheeses.     

Residual clotting activity and ripening properties of vegetable rennet from Cynara cardunculus in La Serena cheese

Vegetable rennet extracted from Cynara cardunculus flowers is traditionally used in the manufacture of La Serena cheese. High levels of proteolytic enzymes of the flowers are responsible for its clotting activity and strong proteolytic action. The presence of residual coagulant in cheese and whey was measured by adding known amounts of vegetable rennet as internal standard. We found no differences between the residual coagulant activity of La Serena cheese compared with other types of cheese. The coagulant content detected at the end of four cheesemakings (vat of 830 l) in cheese and whey represented 27 and 78%, respectively, of the total amount added to milk. When measurements were carried out in 16 different cheeses, vegetable rennet appeared to be highly stable during cheese ripening. Cheese composition (moisture, pH, NaCl, fat and protein) was kept relatively constant during ripening, which seems to contribute to stability of residual activity. Electrophoretic analyses of water insoluble fractions from cheeses manufactured with vegetable rennet showed that α_s-casein was less susceptible to proteolysis than β-casein. The water soluble nitrogen/total nitrogen (WSN/TN) exhibited higher levels only during the first 30 days of ripening although non-protein nitrogen/total nitrogen (NPN/TN) ratio and amino acid nitrogen (NH₂-N) increased with ripening time.     

Ripening of Cheddar cheese made from blends of raw and pasteurised milk

Cheddar cheeses were made from pasteurised milk (P), raw milk (R) or pasteurised milk to which 10 (PR10), 5 (PR5) or 1 (PR1) % of raw milk had been added. Non-starter lactic acid bacteria (NSLAB) were not detectable in P cheese in the first month of ripening, at which stage PR1, PR5, PR10 and R cheeses had 10⁴, 10⁵, 10⁶ and 10⁷ cfu NSLAB g⁻¹, respectively. After ripening for 4 months, the number of NSLAB was 1–2 log cycles lower in P cheese than in all other cheeses. Urea–polyacrylamide gel electrophoretograms of water-soluble and insoluble fractions of cheeses and reverse-phase HPLC chromatograms of 70% (v/v) ethanol-soluble as well as -insoluble fractions of WSF were essentially similar in all cheeses. The concentration of amino acids were pro rata the number of NSLAB and were the highest in R cheese and the lowest in P cheese throughout ripening. Free fatty acids and most of the fatty acid esters in 4-month old cheeses were higher in PR1, PR5, PR10 and R cheeses than in P cheese. Commercial graders awarded the highest flavour scores to 4-month-old PR1 cheeses and the lowest to P or R cheese. An expert panel of sensory assessors awarded increasingly higher scores for fruity/sweet and pungent aroma as the level of raw milk increased. The trend for aroma intensity and perceived maturity was R>PR10>PP5>PR1>P. The NSLAB from raw milk appeared to influence the ripening and quality of Cheddar cheese.     

Proteolysis and biogenic amine buildup in high-pressure treated ovine milk blue-veined cheese

Penicillium roqueforti plays an important role in the ripening of blue-veined cheeses, mostly due to lactic acid consumption and to its extracellular enzymes. The strong activity of P. roqueforti proteinases may bring about cheese over-ripening. Also, free amino acids at high concentrations serve as substrates for biogenic amine formation. Both facts result in shorter product shelf-life. To prevent over-ripening and buildup of biogenic amines, blue-veined cheeses made from pasteurized ovine milk were high-pressure treated at 400 or 600 MPa after 3, 6, or 9 wk of ripening. Primary and secondary proteolysis, biogenic amines, and sensory characteristics of pressurized and control cheeses were monitored for a 90-d ripening period, followed by a 270-d refrigerated storage period. On d 90, treatments at 400 MPa had lowered counts of lactic acid bacteria and P. roqueforti by less than 2 log units, whereas treatments at 600 MPa had reduced lactic acid bacteria counts by more than 4 log units and P. roqueforti counts by more than 6 log units. No residual α-casein (CN) or κ-CN were detected in control cheese on d 90. Concentrations of β-CN, para-κ-CN, and γ-CN were generally higher in 600 MPa cheeses than in the rest. From d 90 onwards, hydrophilic peptides were at similar levels in pressurized and control cheeses, but hydrophobic peptides and the hydrophobic-to-hydrophilic peptide ratio were at higher levels in pressurized cheeses than in control cheese. Aminopeptidase activity, overall proteolysis, and free amino acid contents were generally higher in control cheese than in pressurized cheeses, particularly if treated at 600 MPa. Tyramine concentration was lower in pressurized cheeses, but tryptamine, phenylethylamine, and putrescine contents were higher in some of the pressurized cheeses than in control cheese. Differences in sensory characteristics between pressurized and control cheeses were generally negligible, with the only exception of treatment at high pressure level (600 MPa) at an early ripening stage (3 wk), which affected biochemical changes and sensory characteristics.     

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

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<0.05) increases in pH, moisture and protein contents and decreases in the concentration of moisture in the non-fat substance. The increase in pH as the fat content increased was attributed to the concomitant decrease in the lactate-to-protein ratio. Polyacrylamide gel electrophoresis showed that the concentration of intact casein decreased in all cheeses during ripening and that the rate of decrease was not affected by the fat content. However, for a given concentration of casein, 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.     

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.     

Evolution of lipolysis during the ripening of traditional Feta cheese

Lipolysis was studied during ripening of traditional Feta cheese produced in two small dairies, A and B. The cheeses were made from a thermized mixture of ewes’/goats’ milk by using yoghurt as starter and artisanal rennet from lambs’ and kids’ abomasa (cheese A) or mixed artisanal rennet with calf rennet (cheese B).The acid degree value and the free fatty acids (FFA) contents in both cheeses increased sharply up to 18 d (pre-ripening period at 15 °C) and continued to increase throughout ripening. In both mature cheeses, acetic acid was found at high levels (13–18% of the total FFAs). However, except for this, all FFA contents differed significantly (P < 0.05) between the two cheeses throughout ripening. The levels of individual and total C2:0–C8:0, C10:0–C14:0 and C16:0–C18:2 fatty acids were significantly higher (P < 0.05) in cheese A than in cheese B. Presumably the difference, especially in the C2:0–C8:0 content, was due mainly to the type of the rennet used. Butyric acid was the dominant FFA in cheese A (20% of the total FFAs at 120 d), while the most abundant FFAs in cheese B were capric (18%) and lauric acid (18%). In general, the lipolysis degree of the two cheeses was higher than those reported for the industrially-made Feta cheese.In organoleptic evaluation, cheese A had a piquant taste that was attributed to its high content of butyric acid and showed a significantly (P < 0.05) higher total score than cheese B.     

Effects of high pressure on proteolytic enzymes in cheese: relationship with the proteolysis of ewe milk cheese

Ewe milk cheeses were submitted to 200, 300, 400, and 500 MPa (2P to 5P) at 2 stages of ripening (after 1 and 15 d of manufacturing; P1 and P15). The high-pressure-treated cheeses showed a more important hydrolysis of β-casein than control and 2P1 cheeses. Degradation of α_s1-casein was more important in 3P1, 4P1, and P15 cheeses than control and 2P1 cheeses. The 5P1 cheeses exhibited the lowest degradation of α_s-caseins, probably as a consequence of the inactivation of residual chymosin. Treatment at 300 MPa applied on the first day of ripening increased the peptidolytic activity, accelerating the secondary proteolysis of cheeses. The 3P1 cheeses had extensive peptide degradation and the highest content of free amino acids. Treatments at 500 MPa, however, decelerated the proteolysis of cheeses due to a reduction of microbial population and inactivation of enzymes.     

Evolution of chemico-physical characteristics during manufacture and ripening of Castelmagno PDO cheese in wintertime

Biochemical, volatile and textural profiles during manufacture and ripening were determined in samples of Castelmagno PDO cheese obtained from three different batches in the main artisan cheese plant of Castelmagno PDO production area. At the end of manufacture, samples were characterised by a pH of 6.57% and 52.4% moisture content. The HPLC analysis of organic acids and sugars showed the exhaustion of lactose content, while Urea-PAGE indicated extensive primary proteolysis of both β-casein and α_s1-casein. During ripening, cheeses were characterised by high degradation of β-casein and α_s1-casein, due to bacterial action. RP-HPLC profiles showed a high production of peptides eluted between 20 and 30 min. In total, 92 volatile compounds were identified in cheese headspace. Texture profiles showed an increase in hardness, gumminess, chewiness and adhesiveness values, as well as a decrease in cohesiveness during ripening.     

Microbiological and biochemical characteristics of Kashkaval cheese produced using pasteurised or raw milk

Microbiological quality and biochemical changes of Kashkaval cheese manufactured using sheep's raw milk without starter addition or pasteurised milk with an added commercial starter were studied. Mature cheeses had pH values 5.0–5.3, salt content 2.1–2.7%, protein content 23.3–25.1%, moisture content 36.8–39.5%, fat content 28.0–32.2%, and ash content around 5.0%. In raw milk cheeses, mesophilic non-starter lactobacilli prevailed followed by enterococci. In pasteurised milk cheeses Lactococcus lactis starter prevailed. All cheeses were safe according to the criteria in Regulation (EC) 1441/2007. The proteolysis index was around 20%. Butyric, myristic, palmitic, stearic and oleic were the principal free fatty acids in both cheeses. Ketones were abundant in pasteurised milk cheeses and esters in mature raw milk cheeses. Pasteurisation did not affect (P > 0.05) the physicochemical composition and the proteolysis of cheeses. Raw milk cheeses showed higher levels (P < 0.05) of lipolysis than pasteurised milk cheeses.     

Proteolysis during the ripening of goats’ milk cheese made with plant coagulant or calf rennet

Powdered plant coagulant (PPC) obtained from the cardoon (Cynara cardunculus) was compared with calf rennet (CR) for the manufacture of goats’ milk cheese, by determining difference in the proteolysis throughout ripening. There were no substantial differences between the compositions of cheeses made using the two types of coagulants. However, cheeses manufactured with PPC exhibited higher levels of pH 4.6-SN than cheese made using CR. The extent of breakdown of α_s-casein, as measured by urea-PAGE, was greater in cheese made using PPC than cheese made using CR. The formation of hydrophobic peptides and the ratio of hydrophobic/hydrophilic peptides throughout the ripening were higher in cheeses made with PPC than in cheeses made with CR. Principal component analysis (PCA) of peak heights of RP-HPLC peptide profiles of the ethanol-soluble and ethanol-insoluble fractions distributed the samples according to the coagulant used in their manufacture. Quantitative differences in several peptides were evident between the two types of cheese.