Proteolytic and lipolytic changes during the ripening of a Spanish craft goat cheese (Armada variety)

The proteolytic and lipolytic changes during the ripening process were investigated in four batches of Armada goat's milk cheese (an artisanal variety produced in the North of Spain), by determining the classical nitrogen fractions, caseins and their degradation products, free amino acids, as well as the acidity of the fat, thiobarbituric acid (TBA) number and free fatty acids. Values obtained for the nitrogen fractions and for caseins and their degradation products show that this cheese undergoes very little protein degradation. A low free amino acids content was observed throughout the ripening process with a predominance of Pro followed by Leu+Ile, Glu acid, Phe, His+Lys and Val. The lipid degrada-tion was very intense from the second month of ripening, only comparable to that reported for cheeses ripened by moulds. The average free fatty acids content increased 20-fold during ripening, reaching final values of 44·5 g kg⁻¹. All the free fatty acids increased considerably during ripening, resulting in a predominance of saturated and unsaturated long-chain acids, followed by medium-chain acids, C₁₀ principally. Short-chain fatty acid content by the end of ripening was higher than that presented in other cheese varieties with a similar high degree of lipolysis. © 1997 SCI.     

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.     

不同包装方式对半硬质干酪成熟期间蛋白质降解的影响

对真空包装和涂蜡包装的半硬质干酪成熟过程中蛋白降解进行了研究。结果表明:2种包装的干酪在成熟过程中pH 4.6SN含量和12%TCA-SN含量都随着时间的延长逐渐增大,且2组数据之间差异显著(P<0.05);2种包装的干酪中游离氨基酸总量随成熟时间的延长而逐渐增加,各种氨基酸含量变化的显著性不同;SDS-PAGE电泳图谱显示2种干酪在成熟期内蛋白质都发生了明显的降解,且涂蜡包装的干酪蛋白降解程度较真空包装的深,在成熟45 d后较为明显。     

Comprehensive analysis of proteolysis during 8 months of ripening of high-cooked Old Saare cheese

We applied capillary electrophoresis, liquid chromatography coupled with tandem mass-spectrometry (MS/MS), and ultra-performance liquid chromatography to determine the composition of water-insoluble and water-soluble proteinaceous fractions of the cheese and to study in detail the degradation of caseins during 8 mo of ripening of Estonian high-temperature cooked hard cheese Old Saare. The application of high-resolution and high-accuracy MS/MS enabled identification of more than 3,000 small peptides, representing a fairly full casein peptidome containing peptides of 4 to 25 AA in length: 1,049 from β-casein (CN), 944 from α_S1-CN, 813 from α_S2-CN, and 234 from κ-CN. The majority of β-CN- and α_S1-CN-derived peptides originated from the N-terminal parts of the molecule, f6-93 and f1-124, respectively; peptides from α_S2-CN arose predominantly from the C-terminal end f100-162. At the beginning of ripening, we found a relatively high amount of peptides originating from the glycomacropeptide part of κ-CN, whereas peptides from para-κ-CN prevailed during the later stages of ripening of the cheese. The cleavage patterns of β-CN, α_S2-CN, as well as α_S1-CN, showed that primary proteolysis was started mainly by plasmin, although a low proteolytic activity of chymosin was also evident. Based on the analysis of cleavage sites, we observed a significant participation of proteolytic enzymes, including amino- and carboxypeptidases, of both mesophilic and thermophilic starter bacteria in further hydrolysis of oligopeptides during the ripening. Several new phosphopeptides were detected in the result of MS/MS data analysis. The profiles of the estimated concentrations of phosphopeptides revealed that those originating from β-CN and α_S1-CN accumulated during cheese maturation. In contrast, we did not notice any generation of phosphopeptides from the highly phosphorylated part of α_S2-CN, f25-80, presumably due to the inaccessibility of this region to the action of plasmin and chymosin. The analysis of cleavage sites and the combination of principal component and clustering analyses provided a characterization of the complex dynamics of formation and degradation of peptides during cheese maturation. We made an attempt to obtain a comprehensive picture of proteolysis during Old Saare cheese ripening on the basis of the detailed peptidomic data, including also the less abundant peptides determined by MS/MS, and complemented by the data on intact caseins and free AA and reported the results in the paper.     

Hard ewe's milk cheese manufactured from milk of three different groups of somatic cell counts

As ovine milk production increases in the United States, somatic cell count (SCC) is increasingly used in routine ovine milk testing procedures as an indicator of flock health. Ovine milk was collected from 72 East Friesian-crossbred ewes that were machine milked twice daily. The milk was segregated and categorized into three different SCC groups: < 100,000 (group I); 100,000 to 1,000,000 (group II); and > 1,000,000 cells/ ml (group III). Milk was stored frozen at -19 degrees C for 4 mo. Milk was then thawed at 7 degrees C over a 3-d period before pasteurization and cheese making. Casein (CN) content and CN-to-true protein ratio decreased with increasing SCC group 3.99, 3.97, to 3.72% CN, and 81.43, 79.72, and 79.32% CN to true protein ratio, respectively. Milk fat varied from 5.49, 5.67, and 4.86% in groups I, II, and III, respectively. Hard ewe's milk cheese was made from each of the three different SCC groups using a Manchego cheese manufacturing protocol. As the level of SCC increased, the time required for visual flocculation increased, and it took longer to reach the desired firmness for cutting the coagulum. The fat and moisture contents were lower in the highest SCC cheeses. After 3 mo, total free fatty acids (FFA) contents were significantly higher in the highest SCC cheeses. Butyric and caprylic acids levels were significantly higher in group III cheeses at all stages of ripening. Cheese graders noted rancid or lipase flavor in the highest SCC level cheeses at each of the sampling points, and they also deducted points for more body and textural defects in these cheeses at 6 and 9 mo.     

提高成熟温度加快Mozzarella干酪成熟的研究

制作2批Mozzarella干酪A和B，分别在4℃(A)和7℃(B)下成熟，观察其在成熟期间的变化及测定可溶性N的含量等指标，可知在7℃下成熟的干酪在制作后30d的蛋白水解性、功能特性等和4℃下成熟50d的干酪无显著差异，而和7℃下成熟50d的干酪有显著差异。说明成熟温度显著影响干酪的蛋白水解性。在7℃下贮藏的Mozzarella干酪成熟30d可达到4℃下贮存50d的成熟度，即将成熟温度从4℃提高到7℃，可将成熟期缩短20d左右。     

Protein profiles and total antioxidant capacity of water‐soluble and water‐insoluble fractions of white brined goat cheese at different stages of ripening

This study deals with proteolysis and total antioxidant capacity of proteins of white brined cheese prepared from overheated goat milk and ripened for fifty days. Proteolytic changes were reflected through the relatively low level of soluble nitrogen (50 days ripened cheese had 15.32 g/100 g of water‐soluble nitrogen, 8.1 g/100 g of TCA‐soluble nitrogen and 2.69 g/100 g of PTA‐SN), intensive proteolysis of α_s2‐CN during initial 10 days of ripening (up to 50.70% of initial content) and its much slower degradation through further 40 days, slow but continual decrease of β‐CN content (up to 85.14% of residual content) and high level of proteolytic products tightly bounded into gel network. Total antioxidant capacity of water‐soluble and water‐insoluble fractions increased after cheese ripening. These findings could be useful for better understanding and control over the production of white brined goat cheese as highly valuable functional product.     

Lipolysis and proteolysis in Ragusano cheese during brine salting at different temperatures

The influence of temperature (12, 15, 18, 21, and 24 degrees C) of saturated brine on lipolysis and proteolysis in 3.8-kg blocks of Ragusano cheese during 24 d of brining was determined. Twenty-six 3.8-kg blocks were made on each day. The cheese making was replicated on 3 different days. All blocks were labeled and weighed prior to brining. One block was sampled and analyzed prior to brine salting. Five blocks were placed into each of 5 different brine tanks at different temperatures. One block was removed from each brine tank after 1, 4, 8, 16, and 24 d of brining, weighed, sampled, and analyzed. Both proteolysis and lipolysis in Ragusano cheese increased with increasing brine temperature (from 12 to 24 degrees C), with the impact of brine temperature on proteolysis and lipolysis becoming progressively larger. Proteolysis was highest in the interior of the blocks where salt in moisture content was lowest and temperature had more impact on proteolysis in the interior position of the block than the exterior position. However, the opposite was true for lipolysis. The total free fatty acid content was higher and temperature had more impact on lipolysis at the exterior position of the block where salt in moisture was the highest. This effect of increased salt concentration on lipolysis was confirmed with direct salted cheeses in a small follow-up experiment. Lipolysis increased with increasing salt in the moisture content of the direct salted cheeses. It is likely that migration of water-soluble FFA from the brine into the cheese and from the interior portion of the cheese to the exterior portion of the cheese also contributed to a higher level of FFA at the exterior portion of the blocks. As brine temperature increased the profile of individual free fatty acids released from triglycerides changed, with the proportion of short-chain free fatty acids increasing with increasing brine temperature. This effect was largest at high salt in moisture content.     

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.     

Yield and aging of Cheddar cheeses manufactured from milks with different milk serum protein contents

Whey proteins in general and specifically β-lactoglobulin, α-lactalbumin, and immunoglobulins have been thought to decrease proteolysis in cheeses manufactured from concentrated retentates from ultrafiltration. The proteins found in whey are called whey proteins and are called milk serum proteins (SP) when they are in milk. The experiment included 3 treatments; low milk SP (0.18%), control (0.52%), and high milk SP (0.63%), and was replicated 3 times. The standardized milk for cheese making of the low milk SP treatment contained more casein as a percentage of true protein and more calcium as a percentage of crude protein, whereas the nonprotein nitrogen and total calcium content was not different from the control and high SP treatments. The nonprotein nitrogen and total calcium content of the milks did not differ because of the process used to remove the milk SP from skim milk. The low milk SP milk contained less free fatty acids (FFA) than the control and high milk SP treatment; however, no differences in FFA content of the cheeses was detected. Approximately 40 to 45% of the FFA found in the milk before cheese making was lost into the whey during cheese making. Decreasing the milk SP content of milk by 65% and increasing the content by 21% did not significantly influence general Cheddar cheese composition. Higher fat recovery and cheese yield were detected in the low milk SP treatment cheeses. There was more proteolysis in the low milk SP cheese and this may be due to the lower concentration of undenatured β-lactoglobulin, α-lactalbumin, and other high molecular weight SP retained in the cheeses made from milk with low milk SP content.     

Evolution of free amino acids during ripening of Caciocavallo cheeses made with different milks

The evolution of free amino acids (FAA) in Caciocavallo cheeses, made with cow milk (CC) and cow milk mixed with ewe (CE) and goat (CG) milk, was studied throughout ripening. In all Caciocavallo cheeses produced, the total free amino acid (TFAA) content increased during ripening. In general, the highest TFAA content was found in cow cheeses, and the lowest in CG cheeses, whereas CE cheeses ranged over an intermediate level. In all the analyzed samples, during ripening, the content of the individual FAA increased with the exception of arginine. Tyrosine and histidine were found only in CE samples from the middle to the end of ripening. The major FAA found throughout the whole ripening period, in all types of cheese, were leucine, phenylalanine, lysine, valine, asparagine, γ-aminobutyric acid, and ornithine. The TFAA and several AA showed significant differences in ripening time, whereas tyrosine and histidine showed significant differences in kinds of milk.     

Proteolysis and texture changes of a Spanish soft cheese (‘Torta del Casar’) manufactured with raw ewe milk and vegetable rennet during ripening

Proteolysis and textural changes of the Spanish ewe raw milk soft cheese of the Protected Designation of Origin Torta del Casar were studied in four different stages of ripening, with 1, 30, 60 and 90 days. In general, proteolysis in Torta del Casar cheese was weak at 1 and 30 days and it was more intense between the 30–60 days of ripening. Soluble nitrogen non‐protein nitrogen, polypeptide N and free amino acids values significantly increased during cheese ripening. Protein and casein nitrogen decreased significantly after 60 days of ripening resulting in the increase of the other nitrogen fractions measured. Caseins changes determined by capillary zone electrophoresis showed that proteolysis of β‐casein occurred faster than αs1‐casein but the latter suffered higher proteolytic degradation at the end of ripening (day 90). This pattern of degradation of caseins is reversed in other cheeses made with animal rennet. Texture analysis showed that firmness and consistency decreased along ripening while adhesiveness increased. Highly significant correlations were found between textural parameters, residual caseins levels and nitrogen fractions during maturation, which shows the importance of proteolytic changes for an optimal texture formation.     

Changes of free amino acid content of Teleme cheese made with different types of milk and culture

The evolution of concentration of free amino acids in Teleme cheese made from sheep, goat or cow milk, using a thermophilic, mesophilic or a mixture of a thermophilic, a mesophilic culture throughout ripening was studied. The total free amino acid (TFAA) content increased at all stages of ripening, regardless of the milk and culture used. In general, the TFAA content was higher in cheeses made from cow’s milk than that of the cheeses made from ewe’s milk; cheese from goat’s milk ranged over intermediate levels. Also, higher concentrations of TFAA were found in cheeses made with the thermophilic than with the mesophilic culture. Cheeses made with the mixture of thermophilic–mesophilic culture ranged over intermediate levels. The results of this study have shown that Leu, Glu, Phe, Val and Lys were the major FAA of Teleme cheese at all stages of ripening, regardless of the type of milk and culture used.     

Microbial dynamics during the ripening of a mixed cow and goat milk cheese manufactured using frozen goat milk curd

To overcome the seasonal shortage of goat milk in mixed milk cheese manufacture, pasteurized goat milk curd and high-pressure-treated raw goat milk curd manufactured in the spring were held at −24°C for 4 mo, thawed, and mixed with fresh cow milk curd for the manufacture of experimental cheeses. Control cheeses were made from a mixture of pasteurized cow and goat milk. The microbiota of experimental and control cheeses was studied using culture-dependent and culture-independent techniques. Bacterial enumeration by classical methods showed lactic acid bacteria to be the dominant population in both control and experimental cheeses. In total, 681 isolates were grouped by partial amplified rDNA restriction analysis (ARDRA) into 4 groups and identified by 16S rRNA gene sequencing as Lactococcus lactis ssp. lactis (563 isolates), Leuconostoc pseudomesenteroides (72 isolates), Lactobacillus spp. (34 isolates), and Lc. lactis ssp. cremoris (12 isolates). Temporal temperature gradient gel electrophoresis (TTGE) analysis of cheese showed (1) the predominance of Lc. lactis in all cheeses; (2) the presence of Leu. pseudomesenteroides population in all cheeses from d 15 onward; (3) the presence of a Lactobacillus plantarum population in control cheese until d 15 and in experimental cheeses throughout the ripening period. Due to the most diverse and complete set of peptidases present in the genus Lactobacillus, the prevalence of this population in experimental cheeses could give rise to differences in cheese flavor between experimental and control cheeses.     

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.     

Variation in organic acids content during ripening of pickled white cheese

Nine organic acids (formic, pyruvic, lactic, acetic, orotic, citric, uric, propionic, and butyric) were analyzed during ripening of pickled White cheese for 12 mo by high-performance liquid chromatography with a reverse phase C18 (120x 5-mm) column and UV detector. The level oftotal organic acids showed an increase along the ripening period, but its composition varied during the process. Initially, lactic acid accounted for 95% of the total, after 9 and 12 mo of ripening, butyric acid constituted 20 and 27% of the total, respectively. Each organic acid presented a characteristic pattern of change during ripening. Discriminant analysis classified cheeses according to their age. Stepwise regression analysis allowed estimation of the ripening time of samples according to their organic acid levels.     

Influence of selected lab cultures on the evolution of free amino acids, free fatty acids and Fiore Sardo cheese microflora during the ripening

Fiore Sardo Protected Denomination of Origin is a traditional Sardinian (Italy) hard cheese produced exclusively from whole raw ovine milk and coagulated with lamb rennet paste. Currently, Fiore Sardo is still produced by shepherds at the farmhouse level without the addition of any starter culture and the cheese-making process is characterized by significant waste. The first objective of the present work was to investigate the autochthonous microflora present in milk and Fiore Sardo cheese in order to select lactic acid bacterial (LAB) cultures with suitable cheese-making attributes and, possibly reduce the production waste. Secondly, the ability of selected cultures to guarantee cheese healthiness and quality was tested in experimental cheese-making trials. In this study, we show that the typical lactic microflora of raw ewe's milk and Fiore Sardo cheese is mostly composed of mesophilic LAB such as Lactococcus lactis subsp. lactis, Lactobacillus plantarum and Lactobacillus casei subsp. casei. Moreover, strains belonging to the species were selected for cheese-making attributes and used in experimental cheese-making trials carried out in different farms producing Fiore Sardo. The evolution of the cheese microflora, free amino acids and free fatty acids during the ripening showed that the experimental cheeses were characterized by a balanced ratio of the chemical constituents, by a reduced number of spoilage microorganisms and, remarkably, by the absence of production waste that were significant for the control cheeses.     

Effect of proteolysis during Cheddar cheese aging on the detection of milk protein residues by ELISA

Cow milk is a common allergenic food, and cow milk-derived cheese retains an appreciable level of allergenicity. The specific and sensitive detection of milk protein residues in foods is needed to protect milk-allergic consumers from exposure to undeclared milk protein residues contained in foods made with milk or milk-derived ingredients or made on shared equipment or in shared facilities with milk or milk-derived ingredients. However, during cheese ripening, milk proteins are degraded by chymosin and milk-derived and bacterial proteases. Commercial allergen-detection methods are not validated for the detection of residues in fermented or hydrolyzed products. The objective of this research was to evaluate commercially available milk ELISA kits for their capability to detect milk protein residues in aged Cheddar cheese. Cheddar cheese was manufactured at a local dairy plant and was aged at 5°C for 24 mo, with samples removed at various time points throughout aging. Milk protein residues and protein profiles were measured using 4 commercial milk ELISA kits and sodium dodecyl sulfate-PAGE. The ELISA data revealed a 90% loss of milk protein residue signal between the youngest and oldest Cheddar cheese samples (0.5 and 24 mo, respectively). Sodium dodecyl sulfate-PAGE analysis showed protein degradation throughout aging, with the highest level of proteolysis observed at 24 mo. Results suggest that current commercial milk ELISA methods can detect milk protein residues in young Cheddar cheese, but the detection signal dramatically decreases during aging. The 4 evaluated ELISA kits were not capable of detecting trace levels of milk protein residues in aged cheese. Reliable detection of allergen residues in fermented food products is critical for upholding allergen-control programs, maintaining product safety, and protecting allergic consumers. Furthermore, this research suggests a novel use of ELISA kits to monitor protein degradation as an indication of cheese ripening.