Acceleration of cheese ripening at elevated temperature. An estimation of the optimal ripening time of a traditional Argentinean hard cheese

The effect of elevated ripening temperature on physicochemical, biochemical and sensory characteristics in Reggianito Argentino cheese was evaluated to determine the optimal time for cheese ripening at 18 °C that ensures typical cheese characteristics. Cheeses ripened at 12 or 18 °C and 85% relative humidity were analysed at 2, 4 and 6 months. Seventy-eight variables (as determined by urea-PAGE, RP-HPLC of the water-soluble at pH 4.6 fraction, free amino acids, free fatty acids and sensory analysis) were considered for the principal component analysis. The statistical analysis allowed determination of the optimal time for ripening Reggianito Argentino cheese at 18 °C, which was ranged between 2 and 3 months. In conclusion, the results obtained were not only useful in characterising the ripening of an Argentinean hard cheese, but also in evaluating the effect of an increase of ripening temperature on the main physicochemical, biochemical and sensory changes of Reggianito Argentino cheese.     

Effects of the freezing process on proteolysis during the ripening of Port Salut Argentino cheeses

The effect of freezing at −30 °C, frozen storage at −22 °C for 30 days and thawing at 5 °C on proteolysis during ripening of Port Salut Argentino cheese was studied. Cheeses were sampled at different ripening times (1, 6, 13, 27 and 56 days) and two sampling zones (central and external). Moisture content, salt concentration and RP-HPLC of the nitrogenous fractions (water-insoluble fraction, water-soluble fraction and free amino acids in the sulfosalicylic acid-soluble fraction) were analysed. The freezing process did not affect moisture and salt contents at the beginning of the ripening period nor moisture and salt redistribution during the ripening period studied. However, the freezing process affected proteolysis during ripening of Port Salut Argentino cheeses that had been frozen prior to ripening. There was increased breakdown of α_s1-casein and α_s1-I-casein, and increased breakdown of peptides of the water-soluble fraction (including α_s1-CN (f1-23)) along with an early development of free amino acids.     

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.     

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.     

The influence of ripening temperature and sampling site on the lipolysis in Reggianito Argentino cheese

The effect of ripening temperature elevation and sampling site on lipolysis in Reggianito Argentino cheese was evaluated. Cheeses ripened at 12 or 18 °C and 85% relative humidity were assayed at 2, 4 and 6 months at two sampling zones (central and external). Samples were analysed to determine physicochemical parameters and the concentration of nine free fatty acids (FFAs) (C_6:0–C_18:2). Myristic, palmitic, stearic and oleic acids were found in higher concentration. While ripening time and temperature significantly affected the concentrations of the nine FFAs analysed, sampling zone significantly affected only two FFAs. Ripening temperature increased the lipolytic process, but it seems to have no effect on the pathways of lipolysis in Reggianito Argentino cheese.     

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.     

The effect of exposure to pressure of 50 MPa on Cheddar cheese ripening

The possibility of acceleration of commercial Cheddar cheese ripening by exposure to a high pressure (HP) treatment of 50 MPa for 3 days at 25°C at different stages of ripening was investigated. Proteolysis was examined in the treated and untreated cheeses by measurement of pH 4.6 water soluble nitrogen, expressed as g/100 g total N (pH 4.6 SN/TN), urea-PAGE, reverse phase (RP) HPLC, analysis of molecular mass distribution by gel permeation and measurement of free amino acids (FAA) in the pH 4.6 SN. There was an immediate increase in pH 4.6 SN/TN and FAA in cheese HP-treated at 2 days of age, although this effect decreased with cheese age. Urea-PAGE analysis of cheese samples indicated that HP treatment accelerated degradation of α_s1-casein and accumulation of α_s1-I-casein (f 24-199). RP-HPLC profiles indicated quantitative but not qualitative differences between treated and non-treated samples. Confocal laser scanning microscopy did not indicate any gross structural changes in the cheese matrix as a result of exposure to 50 MPa for 3 days at 25°C. It was concluded that the enhancement of proteolysis observed may be attributed to a combination of the temperature and pressure used in the treatment.     

Primary and Secondary Proteolysis in Eleven Turkish Cheese Varieties

Levels of proteolysis of 75 samples belonging to 11 Turkish cheese varieties, including Civil, Canak, Dil, Divle Tulum, Ezine, Hellim, Malatya, Mihalic, Orgu, Urfa, and Van Otlu, were comparatively studied. The cheeses were mainly produced using traditional methods; however, some varieties were industrially produced. Chemical composition and the levels of soluble nitrogen fractions of the cheeses varied depending on the cheese variety. Gel electrophoresis of the cheeses showed that the samples presented different gel patterns with α_s1-casein being extensively degraded in many cheeses; whereas the hydrolysis of α_s1-casein in Malatya and Hellim was observed to be limited. Peptide profiles by RP-HPLC of the water-soluble fractions were largely different for many of the samples, but some similarities were visualized. Multivariate analysis of the RP-HPLC data grouped the cheeses according to their peptide profiles. The results suggested that each variety of cheese had different levels of proteolysis. The manufacturing technique and ripening conditions employed have played a determinative role on the proteolytic patterns of the cheeses analyzed.     

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.     

一种毛霉表面成熟干酪的蛋白质水解作用评价

从毛豆腐中分离出一株毛霉,并应用于表面成熟干酪,以研究干酪成熟过程中所发生的蛋白质水解作用。在90d 的成熟过程中,干酪的pH 值增加;蛋白质水解作用的评价指标,如干酪外层的水溶性氮- 总氮比、pH4.6水溶性氮- 总氮比、12g/100mL 三氯乙酸可溶性氮- 总氮比,在成熟90d 后分别增加至(23.68 ± 1.07)%、(19.38 ± 1.32)%和(8.61 ± 0.85)%,并且高于干酪的内部相应指标。SDS-PAGE 和毛细管电泳分析干酪的pH4.6 不溶性组分,结果表明酪蛋白在干酪成熟过程中被降解。对干酪成熟过程中分离出的水溶性组分进行RP-HPLC 分析,结果显示成熟过程中蛋白质被水解以及形成一些新肽分子。     

Phosptiopeptides from Comté Cheese: Nature and Origin

Thirteen low-molecular-weight phosphopeptides were isolated from the water-soluble fraction of Comté cheese. The sample was fractionated and purified by gel permeation chromatography and reverse-phase HPLC. The peptide sequences were identified by Edman degradation and primary molecular structure was confirmed by mass spectrometry. The different peptides purified correspond to fragments of the sequence Val13-Lys 28 of β-casein and of the sequence Glu S-Lys 21 of α₃₂ casein. These fragments probably originated from an initial proteolysis of the two caseins by plasmin, followed by further endopeptidase aminopeptidase and, possibly, carboxypeptidase digestions. Partial dephosphorylation of some β-casein fragments was observed. These peptides probably influence the flavor profile of comté cheese.     

Kinetics of Casein Degradation during Ripening of Fynbo Cheese Salted with NaCl/KCl Brine

First-order kinetics with respect to the α_s1-casein concentration was used to study casein degradation during low-fat Fynbo cheese ripening. Effects of partial NaCl replacement by KCI during cheese salting were studied by statistical treatment of the casein degradation results. Four zones from cheeses at 1, 5, 10, 20, and 30 ripening days were analyzed by a polyacrylamide gel electrophoresis method. Similar kinetic parameters were obtained for a cheese salted with a NaCl/KCl brine and for a control cheese during ripening. Results were more affected by salt concentration than by salt substitution. KCl did not strongly influence kinetics of Fynbo cheese proteolysis.     

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.     

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.     

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.     

Proteolysis in goat cheese made from raw,pasteurized or pressure-treated milk

Primary and secondary proteolysis of goat cheese made from raw (RA), pasteurized (PA; 72 °C, 15 s) and pressure-treated milk (PR; 500 MPa, 15 min, 20 °C) were examined by capillary electrophoresis, nitrogen fractionation and HPLC peptide profiles. PA milk cheese showed a more important hydrolysis (P<0.05) of α_s1-casein than RA milk cheese at the first stages of ripening (15 days), while PR milk cheese had a level between those seen in PA and RA milk cheeses. Degradation of β-casein was more important (P<0.05) in PA and PR than in RA milk cheeses at 15 days of ripening. However, from thereon β-casein in PR and RA milk cheeses was hydrolyzed at essentially similar rates, but at lower rates (P<0.05) than in PA milk cheeses. Pressure treatment could induce proteolysis of β-casein in a way, which is different from that produced by heat treatment. There was an increase in 4.6-soluble nitrogen (WSN) and in trichloroacetic acid (TCASN) throughout ripening in cheeses, but higher contents (P<0.05) in PA and PR milk cheeses at the end of ripening were observed. PR milk cheeses contained considerably higher content (P<0.05) of free amino acids than RA or PA milk cheeses. In general, heat and pressure treatments had no significant effect on the levels of hydrophobic and hydrophilic peptides.     

Proteolysis on Reggianito Argentino cheeses manufactured with natural whey cultures and selected strains of Lactobacillus helveticus

Reggianito Argentino cheese is traditionally manufactured with whey starter cultures that provide typical and intense flavor but can cause poor quality standardization. In this study, the influence of natural and selected starters on Reggianito Argentino cheese proteolysis was investigated. Cheeses were manufactured with three strains of Lactobacillus helveticus (SF133, SF138 and SF209) cultured individually in sterile whey and used as single or mixed starters. Control cheeses were made with natural whey starter culture. Cheeses were analyzed to determine gross composition, as well as total thermophilic lactic flora. Proteolysis was assessed by N fractions, electrophoresis and liquid chromatography. Gross composition of the cheeses did not significantly differ, while viable starter cell counts were lower for cheeses made with strain SF209 alone or combined with other strains. Soluble N at pH 4.6 was the same for cheeses made with natural or selected starters, but soluble N in 12% trichloroacetic acid and 2.5% phosphotungstic acid was significantly higher in cheeses made with starters containing strain SF209. Nitrogen fractions results indicated that natural whey starter cultures could be replaced by several starters composed of the selected strains without significant changes to proteolysis patterns. Starter cultures prepared only with SF209 or with the three selected L. helveticus strains produced cheese products with significantly more proteolysis than control cheeses. Chromatographic profiles analyzed by principal components showed that three main peaks on chromatograms, presumptively identified as Tyr, Phe, and Trp, explained most of variability. Principal component scores indicated that cheese samples were grouped by ripening time, which was confirmed by linear discriminant analysis. On the contrary, samples did not cluster by Lactobacillus strain or type of starter.     

Study of the biochemical changes during ripening of Ahumado de Áliva cheese: a Spanish traditional variety

The changes in chemical composition, main physico-chemical parameters, classical nitrogen fractions, caseins and their degradation products, and some fat characteristics were studied during the ripening of ten batches of Ahumado de Áliva cheese, a traditional variety made in the north of Spain. The values of the different compositional and physico-chemical parameters at the end of the ripening did not differ significantly from those found in other cows' milk cheeses elaborated by similar technology. The low pH values are outstanding. The presence of residual lactose at the end of ripening is also relevant. Total soluble nitrogen and non-protein nitrogen increased very little during ripening. The evolution of the values of the different nitrogen fractions show that this cheese undergoes very little proteolysis and that the rennet is the main proteolytic agent. Using PAGE, it was possible to show that, throughout ripening, only 22% of α_s-casein and 9% of β-casein were degraded. The TBA value indicated that the fat of Ahumado de Áliva cheese does not undergo noticeable autooxidation during ripening. The acidity index of the fat also indicated that this cheese underwent little lipolysis during ripening.     

Secondary proteolysis of Fynbo cheese salted with NaCl/KCl brine and ripened at various temperatures

Effects of temperature and salt substitution on secondary proteolysis of Fynbo cheese were studied and different peaks of the chromatographic profiles were examined. Cheeses, salted in solutions of NaCl (190 g l⁻¹) and NaCl/KCl (100 g l⁻¹/100 g l⁻¹) and ripened at 5, 12, and 16 °C, were sampled during 90 days at two different zones. Samples were analysed by RP-HPLC of the trichloroacetic acid-soluble fraction. The information was successfully summarized in 2 dimensions, accounting for 86.5% of data variation using principal component analysis. The source of variation explained by PC1 (77.1% VAR) was related to the ripening time. Two groups of chromatographic peaks were distinguished according to the sign of PC2 loading. Total salt concentration and ripening temperature affected secondary proteolysis significantly, while NaCl replacement by KCl had no affect. An important peptide produced during cheese ripening (α_s1-casein (f1-23)) was tentatively identified, taking into account the chromatographic profile and the amino acid composition of the peak isolated.     

Proteolysis in reduced sodium Kefalograviera cheese made by partial replacement of NaCl with KCl

Kefalograviera cheeses (five trials) of different sodium content were made from split lots of curd by varying the salting processes, i.e. brine — and dry — salting, with NaCl (control) or a mixture of NaCl/KCl (3:1 or 1:1, w/w basis). The extent and characteristics of proteolysis in the cheeses were monitored during aging by Kjeldahl determination of soluble nitrogen fractions (water-soluble nitrogen [WSN], trichloroacetic acid [TCA]-SN, phosphotungstic acid [PTA]-SN), the cadmium-ninhydrin method for the determination of total free amino acids (FAA), urea-polyacrylamide gel electrophoresis of cheese proteins, followed by densitometric analysis of the α_s1- and β-casein fractions, reverse-phase high-performance liquid chromatography (HPLC) analysis of the water-soluble extracts of cheeses, and ion-exchange HPLC analysis of FAA. The results showed that proteolysis was similar in control and experimental cheeses at all sampling ages, indicating that the partial substitution of NaCl by KCl in the manufacture of Kefalograviera cheese did not significantly influence the extent and characteristics of proteolysis during cheese aging.