Secondary proteolysis in Serra cheese during ripening and throughout the cheese-making season

 Experimental Serra cheeses were manufactured from raw ewe’s milk and thistle flowers following a two-way factorial design. The content of nitrogen soluble in water (WSN), in 2% trichloroacetic acid (2% TCA-N), in 12% TCA (TCA-N) and in 5% phosphotungstic acid (5% PTA-N), and the pH and salt-in-moisture concentration were measured throughout the ripening period (sampling at 0, 7, 21 and 35 days) and the cheese-making season (sampling in November, February and May). Proteolysis in 35-day-old Serra cheese was quantitatively high [average values of 34.6% and 11.9% for WSN/TN (total nitrogen) and 2% TCA-N/TN, respectively], but qualitatively low (average values of 5.8% and 1.2% for 12% TCA-N/TN and 5% PTA-N/TN, respectively). The ratios WSN/TN and 2% TCA-N/TN were lowest for cheeses ripened in February, whereas the ratio 12% TCA-N/TN was highest for cheeses ripened in November. By 35 days of ripening, the average pH and salt-in-moisture concentration values were 5.2 and 4.8%, respectively. No correlation was found to occur between the measured pH or salt-in-moisture concentration and the values of soluble nitrogen fractions throughout the cheese-making season. Received: 14 February 1996/Revised version: 4 June 1996     

Effect of Free or Encapsulated Recombinant Aminopeptidase of Lactobacillus rhamnosus S93 on Acceleration of Cheddar Cheese Ripening

The use of recombinant aminopeptidase (PepN) from Lactobacillus rhamnosus S93 in free or encapsulated form was investigated to shorten the duration of Cheddar cheese ripening. Proteolysis was determined by measuring the soluble nitrogen as phosphotungstic acid (PTA-N) derivatives and free amino acids (FAA) over a 6-month period. The experimental cheeses received higher scores for sensory properties than the control cheese. The amounts of PTA-N and total FAA in the cheese with the encapsulated enzyme after 2 months of ripening were close to those of the control cheese after 6 months, suggesting the acceleration in proteolysis by about 4 months.     

Effect of freeze–dried kefir culture on proteolysis in feta-type and whey-cheeses

The effect of freeze–dried kefir culture on the proteolysis of feta-type and whey-cheese was investigated. All nitrogen fractions increased during ripening. Although no significant differences were observed in total nitrogen (TN), the levels of water-soluble nitrogen (WSN), pH 4.4-soluble nitrogen (SN), 12% trichloroacetic acid-soluble nitrogen (TCA-SN) and phosphotungstic acid-soluble nitrogen (PTA-SN) were significantly higher in cheeses produced by freeze–dried kefir culture during the later stages of ripening. Content of total free amino acids (FAA) was significantly affected by freeze–dried kefir starter culture and it was continuously increased in kefir-cheese while, in rennet-cheese it was increased up to 30 days of ripening and then slightly decreased. On the other hand, FAA content continuously decreased in kefir-whey-cheese whereas it increased in whey-cheese. The cheese samples produced by freeze–dried kefir as starter culture were characterised as high-quality products during the preliminary sensory evaluation and they were accepted by the panel. Overall, the use of freeze–dried kefir suggested acceleration of cheese ripening and resulted in improved sensory characteristics.     

Amino acid and soluble nitrogen evolution throughout ripening of Serra da Estrela cheese

Four batches of Serra da Estrela cheese originating from as many dairy farms were sampled throughout the ripening period, and assayed for the evolution of free amino acid (FAA) content, total nitrogen content (TN), water-soluble nitrogen content (WSN), trichloroacetic acid-soluble nitrogen content (TCASN) and phosphotungstic acid-soluble nitrogen content (PTASN). The WSN content increased from 1% (on the day of manufacture) up to 43% of TN by 180 d of ripening, thus reflecting the intense proteolytic activity of the enzymes contributed by the plant coagulant utilized. The TCASN was also found to be high in this cheese by the end of ripening (16–20%), which suggests a high extent of FAA release throughout maturation. The major FAA by 180 d of ripening were Glu, Val, Leu and Lys, representing 56–70% of the total in all four dairies sampled. Cheeses produced from refrigerated milk possessed higher amounts of γ-amino-n-butyric acid (Gaba) and lower amounts of Glu when compared with those manufactured with non-refrigerated milk.     

Influence of ripening on proteolysis and lipolysis of Murcia al Vino cheese

The aim of this study was to evaluate the influence of five different manufacturers and two ripening periods on the proteolysis and lipolysis patterns of Murcia al Vino goat cheese. The manufacturers significantly affected the water activity (a_w), pH, dry matter and fat content, several nitrogen fractions: water soluble nitrogen (WSN), trichloroacetic acid (12% w/v) soluble nitrogen (TCASN) and phosphotungstic acid (5% w/v) soluble nitrogen (PTASN); also the free amino acid (FAA) and free fatty acid (FFA) contents, with the exception of C_4:0, C_16:0 and C_18:0. Different ripening periods significantly affected the dry matter content, WSN and PTASN and all FAA, except serine.     

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.     

Effect of proteolysis and calcium equilibrium on functional properties of natural cheddar cheese during ripening and the resultant processed cheese

The changes in proteolysis, calcium (Ca) equilibrium, and functional properties of natural Cheddar cheeses during ripening and the resultant processed cheeses were investigated. For natural Cheddar cheeses, the majority of the changes in pH 4.6 soluble nitrogen as a percentage of total nitrogen (pH 4.6 SN/TN) and the soluble Ca content occurred in the first 90 d of ripening, and subsequently, the changes were slight. During ripening, functional properties of natural Cheddar cheeses changed, that is, hardness decreased, meltability was improved, storage modulus at 70 °C (G'T=70) decreased, and the maximum tan delta (TDmax) increased. Both pH 4.6 SN/TN and the soluble Ca were correlated with changes in functional properties of natural Cheddar cheeses during ripening. Kendall's partial correlation analysis indicated that pH 4.6 SN/TN was more significantly correlated with changes in hardness and TDmax. For processed cheeses manufactured from natural Cheddar cheeses with different ripening times, the soluble Ca content did not show significant difference, and the trends of changes in hardness, meltability, G'T=70, and TDmax were similar to those of natural Cheddar cheeses. Kendall's partial correlation analysis suggested that only pH 4.6 SN/TN was significantly correlated with the changes in functional properties of processed cheeses.     

The effect of ripening and cooking temperatures on proteolysis and lipolysis in Manchego cheese

The influence of ripening temperature on proteolysis and lipolysis was studied on four lots of raw ewe's milk Manchego cheese held for 60 days at 5, 10, 15 or 20°C. Mean levels of pH 4·6, trichloroacetic acid and phosphotungstic acid soluble N in 20°C cheeses were 52%, 78% and 95% higher than the respective levels in 5°C cheeses at the end of the ripening period. Free fatty acids content after 60 days was 90% higher in 20°C cheeses than in 5°C cheeses. Significant effects of the cooking temperature of the curd (30, 36, 38 or 40°C) on pH, moisture and NaCl content were recorded, but levels of nitrogenous fractions or free fatty acids in 60-day cheeses were not affected.     

凝乳酶对牦牛乳硬质干酪成熟期间蛋白降解的影响

以新鲜牦牛乳为原料,采用小牛皱胃酶、木瓜蛋白酶和微生物凝乳酶制作硬质干酪,探讨凝乳酶种类对牦牛乳硬质干酪成熟期间蛋白质降解的影响。结果表明:三种凝乳酶牦牛乳硬质干酪成熟过程中,不同凝乳酶牦牛乳硬质干酪在成熟期间蛋白质降解能力存在较大差异,总氮（TN）、p H4.6水溶性氮（p H4.6-SN/TN）、12%的三氯乙酸氮（12%TCA-N/TN）、5%磷钨酸氮（5%PTA-N/TN）含量、游离氨基酸均随成熟时间延长不同程度的增加,蛋白氮和酪蛋白氮逐渐降低,多肽氮呈先升高后下降趋势,且微生物凝乳酶降解牦牛乳硬质干酪蛋白能力显著（p<0.05）高于木瓜蛋白酶和小牛皱胃酶。      

How milk type,coagulant, salting procedure and ripening time affect the profile of free amino acids in Picante da Beira Baixa cheese

The concentration of total free amino acids (FAA) in Picante cheese increased with ripening time irrespective of the particular protocol used for manufacture (ie ratio of caprine to ovine milks, animal or plant rennet and number of salting steps). The experimental cheeses manufactured with 20% (v/v) caprine milk, coagulated with animal rennet and salted only once exhibited the highest content of total FAA by 120 days of ripening. All four manufacture parameters were statistically significant on the 0.5% level of significance in terms of total concentration of FAA. The dominating free amino acids present in the various experimental cheeses throughout the ripening period were valine, leucine and phenylalanine, each one representing more than 10% (w/w) of the total concentration of FAA. All four manufacture parameters were, in general, statistically significant with respect to the content of every single FAA, with particular emphasis on salting and ripening time. © 1999 Society of Chemical Industry     

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.     

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.     

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.     

Free amino acid profile during ripening of ewe's milk cheese from the Croatian island Krk

The aim of this study was to determine the content of free amino acids (FAA) and their ratio in ewe's milk cheese from the island Krk during its ripening. FAA content was determined by reversed phase HPLC (RP‐HPLC) of cheese aqueous/ethanol extracts after FAA were transformed into their 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate derivatives. Their concentration increased during ripening, reaching the value of 5% in cheese dry matter. The dominant FAA were glutamic acid>leucine>valine>aspartic acid>phenylalanine>serine>proline, and higher content of nonessential vs essential FAA was revealed. Krk cheese has, in relation to other cheeses, higher values for glutamic acid/leucine, glutamic acid/phenylalanine, glutamic acid/proline and smaller values for leucine/aspartic acid, valine/aspartic acid, phenylalanine/aspartic acid ratios, while other ratios are comparable to those of other hard ovine cheeses.     

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.     

Proteolysis in reduced sodium Feta cheese made by partial substitution of NaCl by KCl

Feta cheeses (five trials) of different sodium content were made, using ewes’ milk, from split lots of curd by varying the salting procedure, i.e. dry salting with NaCl (control) or mixtures of NaCl/KCl (3:1 or 1:1, w/w basis) and filling the cans with brine made with NaCl or the above NaCl/KCl mixtures, respectively, in order to study the influence of the partial substitution of NaCl by KCl on the proteolysis during cheese ripening. The extent and characteristics of proteolysis in the cheeses were monitored during aging by using Kjeldahl determination of soluble nitrogen fractions (water-soluble nitrogen, trichloroacetic acid-soluble nitrogen, phosphotungstic acid-soluble nitrogen), 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 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 Feta cheese had no significant effect on the extent and characteristics of proteolysis during cheese aging.     

Effect of ripening temperature on the growth and significance of non-starter lactic acid bacteria in Cheddar cheese made from raw or pasteurised milk

Two cheese-making trials were conducted, each involving four cheeses, two made from raw milk (R1, R8) and two from pasteurised milk (P1, P8), and ripened at 1°C (R1, P1) or 8°C (R8, P8). The 1-day-old R1 and R8 cheese in trials 1 and 2 contained ∼10⁴ non-starter lactic acid bacteria (NSLAB) g⁻¹. In trial 1, no NSLAB were detected in 1-day-old P1 and P8 cheeses while those in trial 2 contained 10² cfu g⁻¹. In both trials, the maximum differences between the number of NSLAB in the cheeses ripened at 1 or 8°C were observed at 4 months, when the number of NSLAB in cheeses ripened at 8°C were 3 log cycles higher than in those ripened at 1°C. At the end of ripening (6-months), the number of NSLAB in P8 and R8 were ∼2 log cycles higher than in P1 and R1 cheeses, respectively. Primary proteolysis in the cheeses was markedly affected by ripening temperature, but not by pasteurisation of the cheese milk. Urea-polyacyrlamide gel electrophoretograms and reverse-phase (RP)-HPLC of the water-soluble fraction showed differences between cheeses made from raw or pasteurised milk and between cheeses ripened at 1 or 8°C. The concentration of amino acids and fatty acids were in the order R8>P8>R1>P1. Commercial graders awarded highest flavour scores to the R1 cheeses during gradings at 4, 5 and 6 months. A sensory panel found that most flavour and aroma attributes and maturity were in the order of R8>P8>R1=P1. The results of this study suggest that NSLAB play an important role in the development of flavour in Cheddar cheese by contributing to the production of amino acids and fatty acids.     

Solid-State 31P NMR, a Relevant Method to Evaluate the Distribution of Phosphates in Semi-hard Cheeses

The potentiality of solid-state ³¹P nuclear magnetic resonance (NMR) to determine in a non-destructive way the different states of phosphates in cheeses was examined. Sixteen semi-hard cheeses of various compositions were studied, and three fractions of phosphates (P) were distinguished according to their mobility: (1) mobile soluble P (ca. 10 % of total P), (2) mobile insoluble P (70 %) and (3) immobile insoluble P (20 %). In accordance with chemical composition and buffering capacities of the cheeses, these fractions could represent respectively (1) soluble inorganic P, (2) inorganic colloidal calcium P and phosphorylated serine residues (Pser) involved in a loose structure and (3) Pser involved in a tight environment. This method was shown to be of sufficient accuracy to evidence the effect of ripening on the different P fractions. It was thus demonstrated that solid-state NMR is an appropriate method to observe the distribution of phosphates in cheese matrix and their evolution during cheese-making.     

Changes of Mineral Concentrations in Serra Cheese during Ripening and Throughout the Cheesemaking Season

Seasonal changes of the ash content and mineral concentrations in Serra cheese were studied over a typical 35-day ripening period. Statistically significant differences (at the 5% level) exist between the ash content and the concentrations of Na, K, Ca, P, Mg and Zn in cheeses during ripening. The highest concentrations of Na was obtained in cheese ripened for 7 days, whereas the concentrations of K, Ca, P, Ng and Zn decreased significantly during ripening. For 35-day-old cheeses, concentrations of Na, K and Cu were lowest and concentration of P was highest for cheeses manufactured in May. The concentration of Ca was lowest for cheeses manufactured in February. On average, the most concentrated minerals (in g kg^-1 of total solids, TS) in 35-day-old Serra cheese were Na (18·56), Ca (9·70) and P (7·92) and, at a lower level, K (1·70) and Mg (0·96). Only trace levels (in mg kg_TS^-1) of Zn (94·33), Cu (2·26) and Mn (1·25) were detected. A high mineral nutrition quality was thus ascribed to 35-day-old Serra cheese based on the average nutritional densities: 4·8 for Ca, 4·0 for P, 1·1 for Mg, 3·4 for Na, 2·4 for Zn, 0·4 for Cu, 0·2 for Mn and 0·2 for K. © 1997 SCI     

A comparison and joint use of VIS-NIR and MIR spectroscopic methods for the determination of some chemical parameters in soft cheeses at external and central zones: a preliminary study

There is a strong tendency towards exploring rapid and low cost methods for determining chemical parameters and degree of the ripening of cheeses. The visible-near infrared (VIS-NIR), mid infrared (MIR) and combination of VIS-NIR and MIR spectroscopic methods for measurements of some selected parameters of soft cheeses were compared. Fifteen traditional and stabilised retail soft cheeses, differing in manufacturing process were studied. Fat, dry matter (DM), pH, total nitrogen (TN) and water soluble nitrogen (WSN) contents were determined by reference methods and scanned with VIS-NIR and MIR spectrophotometers in reflectance mode. Three separate prediction models were developed from the VIS-NIR, MIR and the joint VIS-NIR-MIR spectra using the partial least square (PLS) regression and leave one-out cross-validation technique. Results showed that fat, DM, TN and WSN were the best predicted with the VIS-NIR models providing the lowest values of the root mean square error of prediction (RMSEP) of 1.32, 0.70, 0.11 and 0.10, respectively. The combination of the VIS-NIR and MIR spectral improved slightly the prediction of only the pH. This suggests using the VIS-NIR for the determination of fat, DM, TN and WSN. The pH can also be predicted from the two techniques with approximate quantitative prediction, while a difference between low and high levels of WSN/TN ratio could be determined by the VIS-NIR, MIR or joint use of VIS-NIR-MIR.