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.     

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.     

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.     

Electrophoretic ripening index for the evaluation of proteolysis and the deduction of the age of Parmesan cheese

We have established an electrophoretic method to evaluate proteolysis in Parmesan cheese by means of an objective ripening index. The separation of caseins by alkaline polyacrylamide gel electrophoresis (PAGE) (12% T, 2.6% C, pH 8.9, 5 M urea) is followed by the densitometric analysis of the- and-casein fractions. The relationship between the resulting coefficients (-Cn/-Cn) and the age of reference samples of Original Italian Grana Padano (6–22 months) was linear up to 15 months, allowing an evaluation of the extent of proteolysis and therefore a deduction of the age of the Parmesan samples analysed. Based on this calibration we propose to use a threshold level of 1.3 (-Cn/-Cn) to verify the required age, i.e. 12 months, of Parmesan cheese, retailed as a loaf or prepacked slices. Commercially grated Parmesan samples may contain 20% cheese rind, which has a very low degree of casein breakdown. So a threshold level of 0.8 (-Cn/-Cn) is proposed for all grated Parmesan products. The preparation of reference solutions with defined coefficients (-Cn/-Cn), corresponding to the proteolysis indices of reference samples of different aged Grana Padano is described. The coefficients (-Cn/-Cn) as well as the-casein content of two additional series of reference samples and of 49 commercial Parmesan samples taken from retail outlets in Austria and Italy are presented.     

Caprine cheese with probiotic strains: the effects of ripening temperature and relative humidity on proteolysis and lipolysis

 The effects of ripening temperature, relative humidity and time on chemical and textural characteristics of a 'probiotic' goat's milk cheese were examined. The experimental layout followed a 2³ factorial design, with all possible combinations of 5  °C and 10  °C (ripening temperature), 85% and 95% (ripening relative humidity) and 1 day and 70 days (ripening time). All proteolytic indices measured (water-soluble nitrogen, trichloroacetic acid-soluble nitrogen and phosphotungstic acid-soluble nitrogen) were enhanced with increased ripening temperature to a greater extent than with increased ripening relative humidity; the increase in phosphotungstic acid-soluble nitrogen was the most significant. Free fatty acid concentrations in cheeses were not influenced by ripening relative humidity but increased with ripening temperature and time. A higher ripening temperature and a lower relative humidity gave rise to firmer cheeses. Postulated empirical models have provided a good fit to the experimental data set generated; such models were able to predict a decrease of 25 days in ripening time with no impairment of either proteolytic or lipolytic indices if a cheese were to be ripened at 10  °C (rather than 5  °C) and 95% relative humidity. Received: 23 March 1998     

Study of the chemical composition,proteolysis, lipolysis and volatile compounds profile of commercial Reggianito Argentino cheese: Characterization of Reggianito Argentino cheese

Reggianito is a typical variety of grana-type hard cheese produced in Argentina. It is the most exported and due to its organoleptic characteristics is very appreciated by the consumers. The objective of this study was to characterise the global composition, lipolysis, proteolysis and volatile compound profiles of commercial Reggianito cheeses from different dairy plants.Statistical differences (P ? 0.05) in some physicochemical parameters, nitrogen fractions and FFA levels among commercial brands were detected. The volatile profiles were studied by SPME–GC–MS/FID. A total of 53 compounds were identified, the majority belonging to the groups of ketones, alcohols, acids, esters and aldehydes. All these compounds have been reported in Italian grana-type cheeses. Visualization of the analytical results was performed by principal component analysis. This analysis clustered cheese samples according to dairy plants. This fact could be, among other factors, consequences of differences in technologies and ripening time of different manufacturers.     

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     

Microbiological quality of Port Salut Argentino cheese stored at two temperature treatments

Microbiological quality of Port Salut Argentino cheese was studied during 10 days (after ripening) at two storage temperature treatments: (a) 4°C and (b) a temperature combination of both 4 and 20°C (4/20°C), which implied 12 h at 4°C and 12 h at 20°C. Total coliforms were not higher than 10³ cfu/g among samples. E. coli was detected at both treatments. Thirty three percent of the cheese contained Staphylococcus aureus. Listeria spp. and Salmonella spp. were not detected in any treatment. Bacillus spp. incidence was 50% of the cheese, being B. cereus, B. cereus variety mycoides and B. pumilus. Bacillus cereus and Staphylococcus aureus grew at 4/20°C. Mesophilic aerobic bacteria were between 10⁴ and 10⁷ cfu/g. At 4/20°C counts decreased. At 4°C the behaviour was variable. Moulds were lower than 10⁴ cfu/g and yeasts were between 10⁴ and 10⁵ cfu/g. pH, moisture content and tritatable acidity ranges of samples were 5.5-6, 51-52.3% and 1.215-1.935 g/100 g of lactic acid, respectively. Manufacturing of this cheese includes a short heat treatment and starter culture addition; consequently, our results indicate that this processing may be insufficient for achieving hygienic cheese production. The storage at refrigeration temperature will not always guarantee the cheese safety and quality.     

Biochemistry of cheese ripening

Rennet-coagulated cheeses are ripened for periods ranging from about two weeks to two or more years depending on variety. During ripening, microbiological and biochemical changes occur that result in the development of the flavour and texture characteristic of the variety. Biochemical changes in cheese during ripening may be grouped into primary (lipolysis, proteolysis and metabolism of residual lactose and of lactate and citrate) or secondary (metabolism of fatty acids and of amino acids) events. Residual lactose is metabolized rapidly to lactate during the early stages of ripening. Lactate is an important precursor for a series of reactions including racemization, oxidation or microbial metabolism. Citrate metabolism is of great importance in certain varieties. Lipolysis in cheese is catalysed by lipases from various source, particularly the milk and cheese microflora, and, in varieties where this coagulant is used, by enzymes from rennet paste. Proteolysis is the most complex biochemical event that occurs during ripening and is catalysed by enzymes from residual coagulant, the milk (particularly plasmin) and proteinases and peptidases from lactic acid bacteria and, in certain varieties, other microorganisms that are encouraged to grow in or on the cheese. Secondary reactions lead to the production of volatile flavour compounds and pathways for the production of flavour compounds from fatty acids and amino acids are also reviewed.     

Use of mass spectrometry to characterize proteolysis in cheese

A rapid method for characterizing proteolysis in different cheese varieties was developed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). pH 4.6-soluble extracts of different cheese varieties were sub-fractionated using ethanol and 70% ethanol-soluble extracts were analyzed using MALDI-ToF-MS. Sample analysis time was ∼3 min compared to ∼100 min for reversed-phase HPLC; moreover, the method yielded accurate molecular masses of peptides originating as a result of proteolysis. Small peptides (<3500 m/z) were monitored and data for relative intensities of peptides were analyzed using multidimensional scaling (MDS) to demonstrate the similarity between profiles obtained from different cheese varieties. The similarities between the profiles of MALDI-ToF mass spectra were demonstrated in a two-dimensional space by performing MDS on the similarity matrix. Use of MALDI-ToF-MS is thus a fast and effective method for monitoring small peptides produced in cheese as a result of proteolysis.     

Intervarietal comparison of proteolysis in commercial cheese

 Proteolysis in different varieties of cheese, i.e. Cheddar, British, Dutch and Swiss types and Italian varieties, was compared by polyacrylamide gel electrophoresis (PAGE) and reverse-phase high-performance liquid chromatography (RP-HPLC). Urea-PAGE of the water-insoluble fraction (WISF) of cheese appeared to be unable to distinguish between Cheddar and Dutch-type cheeses, whereas it could be used to distinguish Emmental from Parmesan and both of these from Cheddar and Dutch types. Urea-PAGE of the 70%-ethanol-insoluble fraction of the water-soluble extract (WSE) showed large inter-varietal differences but there were also some intra-varietal differences. RP-HPLC of the 70%-ethanol-soluble and -insoluble fractions of the WSEs of cheeses was more effective than urea-PAGE of the WISFs or of the 70%-ethanol-insoluble fraction of the WSEs of cheeses when classifying cheese according to variety. However, analysis of a larger number of cheese samples is required to verify this result. One of the problems with using either urea-PAGE or RP-HPLC to identify cheese is that the characteristics analysed by these techniques change as ripening progresses. Received: 22 September 1997 / Revised version: 8 June 1998     

Proteolysis levels of white cheeses salted and ripened in brines prepared from various salts

This study was carried out to investigate the effects of salting and ripening periods in brines, prepared from various salts, on the properties of white cheeses. The brines were made from a mixture of NaCl with CaCl₂, KCl and MgCl₂ at 1 : 1 (molar basis) ratio. The NaCl solution alone was used as a control. The effects of different brines on total nitrogen, water-soluble nitrogen, nonprotein nitrogen, casein nitrogen and proteose-peptone nitrogen contents of cheeses were found to be insignificant, whereas the effect of ripening period on these properties was found to be significant ( P  < 0.05).     

Mini soft cheese as a simple model for biochemical studies on cheese-making and ripening

A new miniature cheese model obtained under controlled microbiological conditions was proposed, characterized and tested for reproducibility. Optimal heat treatment of cheesemilk was defined, as well as maximal ripening time. Miniature cheeses were obtained with batch pasteurized milk (65 °C, 30 min) and ripened at 5 °C. Lactic and nonlactic microbial populations were monitored by plate counts. Proteolysis was assessed by nitrogen fractions, electrophoresis and liquid chromatography, and a sniffing test was applied to evaluate aroma. Coliform bacteria decreased during ripening but moulds and yeasts increased up to 10⁴ cfu/g after 60 d, which defined the end of ripening period. Starter population remained constant during all ripening (10⁹ cfu/g), while nonstarter lactic acid bacteria increased from ∼10² to 10⁴ cfu/g. Soluble nitrogen levels at pH 4.6, in trichloracetic acid (0.73 mol/l) and in phosphotungtic acid (0.009 mol/l) were 151, 67, and 10 g/1000 g of the total nitrogen, respectively, after 60 d of ripening, which are usual values for soft cheeses. Proteolytic patterns as measured by electrophoresis were also similar to those of standard cheeses, as well as the aroma of the products. Peptide profiles revealed that the areas of most peaks increased with ripening time. The proposed model showed to be suitable for the production of mini cheese specimens for laboratory testing of cultures and enzymes in similar conditions to their real environment in the food matrix.     

Influence of residual milk-clotting enzyme on alpha(s1) casein hydrolysis during ripening of Reggianito Argentino cheese

Milk-clotting enzyme is considered largely denatured after the cooking step in hard cheeses. Nevertheless, typical hydrolysis products derived from rennet action on alpha(s1)-casein have been detected during the ripening of hard cheeses. The aim of the present work was to investigate the influence of residual milk-clotting enzyme on alpha(s1)-casein hydrolysis in Reggianito cheeses. For that purpose, we studied the influence of cooking temperature (45, 52, and 60 degrees C) on milk-clotting enzyme residual activity and alpha(s1)-casein hydrolysis during ripening. Milk-clotting enzyme residual activity in cheeses was assessed using a chromatographic method, and the hydrolysis of alpha(s1)-casein was determined by electrophoresis and high performance liquid chromatography. Milk-clotting enzyme activity was very low or undetectable in 60 degrees C- and 52 degrees C-cooked cheeses at the beginning of the ripening, but it increased afterwards, particularly in 52 degrees C-cooked cheeses. Cheese curds that were cooked at 45 degrees C had higher initial milk clotting activity, but also in this case, there was a later increase. Hydrolysis of alpha(s1)-casein was detected early in cheeses made at 45 degrees C, and later in those made at higher temperatures. The peptide alpha(s1)-I was not detected in 60 degrees C-cooked cheeses. The results suggest that residual milk-clotting enzyme can contribute to proteolysis during ripening of hard cheeses, because it probably renatures partially after the cooking step. Consequently, the production of peptides derived from alpha(s1)-casein in hard cheeses may be at least, partially due to this proteolytic agent.     

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.     

Distribution and stability of aflatoxin M1 during processing, ripening and storage of Telemes cheese

Telemes cheeses were produced using milk that was artificially-contaminated with aflatoxin M1 at the levels of 0.050 and 0.100 microg/l. The cheeses produced in the two cheese-making trials were allowed to ripen for 2 months and stored for an additional 4 months to simulate commercial production of Telemes cheese. Concentrations of aflatoxin M1 in whey, curd, brine, and the produced cheeses were determined at intervals by liquid chromatography and fluorometric detection coupled with immunoaffinity column extraction. Concentrations of aflatoxin M1 in the produced curds were found to be 3.9 and 4.4 times higher than those in milk, whereas concentrations in whey were lower than those in curd and milk. Aflatoxin M1 was present in cheese at higher concentrations at the beginning than at the end of the ripening/storage period, and it declined to concentrations 2.7 and 3.4 times higher than those initially present in milk by the end of the sixth month of storage. Concentrations of aflatoxin M1 in brine started low and increased by the end of the ripening/storage period but only a portion of the amounts of aflatoxin M1 lost from cheese was found in the brine. Results showed that Telemes cheeses produced from milk containing aflatoxin M1 at a concentration close to either the maximum acceptable level of 0.05 microg/l set by the European union (EU) or at double this value, will contain the toxin at a level that is much lower or slightly higher, respectively, than the maximum acceptable level of 0.250 microg of aflatoxin M1/kg cheese set by some countries.     

成熟温度对Mozzarella干酪蛋白水解和质构的影响

研究了温度为4,7,10℃时对干酪成熟过程中蛋白水解和质地的影响。结果表明,随着干酪成熟温度的升高,成熟期间干酪中可溶性氮与总氮的比值增加较快,干酪的硬度下降速度也较快。说明在较高的成熟温度下,干酪在较短的时间内能够达到成熟的状态。     

Relationship between proteolysis and angiotensin-I-converting enzyme inhibition in different cheeses

The inhibition of angiotensin-I-converting enzyme (ACE) by the ethanol (70%)-soluble fraction (ESF) from different cheeses was analysed with an extract from rabbit lung acetone powder as enzyme source and 2-furanacryloyl-1-phenylalanylglycylglycine (FAPGG) as substrate. Proteolysis was assessed by a spectroscopic o-phthaldialdehyde (OPA) assay of the pH 4.6-soluble fraction and ESF. Peptides in the ESF were separated by reverse phase-HPLC. The traditional Norwegian cheese Gamalost had per unit cheese weight higher ACE inhibition potential than Brie, Roquefort and Gouda-type cheese, likely due to the combination of highest protein content and most extensive proteolysis providing a high content of ACE inhibitory peptides. However, ACE-inhibition expressed as IC₅₀ per unit peptide concentration from ESF assessed by the OPA-assay was highest for Kesam, a Quark-type cheese with a low degree of proteolysis.     

The effect of plasmin activity and cold storage of cheese milk on the composition, ripening and functionality of mozzarella-type cheese

The effects of casein hydrolysis by plasmin on composition, ripening and functionality in mozzarella-type cheese were studied. Milk was stored at 4°C for 0, 24 and 48 h, with and without added plasmin, before batch pasteurization and the manufacturing of mozzarella-type cheese. In parallel experiments, the effects of incubation of milk with added plasmin for 2 h at 37°C prior to cheese manufacture and of an increase in the level of rennet added during cheesemaking were studied. Plasmin addition adversely affected the rennet coagulation properties of the milk and led to increased (two- to three-fold) plasmin activity in cheese and greater plasmin-associated hydrolysis of β-casein to γ-caseins during ripening. Melted cheese flow rate and stretchability increased over ripening and melting time decreased, but functional properties were not affected by either plasmin activity or cold storage. Overall, neither plasmin activity (at the levels studied) nor cold storage greatly affected the composition or functionality of mozzarella-type cheese.     

Conjugated linoleic acid content and isomer distribution during ripening in three varieties of cheeses protected with designation of origin

Cheeses have been identified as important sources of conjugated linoleic acid (CLA), a mixture of positional and geometric isomers with potential anticarcinogenic activity and other beneficial properties. The objectives of this study were to examine the effects of ripening on the overall CLA content as well as on the isomers profile using GC and Ag⁺-HPLC. Three Spanish cheeses Protected with Designation of Origin (Mahón, Manchego and Cabrales) were manufactured in different cheesemaking plants and monitored at different times during the ripening period. Total CLA content varied from 3 to 9 mg/g of total fatty acids and rumenic acid (9-cis, 11-trans C18:2, RA) represented more than 75% of total CLA. After RA, 7–9 (cis/trans plus trans/cis), 11-trans, 13-trans and 11-trans, 13-cis C18:2 were the main CLA isomers. The results achieved confirm that the effect of ripening on the total CLA concentration and isomer distribution was negligible.