Y. Barcina

Identification and characterization of proteolytic activity of Enterococcus spp. isolated from milk and Roncal and Idiazábal cheese

Roncal and Idiazábal cheeses are manufactured from ewes milk in the Autonomous Region of Navarre in Spain. Levels of enterococci are high in these cheeses and in other types of ewes-milk cheeses. The present study has identified enterococci present in the milk used and in the Roncal and Idiazábal cheeses after 120 days of ripening. A total of 282 strains were isolated and identified, and the cytoplasmic and extracellular enzymatic activities of some of the strains were assessed. The dominating species were Enterococcus faecalis, which accounted for 85% of the total both in the milk as well as in the two types of cheese, and Enterococcus faecium, Enterococcus durans, and Enterococcus avium which were present in lower proportions. Aminopeptidase and proteinase activity levels in enterococci were low, and considerable variation between strains of the same species was recorded, highlighting the need to study individual strains when selecting the most suitable bacteria as a starter culture. Aminopeptidase activity levels for the enterococci were appreciably higher at pH 7 than at pH 5.5, hence aminopeptidase activity by enterococci would appear to be less significant in the normal manufacturing conditions of the two cheeses.

Formation of biogenic amines in Idiazabal ewe's-milk cheese: effect of ripening, pasteurization, and starter

The importance of biogenic amines stems from their toxicity and their potential as a cause of food poisoning. After fish, cheese has been the foodstuff most often responsible for cases of food poisoning caused by ingestion of biogenic amines. Changes in biogenic amine (histamine, tyramine, putrescine, cadaverine, tryptamine, isopentylamine, spermidine, and phenylethylamine) content during the ripening of a cheese made from raw ewes milk were studied, together with the effects of pasteurization and a commonly used commercial starter and indigenous starter cultures. Biogenic amines were determined by high-pressure liquid chromatography following extraction from the cheese and derivatization with dansyl chloride. Levels of histamine, potentially the most toxic amine, in the semihard Idiazábal cheese were very low. Tyramine, putrescine, and cadaverine were the main amines present in this type of cheese. Levels of those amines increased during ripening. Pasteurization of the milk brought about a reduction in the biogenic amine content of the ripened cheese, irrespective of the starter culture employed. The indigenous starter cultures considered in this study produced higher levels of histamine and cadaverine and lower levels of tyramine and putrescine than the commercial starter culture tested.

Indigenous Lactic Acid Bacteria in Idiazábal Ewes’ Milk Cheese

Abstract Three batches of Idiazabal cheese were studied at different production stages. Counts obtained on three solid media (M17, MRS and MSE agar) showed growth during coagulation, pressing and brining operations and a decrease during the two first months of ripening. While the pH and water activity decreased, dry matter and NaCl increased during ripening. Physiological and biochemical characteristics of lactic acid bacteria were studied for their identification. Of the Lactococcus strains, 61% were identified as Lc. lactis subsp. lactis biovar diacetylactis , 30% as Lc. lactis subsp. lactis , 7% as Lc. lactis subsp. cremoris and 2% Lc. raffinolactis . All Lactobacillus isolated from milk and curd and 19% of those isolated from cheese were identified as Lb. plantarum . The predominant Lactobacillus in cheese was Lb. casei subsp. casei (64%). Leuconostoc lactis was identified in cheese.

Ripening of Ossau-Iraty cheese: determination of free amino acids by RP-HPLC and of total free amino acids by the TNBS method

Abstract The free amino acids and sulphosalicylic acid-soluble N fraction in the French ewes’-milk cheese, Ossau-Iraty, were determined to evaluate the degree of proteolysis during ripening of the cheese. RP-HPLC was used to assess the free amino acids, the trinitrobenzenesulphonic acid method to assess the sulphosalicylic acid-soluble N fraction. The total free amino acids content as determined by RP-HPLC ranged from 200 mg/100 g dry matter on the first day of ripening to 2200 mg/100 g dry matter after 120 d of ripening. The major amino acids were glutamic acid, asparagine, glutamine, valine, leucine, phenylalanine and lysine, which accounted for between 50% (on day 1) and 70% (on day 120) of the total free amino acids. Correlations between all the main free amino acids and ripening time were good. Regression of the sulphosalicylic acid-soluble N fraction as determined by the trinitrobenzenesulphonic acid method on the total free amino acids as determined by RP-HPLC yielded a correlation coefficient value of 0.985.

Evolution of free amino acids during Idiazábal cheese ripening

Abstract As a contribution to knowledge of ripening ewes cheese, the evolution of free amino acids was studied over a year of the ripening period by reversed phase high performance liquid chromatography (RP-HPLC). Total free amino acid content during Idiazabal cheese ripening varied between 140 mg/100 g dry matter the first day and 1500 mg/100 g dry matter at one year. The major amino acids were: glutamic acid, leucine, valine, phenylalanine, lysine and alanine which approximately accounted for 50% of the total free amino acid content, probably indicating the preferential cleavage of peptide bonds by the proteolytic agents in the cheese. Non-protein amino acids such as ornithine, citrulline or γ-aminobutyric acid were also found in proportions near to 9% of the total free amino acids. Amino acids such as arginine, which is responsible for unpleasant tastes, or γ-aminobutyric acid, which is usually detected in low quality cheese, are present in very low concentrations in Idiazabal cheese.

Effect of pasteurization and use of a native starter culture on proteolysis in a ewes’ milk cheese

Abstract The effect of different manufacturing methods on the production of a ewes’ milk cheese was studied. Three batches were made: raw milk without added starter, raw milk with added starter, and pasteurized milk with added starter. The starter culture was composed of Lactococcus lactis subsp lactis biovar diacetylactis and Lactobacillus casei subsp casei . Samples were taken from all three cheese batches on 1, 90, and 180 days of ripening for analysis of the soluble nitrogen (SN) and non-protein nitrogen (NPN). SN and NPN levels were the highest in the batch made from the pasteurized milk, which also exhibited the highest level of degradation of the α s and β-caseins. Conversely, that batch had the lowest total free amino acid (FAA) content. This parameter was the highest in the batch made from the raw milk with added starter. The cheeses made from the pasteurized milk presented the highest serine and asparagine contents, but γ-aminobutyric acid levels were negligible.

Changes in the free amino acid content during ripening of Idiazabal cheese: influence of starter and rennet type

The effect of starter and rennet type on free amino acid release during ripening of Idiazabal cheese was studied. Four batches of cheeses were manufactured depending on the rennet used, commercial calf rennet or artisanal lamb rennet, and the addition or not of starter culture. Cheese samples contained 24 individual free amino acids Leu, Glu, Val and Phe showing the highest contents during the ripening. The results indicated that the release of the free amino acids during ripening was strongly affected by starter added to the cheeses, and that this effect varied markedly with the rennet used for cheesemaking. Total amounts of free amino acids were higher for the cheeses made with commercial calf rennet than for those made with artisanal lamb rennet, regardless of starter addition. Likewise, the highest total free amino acid levels were found in the cheeses made with starter, regardless of the type of rennet used.

Validation of capillary electrophoresis in the analysis of ewe's milk casein

Recent investigations have shown that capillary electrophoresis (CE) can be an alternative to other techniques such as polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the qualitative analysis and separation of the different casein fractions in cows and ewes milk. However, past work has not yet clarified whether that method can achieve good quantifications. The present study has used a commercial whole ovine casein standard and a mixture of the standard and whole casein extracted from ewes milk cheese to test the reliability of the technique. The results show that CE was able to quantify the ewes milk caseins. The areas under four of the most representative peaks on the electrophoretogram for two alpha and two beta-caseins (designated alpha-casein1CE, alpha-casein2CE, beta-casein1CE, and beta-casein2CE in order of elution) were used to validate the method. In relation to linearity, coefficient of determination (r2) values greater than 99% were obtained for the regressions of each of the caseins. Moreover, each casein yielded response factors with a relative standard deviation (R.S.D.) of less than or equal to 5. The coefficients obtained in the day-to-day reproducibility analysis were higher than those for the same-day repeatability, but all the values were within acceptable limits. In the study of accuracy, the percentage recovery rates for the alpha-casein fractions were higher than those for the beta-casein fractions, hence quantification of the latter using this technique would appear to be more accurate under the conditions employed.

Optimization of indirect parameters which affect the extractability of volatile aroma compounds from Idiazábal cheese using analytical supercritical fluid extractions (SFE)

Abstract Various sample preparation techniques and collection procedures for a supercritical fluid extraction (SFE) system were tested and compared. Special attention was paid to parameters which influence the extraction results, but are not part of the extraction process per se. The application of SFE to the extraction of the aroma components from unsmoked ewe’s milk Idiazabal cheese was undertaken for the first time. It was, therefore, necessary to optimize and validate the extraction conditions. The parameters which were optimized included sample preparation (lyophilization and sonication); sample to adsorbent ratio; reconstitution solvent; trap type and trapping temperature. The best extraction results were achieved for a comminuted cheese sample containing added alumina (1:1 ratio with cheese) as the adsorbent and n-hexane:acetone (2:1) as the reconstitution solvent, and a polar octadecylsilica (ODS) trap held at a temperature of −5°C to retain volatile compounds.

Brining time effect on physicochemical and microbiological parameters in Idiazábal cheese

Physicochemical and microbiological parameters were compared for three brining times (12, 24 and 36 h) for fresh, young, semihard and hard Idiazabal cheese. Longer brining time produced higher salt, dry matter and salt-moisture ratio and lower water activity values for all types of cheese according to ripening time, while non-significant changes were observed for pH. In fresh cheese (1-15 days ripening), non-significant differences for microbiological counts in relation to brining time were observed, except for moulds. In young and hard cheeses, Lactobacillus and Leuconostoc showed lower counts with longer brining times. In contrast, Micrococaceae, yeast and moulds were stimulated by higher salt content in matured cheeses. In addition. this work has proved that there are lower water activity values and lower microbiological counts in longer-matured Idiazabal cheeses. For the different brining and ripening times, positive correlations were observed among most of the microbial groups studied, but a different behavior was established for Enterococcus, Clostridium tyrobutyricum, yeast and moulds.