Paloma Torre

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.

Use of RAPD and mitochondrial DNA RFLP for typing of Candida zeylanoides and Debaryomyces hansenii yeast strains isolated from cheese

Summary In order to evaluate the genetic diversity of the yeast flora in Northern Spain cheese, we adapted two molecular techniques devised for Saccharomyces, RAPD and RFLP of mitochondrial DNA, to type 27 Candida zeylanoides strains and 28 Debaryomyces hansenii strains isolated from Roncal and Idiazabal cheese at different stages of manufacture in different dairies. RAPD with (GTG)5 primer, although very reproducible, only defined 2 groups of strains for both species. On the other hand, mtDNA RFLP proved to be more discriminating and defined 5 groups of strains for both species. In addition, the strains from the minor RAPD groups in both species corresponded to specific groups defined by mtDNA RFLP. Overall, this analysis has revealed an important genetic diversity, considering the limited sample of different biotopes tested. Nevertheless, the mtDNA RFLP results indicate that a large majority of the strains appeared closely related, suggesting little variability between cheese and dairies. This work has demonstrated that rapid identification methods (the typing of strains can be achieved in two days) can be applied to these poorly characterized yeast species, allowing a rapid evaluation of genetic diversity of these species in different biotopes.

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.

The effect of a commercial starter culture addition on the ripening of an artisanal goat's cheese (Cameros cheese)

The evolution of physicochemical parameters, and the most important microbial groups, were determined for the following three batches of ‘Cameros’ goats milk cheese during ripening: Batch R elaborated with raw milk, Batch RS elaborated with raw milk and with the addition of a starter culture, and Batch PS elaborated with pasteurized milk and with the addition of the same culture. No differences in total solids (TS) or in the content of NaCl, fat and total nitrogen (expressed as percentages of TS) were found during the ripening. The pH, fat acidity and non‐protein nitrogen (NPN, expressed as a percentage of TN) showed significant differences between the batches. The inoculated batches showed the fastest drop in pH at the beginning of the ripening period, but the cheeses of Batch R showed a higher degree of lipolysis and proteolysis. The addition of a starter influenced the microbiological quality of the cheeses. Differences in the counts of Enterobacteriaceae and faecal coliforms were found between Batches R and RS after 15 days. Staphylococcus aureus increased in number during the early period of ripening and attained a population above 6 log cfu g−1 in Batch R in the period from 5 to 10 days. However, enterotoxins were not detected in this Batch. Batch R showed lower values of lactic acid bacteria at the beginning of the ripening period, but no significant differences were found between batches in the period from 5 to 15 days of ripening. At the beginning of the ripening, Lactococcus was the main lactic acid bacteria, with L. lactis lactis being predominant. After 15 days, the lactic acid bacteria counts decreased in the three batches, especially in the cheeses of Batch PS (only 2·2 log cfu g−1 was found at 60 days), as lactococci (the only lactic acid bacteria present in Batch PS) are incapable of growing under the conditions found in cheeses at the end of their ripening period. At this time, Lactobacillus was the predominant genus in Batches R and RS, with L. plantarum predominant. No lactococci were found from day 30 in Batch R and from day 40 in Batch RS. The cheeses of Batch RS received the most favourable scores from the tasting panel for all attributes judged: cut appearance, colour, aroma, taste, texture and general acceptance.

Seasonal changes in the composition of bulk raw ewe's milk used for Idiazabal cheese manufacture

Seasonal changes in the composition of bulk raw ewes milk used for Idiazabal cheese manufacture and its influence on the cheese yield are reviewed. This review is based on biochemical and microbiological data previously published on the composition of a bulk raw ewes milk collected at three different times of the year. In order to characterize, in a more extensive perspective, the seasonal changes in the composition of this bulk raw ewes milk and its influence on actual cheese yield, multivariate statistical analyses were applied to the compositional data of the milk and the actual cheese yield obtained at the factory. The results show pronounced seasonal changes in the composition of bulk raw ewes milk. Changes in cheese yield, lipolytic activity and microbiological quality of the bulk raw ewes milk are the principal aspects seasonally affected during the cheesemaking period. Also, the seasonal changes observed in the bulk raw ewes milk composition strongly affect the quality of Idiazabal cheeses manufactured over the cheesemaking period.

Free amino acids and volatile compounds in an ewe’s milk cheese as affected by seasonal and cheese-making plant variations

Abstract Analysis of free amino acids (FAAs) and volatile compounds in Roncal cheese throughout the cheesemaking seasons was carried out. The possible relationships between them were studied. Also, the influence of two cheesemaking plants was analysed. Total FAAs was 30–50% higher in cheeses made in summer than in cheeses made in either winter or spring. The main amino acids, which accounted for about 69% of the total FAAs, were Glu (20%), Leu (14%), Val (10%), Lys (9%), Phe (6%), and Pro and Ile (about 5% each). Besides six miscellaneous components, the 64 volatile components identified comprised nine hydrocarbons, 16 alcohols, two sulfur-containing compounds, seven aldehydes, eight ketones, six acids and 10 esters. Season was the first factor affecting the concentration of FAAs, and dairy plant was the principal one influencing the level of volatile compounds. Significant interactions between both factors found for the volatile components made very difficult to correlate their formation with the precursor amino acids.

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.

Evaluation of the effect of rennet type on casein proteolysis in an ovine milk cheese by means of capillary electrophoresis.

Capillary electrophoresis was used to study the evolution of casein throughout the ripening process of Roncal Denomination of Origin ewes milk cheese and to assess the type of rennet in its hydrolysis. Two manufactures were prepared, each with four vats; two of them had added lamb artisan rennet, batch A [clotting activity of 97.54 rennet units (RU) ml(-1)] and batch B [clotting activity of 16.26 RU ml(-1)]; one vat included calf industrial rennet, batch I (clotting activity of 45.70 RU ml(-1)); and the fourth vat had added mixed rennet, batch M, a 50:50 mixture of lamb (batch A) and calf (batch I) (clotting activity of 77.53 RU ml(-1)). The content of casein nitrogen in fractions alpha-casein1CE, alpha-casein2CE, beta-casein1CE and beta-casein2CE was quantified in cheese after 1, 15, 30, 60, 120 and 180 days of ripening. Beta-casein fractions undergo lesser degradation during the ripening time than alpha-casein proteins. The degradation of alpha-caseins is very much influenced by the clotting activity of the rennet used, so that the more active the clotting activity the greater the hydrolysis of those caseins. Nevertheless, it is at the level of beta-caseins that we observe the evidence of the influence of the type of rennet, thus noting a less intense proteolytic activity in the batch made with calf rennet, batch I.

Influence of rennet milk-clotting activity on the proteolytic and sensory characteristics of an ovine cheese

Abstract Roncal cheese (regulated by an Apellation of Origin) is a traditional hard cheese manufactured from raw ewes milk in the region of Navarre in Spain. Roncal cheeses, manufactured using two lamb rennets with different milk-clotting activity levels, were evaluated to compare their chemical, proteolytic, and sensory characteristics. A preliminary study of samples of lamb rennets indicated that a large proportion of such rennets did not fulfil current microbiological requirements and likewise revealed considerable variation in the milk-clotting activity of the samples examined. Trends in the overall physicochemical parameter values (pH, dry matter, fat, and protein) were similar in both cheese batches. Proteolysis of the nitrogen fractions was observed to take place at a faster rate in the cheeses made using the rennet with the higher milk-clotting activity (soluble nitrogen, non-protein nitrogen, and amino acid nitrogen values around 13–20% higher than in the cheeses made using the rennet with the lower milk-clotting activity after 180 days of ripening). Urea-PAGE electrophoretic analysis of the caseins from the cheeses manufactured using both types of rennet showed that the β-caseins were less susceptible to proteolysis than the α s -caseins. The effect of the different milk-clotting activity levels was most pronounced on the α s -caseins, in which the rennet with the higher milk-clotting activity gave higher breakdown. Nevertheless, the differences in the proteolysis rates did not yield any appreciable sensory differences.

PCR and RAPD identification of L. plantarum strains isolated from ovine milk and cheese. Geographical distribution of strains

Lactobacilli, and specifically Lactobacillus plantarum, are an important group of microorganisms in ovine cheeses, even though they are not ordinarily included in the starter cultures added. The present study effected counts of lactobacilli in Roncal Protected Designation of Origin (PDO) milk and cheese samples and isolated a total of 1026 strains. The strains were identified to species level by polymerase chain reaction (PCR) using L. plantarum-specific oligonucleotide primers, and the strains belonging to this species were then characterized by randomly amplified polymorphic DNA (RAPD). The percentage of L. plantarum present in the cheeses depended on the plant where the cheese was manufactured. Cluster analysis of the RAPD profiles obtained revealed seven main clusters. On comparing the strains, most of the strains present in the cheese were found not to have come from the raw milk.