Study of Enterobacteriaceae during the manufacture and ripening of San Simón cheese

The aim of this work was to study the evolution of the population of Enterobacteriaceae in one of the traditional Spanish cheeses, San Simón cheese, during the manufacture and ripening processes and its interrelation with the changes in some physico–chemical parameters.The evolution of the Enterobacteriaceae counts (VRBGA medium) and coliform counts (VRBA medium) was studied from samples of milk, curd and inner and surface zones of the cheese at different stages of ripening from five batches of traditionally manufactured artisan cheese. The counts obtained were very similar in both media and in general one log unit higher in the inner portion of the cheeses than on the surface.TheEnterobacteriaceae counts in milk were 10²–10³cfu g⁻¹and the counts increased during the first week of ripening reaching 10⁶–10⁷cfu g⁻¹in the inner portion of the cheese. From this time onwards, the counts slowly decreased to the end of ripening without disappearing completely.The most abundant species in the milk were Klebsiella oxytoca (36% of the isolated strains), Enterobacter cloacae (24%) and Klebsiella pneumoniae (20%). Escherichia coli, constituted the dominant species from the inner portion of the cheeses at the end of ripening (56% of the isolated strains), followed by Hafnia alvei (44%). However, in the samples of the surface portion of the cheese the dominant species at the end of ripening were K. oxytoca (40%), H. alvei (35%) and E. cloacae (20%).     

Stability of α-tocopherol, γ-tocopherol and β-carotene during ripening of pasta-filata cheese made from raw and pasteurised milk with different vitamin contents

Ripening stability of α-tocopherol, γ-tocopherol and β-carotene in cheese was evaluated in relation to different milk vitamin content and to the use of either pasteurised or raw milk. Milk from two farms with different management systems was used to obtain different vitamin content. Milk was divided into two parts, of which only one was pasteurised. Four blocks of cheese were made from each batch and ripened for 0, 15, 30, or 60 days at 14–16 °C. There was a notable variation in cheese vitamin levels, with the differences in milk vitamin content due to farm management having the highest impact. Pasteurisation had no effect on cheese vitamin content. Cheese γ-tocopherol and β-carotene content decreased after 30 and 60 days, respectively, whereas α-tocopherol content remained stable. γ-Tocopherol appeared to be the most efficient antioxidant in cheese, followed by β-carotene. Vitamin stability was not influenced by milk vitamin content or pasteurisation.     

Temporal differences in microbial composition of Époisses cheese rinds during ripening and storage

Époisses is a protected designation of origin smear-ripened cheese from the Burgundy region in France. It has an orange color and a strong flavor, both of which are generated by surface microorganisms. The objective of the present study was to investigate the microbial dynamics at the surface of Époisses cheese during ripening and postmanufacturing storage at low temperatures. Rind samples were analyzed by enumeration on agar plates and by 16S rRNA gene and internal transcribed spacer amplicon sequencing. During most of the ripening process, the counts of yeasts, which corresponded to the species Debaryomyces hansenii and Geotrichum candidum, were higher than those of the aerobic acid-sensitive bacteria. Debaryomyces hansenii reached a level of about 3 × 10⁸ cfu/cm², and its viability strongly decreased in the late stage of ripening and during storage at 4°C. Two of the inoculated bacterial species, Brevibacterium aurantiacum and Staphylococcus xylosus, did not establish themselves at the cheese surface. At the end of ripening, among the 18 most abundant bacterial species detected by amplicon sequencing, 14 were gram-negative, mainly from genera Psychrobacter, Vibrio, Halomonas, and Mesonia. It was hypothesized that the high moisture level of the Époisses rinds, due the humid atmosphere of the ripening rooms and to the frequent washings of the curds, favored growth of these gram-negative species. These species may be of interest for the development of efficient ripening cultures. In addition, because the orange color of Époisses cheeses could not be attributed to the growth of Brevibacterium, it would be interesting to investigate the type and origin of the pigments that confer color to this cheese.     

Influence of vegetable and animal rennet on proteolysis during ripening in ewes’ milk cheese

The renewed interest in using enzymes from thistles of the genus Cynara in the making of traditional ewes’ milk cheese prompted us to investigate the effect of vegetable and animal rennet on proteolysis during ripening of Los Pedroches cheese. Casein hydrolysis was found to be much more extensive and faster in cheese made by using vegetable rennet (the amount of soluble nitrogen at 60, 80 and 100 days of ripening was more than 28% greater than that in cheese produced using animal rennet). The levels of insoluble Tyr and Trp were higher in cheese produced with vegetable rennet. PAGE, using gels containing 7 M urea, revealed decreased contents in residual α_s-CN and β-CN, as well as markedly increased levels of the more mobile components in cheese produced from vegetable rennet at the end of ripening. On the other hand, the degree of proteolysis in terms of NPN or its main components (peptides, amino acids and ammonia) was similar in cheese produced using animal or vegetable rennet.     

Use of PCR-based methods and PFGE for typing and monitoring homofermentative lactobacilli during Comté cheese ripening

In order to obtain a better understanding of the biochemical events taking place in Saccharomyces cerevisiae during the lag phase, the proteins expressed during the first hours after inoculation were investigated by two-dimensional (2-D) gel electrophoresis and compared to those expressed in late respiratory growth phase. The studies were performed on a haploid strain (S288C) grown in defined minimal medium. Some of the abundant proteins, whose expression relative to total protein expression was induced during the lag phase, were identified by MALDI MS, and the expression of the corresponding genes was assessed by Northern blotting. The rate of protein synthesis was found to increase strongly during the lag phase and the number of spots detected on 2-D gels increased from 502 spots just after inoculation to 1533 spots at the end of the lag phase. During the first 20 min, the number of detectable spots was considerably reduced compared to the number of spots detected from the yeast in respiratory growth just prior to harvest and inoculation (747 spots), indicating an immediate pausing or shutdown in synthesis of many proteins just after inoculation. In this period, the cells got rid of most of their buds. The MALDI MS-identified, lag phase-induced proteins were adenosine kinase (Ado1p), whose cellular role is presently uncertain, cytosolic acetaldehyde dehydrogenase (Ald6p) and (DL)-glycerol-3-phosphatase 1, both involved in carbohydrate metabolism, a ribosomal protein (Asc1p), a fragment of the 70-kDa heat shock protein Ssb1, and translationally controlled tumour protein homologue (Yk1056cp), all involved in translation, and S-adenosylmethionine synthetase I involved in biosynthesis reactions. The level of mRNA of the corresponding genes was found to increase strongly after inoculation. By pattern matching using previously published 2-D maps of yeast proteins, several other lag phase-induced proteins were identified. These were also proteins involved in carbohydrate metabolism, translation, and biosynthesis reactions. The identified proteins together with other, yet unidentified, lag phase-induced proteins are expected to be important for yeast growth initiation and could be valuable biological markers for yeast performance. Such markers would be highly beneficial in the control and optimisation of industrial fermentations.     

Effect of natamycin-containing coating on the evolution of biochemical and microbiological parameters during the ripening and storage of ovine hard-Gruyère-type cheese

The effect of a coating containing natamycin on the ripening course of the hard-Gruyère-type cheese Graviera Kritis was assessed. A single treatment at an early stage of ripening was carried out; samples from natamycin-treated (NT) and control cheeses (CTR) were then taken throughout a 12 month ripening and storage period. Coating gave a statistically significant reduction in the yeasts and moulds counts in the cheese rind. It did not influence the counts and the evolution of the thermophilic bacteria related to starter or of the propionic acid bacteria, nor did it affect the associated aminopeptidase activities. Gross composition of NT cheeses did not differ significantly from that of the control cheeses; the same was also true for proteolysis. Natamycin in the cheese rind after the removal of coating was lower than 0.1 mg dm⁻² at all stages of ripening and no migration to the cheese interior was observed.     

Formation of the aroma of a raw goat milk cheese during maturation analysed by SPME–GC–MS

The volatile profile of the Spanish goat raw milk cheese of the protected designation of origin (PDO) “Queso Ibores” was studied at four stages of maturation (day 1, 30, 60, and 90) by the method of solid-phase micro-extraction–gas chromatography–mass spectrometry (SPME–GC–MS) to determinate the characteristic volatile compounds of this cheese and to know the changes in the volatile profile of this cheese during maturation. According to the PDO, Ibores cheese aroma varies between sweet and mild and it has a strong taste, slightly tart. A total of 64 compounds were detected: 14 acids, 18 alcohols, 13 esters, 6 ketones and 13 compounds which could not be classified in these groups. Carboxylic acids were the most abundant volatile compounds in the headspace of Ibores cheese. Content of volatile compounds was significantly modified (P < 0.05) during ripening. The relative total amounts of acids, esters and ketones increased during the first 60 days of maturation. The most characteristic compounds of Ibores cheese aroma were butanoic, hexanoic and octanoic acids, some alcohols (2-butanol and 2-heptanol), ethyl esters of hexanoic and butanoic acids, some methyl ketones (2-butanone, 2-pentanone and 2-heptanone) and δ-decalactone.     

Effects of Lactobacillus strains on the ripening and organoleptic characteristics of Arzúa-Ulloa cheese

Seven batches of Arzúa-Ulloa, a short-ripened soft cow's milk cheese produced in Galicia (NW Spain), were prepared from pasteurized milk. Two control batches of cheese (CB) were made with an acid-aromatic starter containing Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis var. diacetylactis, isolated from raw-milk Arzúa-Ulloa cheeses. Five batches of cheese (LB) were made with the acid-aromatic starter plus one of five strains of mesophilic homofermentative Lactobacillus spp.: four of them isolated from raw-milk Arzúa-Ulloa cheese (characterized in previous works) and the remaining was a commercial Lactobacillus strain. Higher counts of mesophilic viable bacteria, lactic acid bacteria and citrate-fermenting bacteria were found on days 1 or 15 of ripening, while higher counts of lactobacilli were found on day 30 of ripening. On day 1 of ripening the highest diacetyl-acetoin content was noted in the CB, but after day 15 the diacetyl-acetoin content was similar or higher in three of the five LB. The mean degradation of beta-casein in CB was higher than in LB, while the degradation of alpha(s1)-casein was higher in LB. The mean contents of nitrogen-soluble fractions were slightly higher in the LB than in the CB. Volatile free fatty acid (VFFA) contents were, in general, greater in LB than in CB and maximum amounts were determined on day 15 of maturation. Sensorial analysis indicated a more acid taste was in LB, while bitter and astringent tastes were more intense in CB. A positive correlation was found between beta-casein degradation and bitter taste. Yogurt and butter aromas were more intense in CB and in two of the five LB. Firmness was lower in LB and a negative correlation was found between this parameter and alpha(s1)-casein degradation. Crumbliness showed a positive correlation with beta-casein degradation. The use of the Lactobacillus strains assessed in this study is recommended for Arzúa-Ulloa cheese manufacture, in order to enhance the desirable characteristics of this cheese variety, i.e., a soft texture due to alpha(s1)-casein proteolysis but without the bitter taste due to beta-casein degradation and a spicy and slightly rancid aroma and taste.     

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.     

Enrichment of dry-cured ham with α-linolenic acid and α-tocopherol by the use of linseed oil and α-tocopheryl acetate in pig diets

The use of α-linolenic acid and α-tocopherol enriched pork on the fatty acids and the sensory characteristics of Spanish dry-cured hams have been studied. Five batches of hams were manufactured using the posterior legs of pigs fed on diets with the same ingredients except for the oil source: sunflower (C), linseed (L) or linseed and olive (1/1, w/w, LO). Two different α-tocopheryl acetate concentrations [20 (C, L and LO) or 220 (LOE and LE)mg/kg diet] were used. Biceps femoris and Semitendinosus/Semimembranosus muscles from hams with low polyunsaturated fatty acid n-6/n-3 ratio (less than 3) were obtained from animals fed on linseed and linseed/olive oil enriched diets. However, hams from animals fed on diets added with linseed and α-tocopheryl acetate (20mg/kg diet) (batch L) were rejected by consumers because of less acceptable sensory characteristics and higher TBARs. The remaining hams had satisfactory sensory and nutritional characteristics.     

Formation of toxic 2-nonyl-p-benzoquinones from α-tertiary 4-nonylphenol isomers during microbial metabolism of technical nonylphenol

In many environmental compartments, microbial degradation of α-quaternary nonylphenols proceeds along an ipso-substitution pathway. It has been reported that technical nonylphenol contains, besides α-quaternary nonylphenols, minor amounts of various α-H, α-methyl substituted tertiary isomers. Here, we show that potentially toxic metabolites of such minor components are formed during ipso-degradation of technical nonylphenol by Sphingobium xenophagum Bayram, a strain isolated from activated sewage sludge. Small but significant amounts of nonylphenols were converted to the corresponding nonylhydroquinones, which in the presence of air oxygen oxidized to the corresponding nonyl-p-benzoquinones-yielding a complex mixture of potentially toxic metabolites. Through reduction with ascorbic acid and subsequent analysis by gas chromatography-mass spectrometry, we were able to characterize this unique metabolic fingerprint and to show that its components originated for the most part from α-tertiary nonylphenol isomers. Furthermore, our results indicate that the metabolites mixture also contained several α, β-dehydrogenated derivatives of nonyl-p-benzoquinones that originated by hydroxylation induced rearrangement, and subsequent ring and side chain oxidation from α-tertiary nonylphenol isomers. We predict that in nonylphenol polluted natural systems, in which microbial ipso-degradation is prominent, 2-alkylquinone metabolites will be produced and will contribute to the overall toxicity of the remaining material.     

Microbiological and biochemical changes during ripening of salchichón,a Spanish dry cured sausage

Development of the microflora and changes in the lipid and protein fractions during the ripening of salchichón, a kind of Spanish dry cured sausage, were studied. A commercial mixture of Pediococcus pentosaceus andMicrococcus varians was used as starter culture. Total aerobic bacteria, lactic acid bacteria, Micrococcaeae, Staphylococcus aureus, Entero-bacteriaceae , sulphite reducer Clostridium , yeasts and moulds were determined. The sarcoplasmic and myofibrillar solubility, denaturation and proteolysis index, parameters related to fat stability and the free fatty acid composition were analysed throughout the curing process: after mincing, after the fermentation stage, after the 2nd week of drying and in the final product (4th week of drying). The major changes in the characteristics of salchichón took place during the fermentation stage. This was due to proteolysis and lipolysis phenomena derived from high counts of lactic acid bacteria and Micrococcaeae in this stage. These changes were shown in the increase of the total free amino acid content, the peroxide index and the carbonyl compound content, and in the decrease of the protein solubility and the percentage of polyunsaturated free fatty acids, thus achieving the organoleptic characteristics of the final product.     

Characterisation by SPME–GC–MS of the volatile profile of a Spanish soft cheese P.D.O. Torta del Casar during ripening

The volatile profile of the Spanish soft cheese of the Protected Designation of Origin (PDO) Torta del Casar, made from raw ewes’ milk, was studied in four different stages of ripening (1, 30, 60 and 90 days) by the method of SPME–GC–MS. A total of 46 compounds were detected: 13 acids, 9 esters, 4 ketones, 7 alcohols, 3 aldehydes, 7 aromatic compounds and 3 compounds which could not be classified into those groups. Carboxylic acids were the most abundant group isolated; their levels significantly increased during ripening and comprised 61.5% of the total aroma extract at the end of ripening. At day 90, acids of microbial origin were the most abundant, followed by acids derived from amino acids while acids from lipolysis of triglycerides were the least abundant. Esters were the next most important group and their amount also increased significantly during maturation, as did ketones, while alcohols content slightly decreased at the end of the ripening. At day 90, the compounds at highest levels were acetic acid, 3-methylbutanoic acid, butanoic acid propyl ester and 2-butan-one; so these compounds, due to their high levels and their low threshold value could play an important role in the final aromatic profile of this cheese.     

Changes in physico-chemical, microbiological, textural and sensory attributes during ripening of dry-cured foal salchichón

The changes in the physico-chemical, microbiological, textural and sensory attributes of foal salchichón were followed during ripening. Foal salchichón samples were taken at 0 days (mix before stuffing), and after 7, 14, 21, 28, 35, 42 and 49 days of ripening. The final a_w was 0.82, whereas pH values stayed around their initial values. TBAR'S values increased significantly (P < 0.001) during processing, from 0.44 to 2.26 mg/kg of sausage. Ripening time also affected the lightness (L*), redness (a*) and yellowness (b*) (P < 0.001). Hardness, gumminess and chewiness increased (P < 0.001) from 0.96, 0.6 and 0.47 to 46.92 kg/cm², 21.34 kg/cm² and 15.14 kg, respectively during processing, whereas cohesiveness and springiness values decreased during ripening. Lactic acid bacteria increased slowly in number and a large increase of Micrococcaceae was noticed. Regarding sensorial characteristics, foal salchichón samples showed high values for intensity of flavour (7.22 ± 0.44), hardness (7.33 ± 0.71) and dryness (6.67 ± 0.71) and low scores for acid taste (1.67 ± 0.71) and saltiness (3.11 ± 0.6).     

Lipid and protein oxidation of α-linolenic acid-enriched pork during refrigerated storage as influenced by diet supplementation with olive leaves (Olea europea L.) or α-tocopheryl acetate

The objective of this study was to evaluate the effect of diet supplementation with olive leaves or α-tocopheryl acetate on lipid and protein oxidation of raw and cooked n-3 enriched-pork during refrigerated storage. Enrichment of pork with α-linolenic acid through diet supplementation with linseed oil enhanced (p≤0.05) lipid oxidation in both raw and cooked chops but had no effect (p>0.05) on protein oxidation during refrigerated storage while decreasing (p≤0.05) the sensory attributes of cooked pork. Diet supplementation with olive leaves or α-tocopheryl acetate had no effect (p>0.05) on the fatty acid composition of pork but decreased (p≤0.05) lipid oxidation while exerting no effect (p>0.05) on protein oxidation in both raw and cooked α-linolenic acid-enriched chops stored and chilled for 9days. Moreover, olive leaves and α-tocopheryl acetate supplemented at 10g/kg and 200mg/kg diet, respectively, exerted (p≤0.05) a beneficial effect on the sensory attributes of cooked α-linolenic acid-enriched pork chops.     

Effect of added α-ketoglutaric acid,pyruvic acid or pyridoxal phosphate on proteolyis and quality of Cheddar cheese

Cheddar cheese curds were supplemented with 1, 5 or 20 g of α-ketoglutarate or pyruvic acid or 1.2 g pyridoxal-5¹-phosphate/kg cheese curd. The higher levels of keto-acids (5 or 20 g/kg curd) caused undesirable changes in the physico-chemical properties of resultant cheese. All levels of α-ketoglutarate reduced the pH of the cheese and promoted syneresis during pressing, while pyruvic acid increased the pH of the cheese. The numbers of starter and non-starter lactic acid bacteria were not affected by the addition of keto-acids or pyridoxal-5¹-phosphate. α-Ketoglutarate or pyruvic acid, at 1 g/kg, or pyridoxal-5¹-phosphatase, at 1.2 g/kg cheese curd, did not influence primary proteolysis in the cheese. The highest and lowest concentrations of total and individual free amino acids were found in the cheeses treated with pyruvic acid or α-ketoglutarate, respectively. The concentrations of most amino acids were lower in the cheeses treated with pyridoxal-5¹-phosphate than in the control. The results of this study suggest that α-ketoglutarate and pyridoxal-5¹-phosphate enhanced the degradation of most amino acids in Cheddar cheese while pyruvic acid promoted the formation of amino acids. The cheeses treated with α-ketoglutarate were more mature than the control cheese of the same age while pyruvic acid-treated cheese had a better flavour than the control.