Antibacterial potential of Enterococcus faecium strains isolated from ewes’ milk and cheese

The study was conducted to evaluate the antibacterial activity of three bacteriocin-producing Enterococcus faecium strains (TW15, TW20 and TW22) isolated from ewes’ milk and cheese sampled in the Patagonian region of Argentina. The strains were tested against spoilage and pathogens microorganisms showing antimicrobial activity towards 4 strains of Listeria monocytogenes, one strain of Listeria innocua and 2 strains of Staphylococcus aureus. E. faecium TW15, E. faecium TW20 and E. faecium TW22 were sensitive to vancomycin. Furthermore, investigation of virulence factors revealed the absence of the genes encoding them. The bacteriocin-like substances (BLISs) produced by the 3 strains were thermostable, pH resistant and can be expressed even in the presence of NaCl (3.0 g/100 g). Moreover, they prove to have a bactericidal mode of action. Results from physicochemical and biochemical characteristics of BLIS produced by these E. faecium strains make them potential candidates to aid in preservation of foods.     

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.     

Changes in the volatile fraction during ripening of Mahón cheese

Mahón cheeses, manufactured in the Island of Minorca (Spain), were produced and ripened under controlled conditions. Cheeses were sampled and analyzed at different stages of ripening. Twenty-one major aroma components were identified. It was observed that, during ripening until the most commercially valuable product was obtained, (60–90 days) only 16 compounds varied significantly with time: butanoic, isovaleric, hexanoic, octanoic and decanoic fatty acids (p<0.001), together with heptanoic acid (p<0.005), 2-methyl pentanone, 2-methyl heptanone, 2-methyl nonanone, methyl ketones (p<0.001), ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl tetradecanoate, ethyl hexadecanoate and ethyl esters (p<0.001). These changes could be described by means of zero order kinetics. Through a PCA analysis it was found that three factors explained ca. 87% of the total variance. According to the first two components the variables were grouped by their chemical characteristics (fatty acids, methyl ketones, ethyl esters) and the cases by their ripening time (0/18 days, 35 days, 53/67/82 days).     

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%).     

Ripening and seasonal changes in microbial groups and in physico-chemical properties of the ewes’ cheese Pecorino del Poro

Three batches of Pecorino del Poro, ewes’ cheese made from raw milk, were examined throughout a 28-day ripening time at three different seasons. High logarithmic counts per gram of cheese for mesophilic coccal-shaped lactic acid bacteria (6.70–12.45), mesophilic lactobacilli (4.82–11.73), thermophilic coccal-shaped lactic acid bacteria (2.30–9.90), and thermophilic lactobacilli (2.95–8.15) were found. Coccal-shaped lactic acid bacteria were the dominant microorganisms throughout ripening. The microorganisms used as an indicator of hygiene during manufacture of the cheeses, coliforms and Escherichia coli, were considerably lower, as were enterococci and yeasts. Coliforms and E. coli decreased sharply throughout ripening. Physico-chemical parameters such as pH (5.07–7.03), dry matter (46.34–72.79%), ether extract (31.35–51.84% of dry matter), crude protein (29.93–44.73% of dry matter), and chloride content (2.36–4.11% of dry matter) were also determined. Probably, the use of selected autochthonous mesophilic lactococci as a starter would control or suppress the growth of undesirable microorganisms. The results obtained suggest the need for improvements in milking and dairy conditions.     

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.     

Glycosylation of κ-casein: Genetic and nongenetic variation and effects on rennet coagulation properties of milk

The aims of this study were to investigate genetic and nongenetic variation in the degree of glycosylation of κ-casein (κ-CN) and to estimate the effects of glycosylated (G-κCN) and unglycosylated (U-κCN) κ-CN contents on milk coagulation properties of Simmental cows. Measures of contents of the main casein fractions, G-κCN, and U-κCN, and assessment of genotypes at CSN2, CSN3, and BLG were obtained by reversed-phase HPLC analysis of 2,015 individual milk samples. Content of total κ-CN (κ-CNtot, g/L) was the sum of G-κCN and U-κCN, and the glycosylation degree of κ-CN (GD) was measured as the ratio of G-κCN to κ-CNtot. Rennet coagulation time (RCT) and curd firmness were measured by using a computerized renneting meter. Measures of curd firmness were adjusted for RCT before statistical analysis. Variance components of κ-CNtot, G-κCN, U-κCN, and GD were estimated by Bayesian procedures and univariate linear models that included the class effects of the herd-test-day, parity, days in milk, genotypes at milk protein genes, and animal. These class effects, those of G-κCN, U-κCN, and content of other caseins, and the linear effect of milk pH were accounted for by models investigating the influence of κ-CN glycosylation on coagulation properties. The GD ranged from 22 to 76%, indicating that variation in G-κCN depends on the variation both in κ-CNtot and in the efficiency of κ-CN glycosylation. Genotype CSN3 BB exhibited high G-κCN and U-κCN relative to that of CSN3 AA. Heritability of G-κCN, U-κCN, and GD was high and ranged from 0.46 to 0.56. A large proportion of the additive genetic variation in G-κCN and U-κCN was attributable to influence of CSN and BLG, but these genes did not affect variation in GD, and across-genotypes differences in the trait were small or trivial. Average RCT of the milk class having the highest G-κCN was, on average, 2 min (standard deviation 0.5) shorter than that of the lowest class. Conversely, U-κCN and content of other caseins were not associated with any effect on RCT, except for a slight delay in coagulation when U-κCN was very high. Curd firmness increased when the contents of both κ-CN fractions and other caseins increased. This study provides evidence that the positive association between RCT and κ-CN content is exclusively attributable to the glycosylated fraction of the protein. Because exploitable additive genetic variation in G-κCN exists, improvement of κ-CN composition through selective breeding might be an effective way to enhance milk coagulation properties.     

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.     

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.     

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.     

Influence of chemical and biochemical characteristics on the texture of Appenzeller® cheese

Texture properties are very important quality parameters of Appenzeller^® cheese. The Appenzeller^® Cheese Makers Association defines optimal texture quality as being characterised by a softness that lasts at least three months. During winter production, texture-related problems are sporadically encountered. In the present study, ten Appenzeller^® cheese loaves with optimal texture properties aged for 90 days and ten loaves with dry and firm texture of the same age were selected and characterised through a uniaxial compression test and with physicochemical and biochemical methods. All cheeses had been produced in the same week during winter (January) in different cheese factories and ripened for three months. The aim of the study was to determine the main factors responsible for classification into the two groups based on univariate and multivariate statistical analysis tools. The three main parameters influencing the force at 33% deformation in this study were calcium, water, and free amino acids.     

Variations in some heavy metals’ level during processing of soft cheese

The aim of this study was to determine the content of lead, cadmium, aluminum, copper and mercury during Feta cheese manufacturing by using atomic absorption spectrometer. Results revealed that lead and mercury concentrations were higher after curdling and in fresh cheese after salting than other elements. Cadmium was detected at low concentrations in raw milk, pasteurized milk, after curdling and fresh cheese (0.053, 0.10, 0.20 and 0.24 mg/kg, respectively). Aluminum concentration did not change seriously during different steps of cheese manufacturing. On the other hand, the concentration of copper increased from 2.83 ± 0.97 mg/kg in raw milk to 3.25 ± 1.06 mg/kg in fresh cheese. It was concluded that the curdling and cheese after salting are the major technological steps that affect the concentration of some heavy metals rather than heat treatment.     

MRI ‘texture’ analysis of MR images of apples during ripening and storage

Images obtained by magnetic resonance imaging (MRI) of the apple varieties Idared, Redspur and Topaz during ripening and storage were analysed by texture analysis (TA) to determine the correlation between TA parameters and firmness, soluble solids content (SSC) and titratable acids. All varieties differ significantly in SSC, acidity and the firmness of fruits. Three sagital T2w slices measured at 4.7 T (resolution 256×256 pixels) were analysed numerically using the software MaZda 2.11 and the statistical package S-PLUS 4.5. A significant correlation was found between acidity and TA parameters such as sum average, sum variance and sum entropy. The dynamics of TA parameters during the periods of maturation and storage were described by polynomial functions.The following distinctive TA parameters were found: skewness and kurtosis (among histogram-based parameters), variance of absolute gradient (among gradient-based parameters), grey level nonuniformity (among run length matrix-based parameters) and especially some co-occurrence matrix-derived parameters such as correlation, sum average, sum variance and sum entropy (within 1-, 3-, and 5-pixel neighbourhoods). The TA parameter correlation obtained from MR images can be used for characterising fruit ripening.     

Influence of processing conditions on reducing γ-aminobutyric acid content during fortified milk production

The work studied the effects of processing conditions on the γ-aminobutyric acid (GABA) loss during fortified milk production. Bovine milk or their proteins/lactose fractions (0.66% whey protein and 2.6% casein or 4.9% lactose, w/v) containing 0.05–1.0% added γ-aminobutyric acid (w/w, based on bulk milk or these fractions) were subjected to a simulated milk technological process as following the sequential preheating (25–60 °C), homogenization (0–20 MPa), and pasteurization (62 °C/30 min, 72 °C/15 s, 95 °C/5 min, and 138 °C/2 s) or their unit processes to treat GABA. The resulting samples were characterized through GABA and lactose concentrations under various processing conditions. The amine and carboxyl groups and the structural characteristics of the resulting protein (lactose) were also examined through their concentrations (for lactose) and mass/spectral analyses, respectively. The results showed that the increase in temperature significantly promoted a reduction in GABA content. Whey protein fractions than caseins were primarily responsible for inducing GABA, whereas lactose had no remarkable effect on it. The rationale for GABA reduction is potential reactions with milk proteins/lactose, which preliminarily confirmed by the measurement of protein modification and lactose mass spectrometry.