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%).     

Fate of Listeria innocua during production and ripening of smeared hard cheese made from raw milk

The fate of 2 different Listeria innocua strains was analyzed during the production and ripening of smeared raw milk Greyerzer cheese (Gruyère). These strains were used as surrogates for the pathogenic Listeria monocytogenes, as they are physiologically very similar. Bacterial cells were added to the cheese milk at levels of 10⁵ cfu/mL. During the first 24 h of cheese making, the number of the test strains decreased to a level of below 10² cfu/g. Obviously, the cooking temperature of 56°C and the subsequent slight temperature decrease to 50°C within 70 min contributed to a distinct reduction of Listeria counts. The counts in the cheese cores did not exceed 10³ cfu/g within 12 wk of cheese ripening and Listeria was not detectable after 24 wk. In contrast to the cores of the cheeses of the 4 batches in this study, their rinds always contained a high listerial load of approximately 10⁶ to 10⁸ cfu/g throughout the entire ripening period. The smeared surface showed an increase of pH to alkaline values, corresponding to smear microbiota development. Coryneforms and Staphylococcus counts were stable at >10⁷ cfu/cm² over 175 d, whereas yeast counts decreased to about 10⁵ cfu/cm² at the end of ripening. The study shows that the smear culture had no noticeable anti-listerial potential. When removing the rind or portioning such smeared cheese loaves with a cutting device, a postprocess contamination of the core might occur, thus presenting a major hygienic risk.     

Effect of addition of native cultures on characteristics of Armada cheese manufactured with pasteurized milk: A preliminary study

Six batches of Armada cheese were produced, one from raw milk with no added starter, another from pasteurized milk with a commercial mesophilic starter and four from pasteurized milk with experimental starters. These starters included: Lactococcus lactis subsp. lactis and L. lactis subsp. lactis biovar. diacetylactis; or the same strains plus either Enterococcus raffinosus, Leuconostoc mesenteroides subsp. dextranicum or Lactobacillus plantarum, all isolated from Armada cheese made from raw milk. The highest counts of aerobic mesophilic bacteria and populations of lactic acid bacteria for all batches were reached in the first week of ripening. These counts declined later throughout the ripening, although not at identical rates in every batch. Counts of lactobacilli were significantly higher in cheeses made from raw milk and those inoculated with the lactobacilli strain than in the other batches, over the whole ripening period. Added native starters minimized growth of Enterobacteriaceae. Cheeses made with the starter containing the Enterococcus strain had the most favourable sensory attributes throughout ripening.     

Changes in the Microflora of Valdeteja Raw Goat's Milk Cheese throughout Manufacturing and Ripening

Changes in the microbiological flora of Valdeteja, a ripened home-made raw goat's milk cheese produced in northwest Spain, were studied during the manufacture and ripening processes using the spiral plating method. High counts of all microbial groups were observed in milk (7.66, 7.57, 6.13, 6.91, 2.47, 5.18, 3.27 and 3.85 log₁₀ cfu/g for aerobic mesophilic bacteria, lactococci, lactobacilli, leuconostocs, enterococci, Enterobacteriaceae,Micrococcaceae and moulds and yeasts, respectively). Counts increased between 0.76 and 1.96 log units from milk to curd. Lactic acid bacteria (lactococci, lactobacilli and leuconostocs) were the dominant microorganisms throughout ripening. Lactococci dominated over the lactobacilli up to day 27 of ripening. Leuconostocs were stable from day 2 of ripening. The final levels of typical enterococci were 3.62 log₁₀ cfu/g. Aerobic mesophilic bacteria, Enterobacteriaceae andMicrococcaceae reached their maximum in curd and decreased significantly throughout ripening. The decrease ofEnterobacteriaceae was very marked, although they did not completely disappear at the end of the ripening process (2.74 log₁₀ cfu/g). Moulds and yeasts counts increased significantly during ripening, giving final levels of 6.09 log₁₀ cfu/g. A strong correlation (P<0.001) was found between pH and lactobacilli (R=−0.87), leuconostocs (R=−0.82) and moulds and yeasts (R=−0.74) and between a_w and various microbial groups (e.g. EnterobacteriaceaeR=0.83).     

Effect of autochthonous starter cultures isolated from Siahmazgi cheese on physicochemical,microbiological and volatile compound profiles and sensorial attributes of sucuk,a Turkish dry-fermented sausage

The effect of adding autochthonous starter cultures isolated from Siahmazgi cheese, on the physicochemical parameters and microbial counts of sucuk was investigated during the ripening period. SPME–GC/MS was used in volatile compound analysis and a trained group of panelists carried out sensory analysis of the final product. After preliminary screening, three strains of Lactobacillus plantarum, which possess desirable technological properties, were used to prepare three starter cultures: LBP7, LBP10 and LBP14. The addition of LBP7 and LBP14 starter cultures had a significant effect (P < 0.05) on lightness, leading to higher L values compared to control sausages during the ripening period. Both LBP7 and LBP14 sausages showed higher counts of lactic acid bacteria, lower growth of Enterobacteriaceae and Gram-positive catalase-positive cocci and greatly lowered the pH value compared to control sausages throughout the ripening process. At the end of the ripening process, lactic acid bacteria counts were affected (P < 0.05) by the addition of starter culture since higher counts were observed in sausages prepared with LBP7 (9.14 log CFU/g) and LBP14 (8.96 log CFU/g) batches. The decrease of water activity during the ripening of sausages was not affected by the various starters. The texture profiles of all sausages were similar except for LBP10, which showed lower hardness and gumminess during ripening. Under the conditions of the study, volatile compounds were mainly from spices, and no marked differences were found among inoculated sausages. However, sensory evaluation revealed that most of the sensory attributes were scored higher for inoculated sausages than for the control ones. Therefore, LBP7 and LBP14 could be promising candidates for inclusion as starter cultures for the manufacture of sucuk.     

Effect of activating lactoperoxidase system in cheese milk on the quality of Saint‐Paulin cheese

Saint‐Paulin cheese was made from cow’s milk refrigerated at 4 °C for 72 h and preserved by the lactoperoxidase (LP) system. The effect of the LP system on the microbiological, physicochemical and biochemical properties of cheese over a ripening period of 23 days was investigated, using a control (C0), refrigerated LP‐inactivated cow’s milk (C1) and refrigerated LP‐activated cow’s milk (LPA). The LPA treatment showed the least contamination in flora count, particularly salt‐tolerant bacteria at the end of the ripening period. LPA cheese had significantly lower coliform, yeasts and mould counts (P < 0.05) than the other cheeses; this demonstrated the bacteriostatic effect of the LP system. The proteolysis results showed the least value for LPA cheese as compared with the two other samples, as determined by using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of casein fractions extracted from the three samples. The findings indicated that the preservation of cow’s cheese milk by the LP system can be used to improve the microbiological quality, inhibit psychotropic germs, correct the losses of soluble nitrogen fractions in the whey and conserve the cheese yield affected by refrigeration.     

Role of the microbial population on the flavor of the soft-bodied cheese Torta del Casar

The purpose of this work was to investigate the influence of the spontaneous microbial population on the flavor of Torta del Casar cheese. A total of 16 batches of cheeses with different microbial qualities were used. Their physicochemical and microbial characteristics were evaluated during ripening and then related with the volatile compounds, taste, and flavor properties of the finished cheeses. Acids were the most abundant volatile compounds, followed by alcohols and carbonyls. The amount of acetic acid and several alcohols were linked to cheeses with higher counts of lactic acid bacteria (LAB), whereas Enterobacteriaceae counts were associated with semivolatile fatty acids. The gram-positive catalase-positive cocci counts were correlated with esters and methyl ketones. Although the role of the LAB in the flavor development of Torta del Casar is the most relevant, other microbial groups are necessary to impart the flavor of the cheese and to minimize the possible off-flavor derived from excessive concentrations of LAB metabolites, such as acetic acid.     

Influence of native lactic acid bacteria on the microbiological,biochemical and sensory profiles of Serra da Estrela cheese

Cheesemaking from batches of raw ewe's milk was carried out via inoculation with wild strains of Lactococcus lactis subsp. lactis ESB110019 and Lactobacillus plantarum ESB5004 independently, or combined with each other. Those two strains had been isolated from the native microflora of typical Serra da Estrela cheese. One control batch was processed in parallel without addition of any starter. The evolution in viable counts of the main micro-organisms (viz. lactic acid bacteria, Enterobacteriaceae, staphylococci and yeasts), as well as in secondary proteolysis (WSN, 2% TCASN, 12% TCASN and 5% PTASN), was monitored throughout ripening time (over a 63-day period) in cheeses from each batch. The sensory features of the fully ripened cheeses were also assessed. Cheeses manufactured with starter showed significantly lower levels of viable Enterobacteriaceae than those manufactured without starter; viable counts of enterococci and staphylococci did significantly increase after addition of L. lactis or Lb. plantarum, respectively. Proteolysis in terms of WSN and 5% PTASN was not significantly affected by the lactic acid bacteria tested when compared to the control, but L. lactis played a significant role toward increasing the 2% TCASN content of cheeses; both strains led to a statistically significant increase of the 12% TCASN. The scores for flavor and texture of the control cheeses were somewhat above those for the experimental cheeses manufactured with starter.     

Short communication: Antimicrobial activity of pequi (Caryocar brasiliense) waste extract on goat Minas Frescal cheese presenting sodium reduction

Sodium chloride reduction in foods is a significant focus of the dairy industry; however, it can interfere with dairy product quality. Thus, researchers have carried out studies on alternatives to maintain dairy product safety when presenting reduced NaCl content, such as natural antimicrobial addition. Caryocar brasiliense (pequi) is a fruit with high phenolic compound concentrations in the pulp and peel and known antioxidant and antimicrobial properties. This study aimed to define the optimum stage for pequi waste extract addition during cheese manufacturing in order to maintain and prolong the shelf life of reduced-sodium goat Minas Frescal cheese. Four different goat Minas Frescal cheese treatments were carried out: control cheese (without extract; CC), pequi extract addition to milk (CM), pequi extract addition to mass (CS), and cheese immersion in pequi extract (CIE). The treatments were subjected to microbiological (Staphylococcus spp., Escherichia coli, Enterobacteriaceae, coliforms and fecal coliforms, Lactococcus spp., and lactic acid bacteria counts), textural (hardness and consistency), and instrumental color (luminosity, yellow intensity, red intensity, chroma, hue angle, and total color change) analyses. No Enterobacteriaceae, Staphylococcus spp., E. coli, or coliforms and fecal coliforms were detected during storage for any of the assessed samples, including CC. Regarding texture, all samples presented a trend for decreasing rigidity during storage. In addition, lower luminosity values were also observed in cheeses produced with added pequi extract (CM, CS, and CIE) when compared with CC. All cheeses produced with added pequi were stable regarding all evaluated parameters; however, pequi extract addition to milk (CM) was shown to be more efficient, leading to higher textural parameters and better microbiological quality during storage. Thus, the CM treatment is the most recommended for pequi waste extract addition during Minas Frescal cheese manufacture.     

Relationships between flavour and microbiological profiles in Serra da Estrela cheese throughout ripening

Cheeses manufactured in certified dairies in the Portuguese region of Serra da Estrela, using refrigerated and non-refrigerated raw sheep milk, were quantitatively evaluated in terms of indigenous microflora and volatile compounds during a ripening period of up to 6 mo. Viable counts were obtained for lactococci, lactobacilli, leuconostoc, enterococci, yeasts, Enterobacteriaceae and staphylococci; analyses of volatiles were performed by chromatography after solid-phase micro-extraction. Treatment of all analytical data produced by principal component analysis revealed correlations between the major microbial groups present in cheese and patterns of volatiles generated. End products resulting from the degradation of sugars, free amino acids and glycerides constituted the predominant volatiles of Serra da Estrela cheeses. Among volatile, short-chain carboxylic acids detected were acetic, propionic, iso-butyric and iso-valeric acids; these compounds are known to be breakdown products of Gly, Ala and Ser, of Thr, of Val, and of Ile, respectively, following oxidative deamination. Semi-volatile fatty acids and their corresponding ethyl esters appeared in the cheese, probably as a result of the activity of lipases produced by yeasts and Enterobacteriaceae. These ethyl esters, which are responsible for fruity flavours, were especially pronounced in cheeses manufactured from refrigerated milk.     

Probiotic cheese production using Lactobacillus casei cells immobilized on fruit pieces

Lactobacillus casei cells were immobilized on fruit (apple and pear) pieces and the immobilized biocatalysts were used separately as adjuncts in probiotic cheese making. In parallel, cheese with free L. casei cells and cheese only from renneted milk were prepared. The produced cheeses were ripened at 4 to 6°C and the effect of salting and ripening time on lactose, lactic acid, ethanol concentration, pH, and lactic acid bacteria viable counts were investigated. Fat, protein, and moisture contents were in the range of usual levels of commercial cheeses. Reactivation in whey of L. casei cells immobilized on fruit pieces after 7 mo of ripening showed a higher rate of pH decrease and lower final pH value compared with reactivation of samples withdrawn from the remaining mass of the cheese without fruit pieces, from cheese with free L. casei, and rennet cheese. Preliminary sensory evaluation revealed the fruity taste of the cheeses containing immobilized L. casei cells on fruit pieces. Commercial Feta cheese was characterized by a more sour taste, whereas no significant differences concerning cheese flavor were reported by the panel between cheese containing free L. casei and rennet cheese. Salted cheeses scored similar values to commercial Feta cheese, whereas unsalted cheese scores were significantly lower, but still acceptable to the sensory panelists.     

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.     

Effect of milk centrifugation and incorporation of high heat-treated centrifugate on the microbial composition and levels of volatile organic compounds of Maasdam cheese

Centrifugation is a common milk pretreatment method for removal of Clostridium spores which, on germination, can produce high levels of butyric acid and gas, resulting in rancid, gassy cheese. The aim of this study was to determine the effect of centrifugation of milk, as well as incorporation of high heat-treated centrifugate into cheese milk, on the microbial and volatile profile of Maasdam cheese. To facilitate this, 16S rRNA amplicon sequencing in combination with a selective media-based approach were used to study the microbial composition of cheese during maturation, and volatile organic compounds within the cheese matrix were analyzed by HPLC and solid-phase microextraction coupled with gas chromatography–mass spectrometry. Both culture-based and molecular approaches revealed major differences in microbial populations within the cheese matrix before and after warm room ripening. During warm room ripening, an increase in counts of propionic acid bacteria (by ～10^1.5 cfu) and nonstarter lactic acid bacteria (by ～10⁸ cfu) and a decrease in the counts of Lactobacillus helveticus (by ～10^2.5 cfu) were observed. Lactococcus species dominated the curd population throughout ripening, followed by Lactobacillus, Propionibacterium, and Leuconostoc, and the relative abundance of these accounted for more than 99% of the total genera, as revealed by high-throughput sequencing. Among subdominant microflora, the overall relative abundance of Clostridium sensu stricto was lower in cheeses made from centrifuged milk than control cheeses, which coincided with lower levels of butyric acid. Centrifugation as well as incorporation of high heat-treated centrifugate into cheese milk seemed to have little effect on the volatile profile of Maasdam cheese, except for butyric acid levels. Overall, this study suggests that centrifugation of milk before cheesemaking is a suitable method for controlling undesirable butyric acid fermentation without significantly altering the levels of other volatile organic compounds of Maasdam cheese.     

Survival of Escherichia coli, strain W, during the manufacture of cottage cheese.

Cottage cheese was manufactured in 10-liter experimental vats by the direct-acid-set method from milk that was inoculated with a heat resistant strain of Escherichia coli. Growth or survival of Strain W (ATCC 9637) E. coli was determined at various stages of the cheese making operation after the cheese-skim milk was inoculated to give counts of 2.5 X 10(4) or 4.0 X 10(5) cells/ml. Numbers of coliform organisms remained constant at the inoculated concentration in the cheese milk up to a cooking temperature of 43 C. At 43 C, when curd was separated from the whey, the curd (not washed) had coliform counts that were two log cycles greater than the whey. These trends were in milks with both cell counts. Washing of the curd with acid and 10 ppm chlorine reduced the number of coliform cells in the curd at all cooking temperatures as compared with unwashed curd. Acid wash of the curd at pH 5 did not reduce the coliform counts below those of unwashed curd. Cooking temperatures of 54 C were necessary to destroy (less than 1 cell/ml) E. coli Strain W, in either the unwashed or acid-chlorine washed curd. Holding curd with an initial average log count of 6.26 coliform cells/ml at constant temperatures of 38, 43, 49, and 54 C confirmed that 54 C for 50 min was necessary to reduce the average count to less than 1 cell/ml in isolated curd. Coliforms in whey were reduced to that concentration after 10 min at 54 C.     

Ripening of extra-hard cheese made with mesophilic DL-starter

Extra hard cheese is commonly made with thermophilic starters using high temperatures to stimulate expulsion of whey. In this work, microflora, proteolysis and volatiles were investigated in an extra-hard cheese made with mesophilic DL-starter, produced using challenging cooking temperatures for the starter bacteria over several hours. Cheese from six commercially produced vats was investigated over 56 weeks. The number of starter bacteria decreased after three weeks of ripening. Casein breakdown was characterised by chymosin and plasmin activity on α_s1- and β-caseins, respectively. Peptide profiles showed accumulation of Lactococcus derived peptides from α_s1-CN f1–23, and the peptide β-CN 29–93 as a result of joint plasmin and chymosin activity and absence of highly proteolytic thermophilic Lactobacillus, commonly present in extra-hard cheese. The composition of amino acids depended mainly on starter during the first 26 weeks of ripening. The content of volatiles depended both on ripening time and the starter used.     

Efficacy of polyethylene-based antimicrobial films containing principal constituents of basil

The feasibility of low-density polyethylene (LDPE)-based films containing linalool or methylchavicol as antimicrobial (AM) packages to retard microbial growth on food surfaces was investigated. The AM LDPE-based films were tested for inhibition against selected microorganisms. Both compounds retained their AM activity, after an extrusion film-blowing process, against Escherichia coli in solid medium. Cheddar cheese was wrapped with the AM films and the packaged cheese samples were stored at 4 °C. The changes in the mesophilic aerobic bacteria and coliform, as well as yeast and mould counts were monitored. In addition, cheese samples inoculated with E. coli or Listeria innocua were wrapped with the AM films, stored at refrigerated (4 °C) or at abuse (12 °C) temperatures and the count of these microorganisms was monitored as a function of time. The results showed an inhibitory effect of these AM films against microbial growth in naturally contaminated cheese and in inoculated samples. The effect on suppression of E. coli and L. innocua growth was more pronounced at the abuse temperature. Methylchavicol-LDPE-based film exhibited a higher efficacy of inhibition than that of linalool-LDPE-based film. In addition, a sensory evaluation was performed with regards to possible taint in the flavour of the cheese. Taint in flavour as affected by linalool or methylchavicol was not significantly detectable by the panelists at the end of the storage period of 6 weeks. This study shows the potential use of polymeric films containing the principal constituents of basil as the AM components for enhancing quality and safety of cheeses.     

Seasonal diversity of yeasts associated with white-surface mould-ripened cheeses

Yeasts present in Camembert and Brie cheeses during processing were monitored in a single cheese factory during summer and winter, to determine the seasonal diversity of yeasts over a ripening period of 56 days. Lactic acid bacteria predominate during cheese making, but yeasts play a significant role during maturation reaching counts of 6 log units at the later stages of ripening. Sources of yeast contamination that may lead to contamination of the curd were also determined. A diverse variety of 20 yeast species representing 10 genera were present during the winter period associated with the factory environment, during processing and ripening, whereas only seven yeast species representing six genera were isolated during summer. Although a broad spectrum of yeasts was isolated from both cheeses, Debaryomyces hansenii and Yarrowia lipolytica were the most abundant yeast species isolated. Other species encountered were Torulaspora delbrueckii, Rhodotorula mucilaginosa, Rhodotorula minuta and various species of Candida.     

THE FORMATION OF HISTAMINE IN HERBY CHEESE DURING RIPENING

The purpose of this study is to observe the formation of histamine throughout the period of ripening in herby cheese. Herby cheese samples were made from raw milk and buried in soil where they mature in 3 months. Samples were taken on the first, seventh, 15th, 30th, 60th and 90th days of ripening. The moistness, content pH, salt and (total aerobic mesophile bacteria, lactic acid bacteria, coliform bacteria and yeasts and mold) changes were observed. The concentration of histamine are 2.19 mg/100 g on the first day of ripening. It gradually increased and reached 4.62 mg/100 g on the 90th day. Consequently, the histamine that was observed in herby cheese during the ripening appears to be not important for public health.     

Proteolysis and biogenic amine buildup in high-pressure treated ovine milk blue-veined cheese

Penicillium roqueforti plays an important role in the ripening of blue-veined cheeses, mostly due to lactic acid consumption and to its extracellular enzymes. The strong activity of P. roqueforti proteinases may bring about cheese over-ripening. Also, free amino acids at high concentrations serve as substrates for biogenic amine formation. Both facts result in shorter product shelf-life. To prevent over-ripening and buildup of biogenic amines, blue-veined cheeses made from pasteurized ovine milk were high-pressure treated at 400 or 600 MPa after 3, 6, or 9 wk of ripening. Primary and secondary proteolysis, biogenic amines, and sensory characteristics of pressurized and control cheeses were monitored for a 90-d ripening period, followed by a 270-d refrigerated storage period. On d 90, treatments at 400 MPa had lowered counts of lactic acid bacteria and P. roqueforti by less than 2 log units, whereas treatments at 600 MPa had reduced lactic acid bacteria counts by more than 4 log units and P. roqueforti counts by more than 6 log units. No residual α-casein (CN) or κ-CN were detected in control cheese on d 90. Concentrations of β-CN, para-κ-CN, and γ-CN were generally higher in 600 MPa cheeses than in the rest. From d 90 onwards, hydrophilic peptides were at similar levels in pressurized and control cheeses, but hydrophobic peptides and the hydrophobic-to-hydrophilic peptide ratio were at higher levels in pressurized cheeses than in control cheese. Aminopeptidase activity, overall proteolysis, and free amino acid contents were generally higher in control cheese than in pressurized cheeses, particularly if treated at 600 MPa. Tyramine concentration was lower in pressurized cheeses, but tryptamine, phenylethylamine, and putrescine contents were higher in some of the pressurized cheeses than in control cheese. Differences in sensory characteristics between pressurized and control cheeses were generally negligible, with the only exception of treatment at high pressure level (600 MPa) at an early ripening stage (3 wk), which affected biochemical changes and sensory characteristics.