Cheese Surface Research Articles

Development of a device and method for the time-course estimation of low water fluxes and mean surface water activity of food products during ripening and storage

Abstract Accurate measurement of water activity ( a w ) is an important goal for the food industry because a w is a key parameter in microbial growth, biological reaction rates and physical properties. An experimental device was setup using air-product water balance to non-destructively estimate the time-course of mean a w at the food product surface under well-controlled airflow conditions. The device is especially suited for studying the ripening of cheeses and fermented meat products, where water fluxes exchanged between products and air are very low. The validation tests performed with a w -known model products showed that water fluxes of 10 −7 kg s −1 can be estimated with an accuracy better than 2% over very short periods of time, and that surface a w can be estimated with an absolute uncertainty of less than 0.01 a w units. A handful of cheese ripening trials illustrate the potential of the method, highlighting the effects of a low air velocity and high air RH on the water exchanges occurring at a cheese surface, thus demonstrating the strong surface sensitivity to external air conditions.

Surface microbiota analysis of Taleggio, Gorgonzola, Casera, Scimudin and Formaggio di Fossa Italian cheeses

The composition of the bacterial consortia of the smear Italian cheeses and their role on quality and safety is still poorly understood. The objective of this study was to identify and characterize the bacterial communities present on the surface of five traditional Italian cheeses, Casera Valtellina, Scimudin, Formaggio di Fossa, Gorgonzola and Taleggio. DGGE analysis performed using total DNA obtained from cheese surfaces enabled us to identify the dominant bacterial populations. Bands showing different intensity and identified as Staphylococcus, Micrococcus, Psychrobacter, Enterococcus and Brevibacterium species were detected on the surface of cheeses. The cluster analysis showed that Gorgonzola, Taleggio and Formaggio di Fossa cheeses present high similarity in their surface bacterial composition while major differences in the DGGE profiles were observed in Scimudin and Casera. The molecular taxonomical identification among the Gram positive isolates, reveals the presence of the following bacterial genera: Staphylococcus, Micrococcus, Macrococcus, Enterococcus, Lactobacillus, Carnobacterium, Leuconostoc, Brevibacterium, Corynebacterium, Brochothrix, Bacillus. The combination of culture dependent and independent techniques allowed us to obtain information about the bacterial species covering the surface of five different traditional Italian cheeses.

Assessment of the anti-listerial activity of microfloras from the surface of smear-ripened cheeses

The anti-listerial activity of microfloras from the surface of various smear-ripened cheeses was evaluated using four methods that were then compared. Method A measured the anti-listerial potential of supernatants from short-time liquid cultures, whereas in Method B, a model cheese was co-inoculated with the microflora and Listeria innocua test strains. Method C was based on successive propagations of the microfloras on this model cheese, and Method D on successive propagations of the microfloras together with Listeria test strains. Anti-listerial activity considerably depended on the microflora used. Significant correlations were obtained between Methods A and B and Methods C and D. With Methods C and D, the highest anti-listerial activity was obtained with the microflora from a Livarot-type cheese (FC12). To investigate the cause of the anti-listerial activity of FC12, Fourier Transform InfraRed (FTIR) analyses of microbial populations were performed on the original microflora as well as on the microflora after propagations on the model cheese. The composition of FC12 had changed considerably upon propagation, and in the propagated microflora, the population of yeasts was dominated by Yarrowia lipolytica strains, whereas the population of bacteria was dominated by Vagococcus species.

Limited ripening of low-fat UF-cheese due to CaPO4barrier?

The ripening of industrial soft cheeses manufactured using a liquid pre-cheese produced from the ultrafiltration (UF) of milk was observed to be slow in comparison to that of cheese manufactured by a traditional process. Moreover, in the UF-cheeses investigated in this study, which were produced using Penicillium camemberti as the surface flora, several surface defects were observed: the texture was ‘carton’ like and the rind frequently detached from the cheese. To gain a fuller understanding of the development of these surface defects in UF-cheeses, the migration of different minerals and ions, and the study of the rind microstructure by scanning electron microscopy and X-ray mapping were performed. The results suggest that the slower diffusion of lactate, possibly due to the mineral layer at the surface of cheeses, acting as a barrier to its diffusion, may have caused an alteration in the metabolism and growth of the surface mould and may explain the surface defects of these UF-cheeses.

Chemical changes that predispose smoked Cheddar cheese to calcium lactate crystallization

We have observed a high incidence of calcium lactate surface crystals on naturally smoked Cheddar cheese in the retail marketplace. The objective of this study was to identify chemical changes that may occur during natural smoking that render Cheddar cheese more susceptible to calcium lactate crystal formation. Nine random-weight (approximately 300g) retail-packaged samples of smoked Cheddar cheese were obtained from a commercial manufacturer immediately after the samples were smoked for about 6h at 20°C in a commercial smokehouse. Three similarly sized samples that originated from the same 19.1-kg block of cheese and that were not smoked were also obtained. Within 2 d after smoking, 3 smoked and 3 control (not smoked) samples were sectioned into 5 subsamples at different depths representing 0 to 2, 2 to 4, 4 to 6, 6 to 8, and 8 to 10mm from the cheese surface. Six additional smoked cheese samples were similarly sectioned at 4 wk and again at 10 wk of storage at 5°C. Sample sections were analyzed for moisture, l(+) and d(−) lactate, pH, and water-soluble calcium. The effects of treatment (smoked, control), depth from cheese surface, and their interactions were analyzed by ANOVA according to a repeated measures design with 2 within-subject variables. Smoked samples contained signficantly lower moisture and lower pH, and higher total lactate-in-moisture (TLIM) and water-soluble calcium-in-moisture (WSCIM) than control cheeses. Smoked samples also contained significant gradients of moisture, pH, TLIM, and WSCIM, with lower moisture and pH, and higher TLIM and WSCIM, occurring at the cheese surface. Gradients of moisture were still present in smoked samples at 4 and 10 wk of storage. In contrast, the pH, TLIM, and WSCIM equilibrated and showed no gradients at 4 and 10 wk. The results indicate that calcium and lactate in the serum phase of the cheese were elevated because of smoking, especially at the cheese surface immediately after smoking treatment, which presumably predisposes the smoked cheeses to increased susceptibility to calcium lactate surface crystallization.

Bavariicoccus seileri gen. nov., sp. nov., isolated from the surface and smear water of German red smear soft cheese

The phylogenetic position and physiological characters of six hitherto-unknown lactic acid bacterial isolates, which form part of the surface microbiota of German red smear soft cheese, are reported. The coccoid cells are aerotolerant, Gram-positive, catalase-negative and non-motile. The cell-wall peptidoglycan contains alanine, glutamic acid, lysine and aspartic acid and is of the A4alpha type (l-Lys-d-Asp). The sequences of the 16S rRNA, groEL and rpoB genes of the six isolates are identical and reveal that these isolates represent an independent lineage within the radiation of the family Enterococcaceae in the phylum Firmicutes. Their closest phylogenetic neighbour is the lactic acid bacterium Atopobacter phocae M1590/94/2(T), with which they share 94.9 % 16S rRNA gene sequence similarity; representatives of other genera such as Granulicatella, Carnobacterium and Trichococcus are more distantly related. DNA-DNA hybridization studies reveal that the six isolates are members of a single species, and this is confirmed by similarities in biochemical characteristics. The six isolates were assigned four different groups by Fourier-transform infrared and randomly amplified polymorphic DNA typing. Therefore, it is formally proposed that these isolates should be classified in a single novel species of a novel genus and be named Bavariicoccus seileri gen. nov., sp. nov. The type strain of Bavariicoccus seileri is WCC 4188(T) (=DSM 19936(T) =CCUG 55508(T)).

Effect of Packaging Films Releasing Antimicrobial Agents on Stability of Food Products

The study was aimed to (<I>I</I>) test the effect of polyethylene (LDPE) films coated by commercially available polyvinylchloride (PVC) lacquer with addition of nisin preparation Nisaplin® (5% w/w) and/or natamycin preparation Delvocid<sup>TM</sup> (10% w/w) on the growth of target bacteria, yeasts and moulds on the surface of soft cheese Blaťácké zlato produced by company Madeta, a.s., Veselí nad Lužnicí, and (<I>ii</I>) prepare and study the antimicrobial films with incorporated lactic acid or sodium lactate, suitable for packaging of raw chicken meat. The cheese in contact with nisin/natamycin treated film was stored at temperature 4°C and 23°C for 23 days, while chicken meat packaged in lactic acid/sodium lactate releasing films was stored only at 4°C for 7 days. Released antimicrobial agents caused retardation of tested microorganism growth by more than 1 logarithmic cycle on the surface of cheese Blaťácké zlato and by more than 2 logarithmic cycles in raw chicken meat.

Control of pathogenic and spoilage microorganisms from cheese surface by whey protein films containing malic acid, nisin and natamycin

The inhibitory effects of nisin, natamycin and malic acid, incorporated in whey protein films with pH 3, were investigated alone or with addition of sucrose esters, Tween80 or EDTA. Water vapour permeability measurements and mechanical and rheological tests were also assessed. EDTA and Tween80 did not significantly ( P < 0.05) influence the inhibitory activity of films against Pseudomonas aeruginosa and Yarrowia lipolytica in contrast with the improved effect against Listeria monocytogenes, Penicillium commune and Penicillium chrysogenum. Sucrose esters reduced significantly ( P < 0.05) the inhibitory effect for Y. lipolytica and Penicillium spp. The present work provides an antimicrobial film formulation with potential to be a hurdle against spoilage and pathogenic microorganisms isolated from cheese surface.

Catheter-related fungemia caused by Candida intermedia

Candida intermedia is rarely reported as a human pathogen. We report two cases of catheter-related fungemia caused by C. intermedia which were treated successfully with intravenous fluconazole and catheter removal. The isolates were identified by commercial biochemical methods, oligonucleotide array, and partial sequencing analysis of rRNA genes.

Antifungal activity of non-starter lactic acid bacteria isolates from dairy products

The antifungal activity of 81 NSLAB isolates from traditional dairy products against moulds developed on the surface of hard cheeses as well as yeasts was studied. Twenty isolates of facultatively heterofermentative and eleven of obligately heterofermentative lactobacilli from Feta cheese exhibited antifungal activities. These isolates were classified to species level by phenotypic criteria and the SDS–PAGE of whole-cell proteins. The former group also showed a broad spectrum of antibacterial activities with preference towards Listeria monocytogenes and other food-borne pathogens. The extracellular antimicrobial substances were sensitive to proteolytic enzymes, suggesting that the inhibitory activity was due to bacteriocin-like substances.

Assessment of the microbial diversity at the surface of Livarot cheese using culture-dependent and independent approaches

The microbial diversity of the surface of a commercial red-smear cheese, Livarot cheese, sold on the retail market was studied using culture-dependent and independent approaches. Forty yeasts and 40 bacteria from the cheese surface were collected, dereplicated using single-strand conformation polymorphism (SSCP) analysis and identified using rRNA gene sequencing for the culture-dependent approach. The culture-independent approach involved cloning and sequencing of the 16S rRNA gene and SSCP analysis from total DNA extracted from the cheese. The most dominant bacteria were Microbacterium gubbeenense, Leucobacter komagatae and Gram-negative bacteria from the Gamma-Proteobacteria class. Fluorescence in situ hybridization (FISH) analysis was also used to study the cheese microbial diversity with class-level and specific rRNA-targeted probes for bacteria and yeasts, respectively. FISH analysis confirmed that Gamma-Proteobacteria were important microorganisms in this cheese. Four specific FISH probes targeting the dominant yeasts present in the cheese, Candida catenulata, Candida intermedia, Geotrichum spp. and Yarrowia lipolytica, were also designed and evaluated. These probes allowed the detection of these yeasts directly in cheese. The use of the rRNA gene-based approach combined with FISH analysis was useful to investigate the diversity of a surface microbial consortium from cheese.

Effects of Proteus vulgaris growth on the establishment of a cheese microbial community and on the production of volatile aroma compounds in a model cheese

To investigate the impact of Proteus vulgaris growth on a multispecies ecosystem and on volatile aroma compound production during cheese ripening. The microbial community dynamics and the production of volatile aroma compounds of a nine-species cheese ecosystem were compared with or without the presence of P. vulgaris in the initial inoculum. Proteus vulgaris was able to colonize the cheese surface and it was one of the dominant species, representing 37% of total isolates at the end of ripening with counts of 9.2 log(10) CFU g(-1). In the presence of P. vulgaris, counts of Arthrobacter arilaitensis, Brevibacterium aurantiacum and Hafnia alvei significantly decreased. Proteus vulgaris influenced the production of total volatile aroma compounds with branched-chain aldehydes and their corresponding alcohols being most abundant. Proteus vulgaris was able to successfully implant itself in a complex cheese ecosystem and significantly contributed to the organoleptic properties of cheese during ripening. This bacterium also interacted negatively with other bacteria in the ecosystem studied. This is the first time that the impact of a Gram-negative bacterium on cheese microbial ecology and functionality has been described.

Fate of Listeria monocytogenes on fully ripened Greek Graviera cheese stored at 4, 12, or 25 degrees C in air or vacuum packages: in situ PCR detection of a cocktail of bacteriocins potentially contributing to pathogen inhibition.

The behavior of Listeria monocytogenes on fully ripened Greek Graviera cheese was evaluated. Three batches (A, B, and C) were tested. Batches A and C were prepared with a commercial starter culture, while in batch B the starter culture was combined with an enterocin-producing Enterococcus faecium Graviera isolate. Cheese pieces were surface inoculated with a five-strain cocktail of L. monocytogenes at ca. 3 log CFU/cm2, packed under air or vacuum conditions, stored at 4, 12, or 25 degrees C, and analyzed after 0, 3, 7, 15, 30, 60, and 90 days. L. monocytogenes did not grow on the cheese surface, regardless of storage conditions. However, long-term survival of the pathogen was noted in all treatments, being the highest (P < 0.05) at 4 degrees C under vacuum conditions. Overall, the lower the storage temperature, the higher and longer the survival of L. monocytogenes was. Although enterocin A-specific PCR products were detected in situ in cheese batch B, inhibition of L. monocytogenes by the enterocin-producing strain was not enhanced compared with batches A and C, which also contained enterocin A, but in lower amounts. Additionally enterocins B, P, L50A, and L50B; lactococcin G; and plantaricin A genes were detected in all batches, suggesting that indigenous bacteriocin-producing lactic acid bacteria might contribute to Listeria inhibition in cheese. In conclusion, Graviera cheeses that may be accidentally contaminated in retail at the European Union maximal allowable level of 100 CFU/cm2 or g are at low risk regarding a potential outgrowth of L. monocytogenes, which, however, may survive for a long period during cheese storage.

Species composition of food-spoiling mycelial fungi

We investigated the composition of the microflora that spoils foodstuffs (the surface of hard cheeses and sausages) at agribusiness factories. Mycelial fungi, mostly ascomycetes of the order Eurotiales belonging to the genus Penicillium play the main role in spoiling food. Most representatives of these fungi are mesophiles and possess the capacity for utilizing nutrient substrates in surface and submerged cultures.

Growth and aroma contribution of Microbacterium foliorum, Proteus vulgaris and Psychrobacter sp. during ripening in a cheese model medium

The growth and aroma contribution of Microbacterium foliorum, Proteus vulgaris and Psychrobacter sp., some common but rarely mentioned cheese bacteria, were investigated in a cheese model deacidified by Debaryomyces hansenii during the ripening process. Our results show that these bacteria had distinct growth and cheese flavour production patterns during the ripening process. P. vulgaris had the greatest capacity to produce not only the widest variety but also the highest quantities of volatile compounds with low olfactive thresholds, e.g. volatile sulphur compounds and branched-chain alcohols. Such compounds produced by P. vulgaris increased after 21 days of ripening and reached a maximum at 41 days. The three bacteria studied exhibited various degrees of caseinolytic, aminopeptidase and deaminase activities. Moreover, P. vulgaris had a greater capacity for hydrolysing casein and higher deaminase activity. Our results show that P. vulgaris, a Gram-negative bacterium naturally present on the surface of ripened cheeses, could produce high concentrations of flavour compounds from amino acid degradation during the ripening process. Its flavouring role in cheese cannot be neglected. Moreover, it could be a useful organism for producing natural flavours as dairy ingredients.

Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer, against Listeria monocytogenes

Enterococcus faecium WHE 81, a multi-bacteriocin producer, was tested for its antimicrobial activity on Listeria monocytogenes in Munster cheese, a red smear soft cheese. The naturally delayed and superficial contamination of this type of cheese allowed the use of E. faecium WHE 81 at the beginning of the ripening as a surface culture. A brine solution inoculated at 10 5 CFU of E. faecium WHE 81 per mL was sprayed on the cheese surface during the first smearing operation. On day 7, smearing of cheese samples with a brine solution at 10 2 CFU of L. monocytogenes per mL yielded initial cell counts of approximately 50 CFU g −1 of the pathogen on the cheese surface. Although, in some instances, L. monocytogenes could survive (<50 CFU g −1) in the presence of E. faecium WHE 81, it was unable to initiate growth. In control samples however, L. monocytogenes counts often exceeded 10 4 CFU g −1. In other respects, E. faecium WHE 81, which naturally existed in Munster cheese, did not adversely impact on the ripening process.

Characterization of Calcium Lactate Crystals on Cheddar Cheese by Image Analysis

Previous research demonstrated that crystal coverage on the surface of Cheddar cheese can be quantitatively and nondestructively measured using image analysis of digital photographs of the cheese surface. The objective of the present study was to extend image analysis methodology to quantify and characterize additional features of visible crystals on cheese surfaces as they grow over time. A random weight (∼300g) retail sample of naturally smoked Cheddar cheese exhibiting white surface crystals was obtained from a commercial source. The total area occupied by crystals and total number of discrete crystal regions on one of the surfaces (∼55×120mm) was measured at 3-wk intervals for 30 wk using image analysis. In addition, 5 small (∼0.3mm radius) individual crystals on that surface were chosen for observation over the 30-wk period. The crystals were evaluated for area, radius, and shape factor (circularity) every third week using image analysis. The total area occupied by crystals increased in a linear manner (R2=0.95) from about 0.44 to 7.42% of the total cheese surface area over the 30-wk period. The total number of discrete crystal regions also increased but in a nonlinear manner that was best described by a quadratic relationship. Measurement of discrete crystal regions underestimated the true number of crystals present at the cheese surface due to merging of adjacent crystals as they grew and merged into a single crystal region over time. Throughout this period, the shapes of the 5 individual crystals closely approximated perfect circles, except when adjacent crystals merged to form a single irregular crystal region, and the area occupied by each of the 5 crystals increased in a near-linear manner (R2=0.95). Image analysis approaches may be used to evaluate crystal formation and growth rates and morphology on cheese.

Transcriptional Analysis of l -Methionine Catabolism in the Cheese-Ripening Yeast Yarrowia lipolytica in Relation to Volatile Sulfur Compound Biosynthesis

Yarrowia lipolytica is one of the yeasts most frequently isolated from the surface of ripened cheeses. In previous work, it has been shown that this yeast is able to convert L-methionine into various volatile sulfur compounds (VSCs) that may contribute to the typical flavors of several cheeses. In the present study, we show that Y. lipolytica does not assimilate lactate in the presence of L-methionine in a cheeselike medium. Nineteen presumptive genes associated with L-methionine catabolism or pyruvate metabolism in Y. lipolytica were transcriptionally studied in relation to L-methionine degradation. The expression levels of the YlARO8 (YALI0E20977g), YlBAT1 (YALI0D01265g), and YlBAT2 (YALI0F19910g) genes (confirmed by real-time PCR experiments) were found to be strongly up-regulated by L-methionine, and a greater variety and larger amounts of VSCs, such as methanethiol and its autooxidation products (dimethyl disulfide and dimethyl trisulfide), were released in the medium when Y. lipolytica was grown in the presence of a high concentration of L-methionine. In contrast, other genes related to pyruvate metabolism were found to be down-regulated in the presence of L-methionine; two exceptions were the YlPDB1 (YALI0E27005g) and YlPDC6 (YALI0D06930g) genes, which encode a pyruvate dehydrogenase and a pyruvate decarboxylase, respectively. Both transcriptional and biochemical results corroborate the view that transamination is the first step of the enzymatic conversion of L-methionine to VSCs in Y. lipolytica and that the YlARO8, YlBAT1, and YlBAT2 genes could play a key role in this process.

Survival and Acid Resistance of Listeria innocua in Feta Cheese and Yogurt, in the Presence or Absence of Fungi

The objective of the study was to assess the survival of Listeria innocua, alone or coinoculated with fungal isolates, during storage of Feta cheese (pH 4.43 to 4.56) and yogurt (pH 4.01 to 4.27) at 3 to 15°C. The acid resistance of the bacterium during subsequent exposure to pH 2.5 for 3 h was also evaluated in samples stored at 3 and 10°C. In Feta cheese, L. innocua survived better than it did in yogurt at all temperatures. At 5, 10, and 15°C, the pH of cheese increased due to fungal growth, and this enhanced the survival of L. innocua more than during storage at 3°C. Moreover, during storage of Feta cheese, L. innocua was capable of surviving the subsequent exposure for 3 h in broth of pH 2.5, in contrast to cultures not inoculated in the product (control cultures; 24 h at 30°C in broth). In yogurt, L. innocua reduced more than 5 log within 15 days of storage at 5, 10, and 15°C, whereas extended survival was observed at 3°C until day 22, with total reduction of approximately 4.5 log. In contrast to what was observed in Feta cheese, surviving populations of L. innocua in yogurt were eliminated after subsequent exposure for 3 h to pH 2.5. The findings indicate that growth of fungi on the surface of Feta cheese and yogurt may compromise the safety of these products by enhancing survival of the bacterium. Particularly, when fungi increase the pH of Feta cheese, L. innocua demonstrates better survival and prolonged storage may raise concerns for the development of acid-resistant Listeria populations.

InfluenceofCoryneformCulturesIsolated from the Surface of a Farmhouse Smear-Ripened Cheese on the Ripening of Cheddar Cheese

InfluenceofCoryneformCulturesIsolated from the Surface of a Farmhouse Smear-Ripened Cheese on the Ripening of Cheddar Cheese