Cheese Surface Research Articles

Antimicrobial Activity of Natural Agents against Saccharomyces cerevisiae

SUMMARYThe antimicrobial (AM) activity of starch‐based films coated with linalool, carvacrol or thymol against Saccharomyces cerevisiae in vitro and/or inoculated on the surface of Cheddar cheese was investigated. In solid medium using the agar diffusion method and in experiments involving the inoculation of the microorganism on the surface of Cheddar cheese, all the films containing these AM agents in coatings demonstrated an inhibitory effect against S. cerevisiae. The results suggest that the overall inhibitory effect of linalool, carvacrol or thymol increased significantly (p ≤ 0.05) with the concentration of each of the AM agents in the film coating and that the response is linear in the concentration range of 1 to 5% (w/w) of the AM agent. Thymol had the highest AM efficacy followed by carvacrol, whereas linalool had the lowest efficacy amongst the three systems. The zones of inhibition in the agar diffusion test method at 25°C for S. cerevisiae were found to be 7.6, 7.1 and 6.1 mm for thymol, carvacrol and linalool at 1% (w/w) loading and 13.2, 12.2 and 11.2 mm at 5% (w/w) loading of the AM agents, respectively. The death rates of S. cerevisiae on Cheddar cheese wrapped in films coated with thymol, carvacrol and linalool and stored for up to 28 days at 15°C were found to be 0.044, 0.043 and 0.038 per day at 1% (w/w) loading and 0.077, 0.073 and 0.063 per day at 5% (w/w) loading of the AM agents, respectively. Copyright © 2011 John Wiley & Sons, Ltd.

Incidence of Listeria monocytogenes in cheese manufacturing plants from the northeast region of São Paulo, Brazil.

The incidence of Listeria monocytogenes in three cheese manufacturing plants from the northeastern region of São Paulo, Brazil, was evaluated from October 2008 to September 2009. L. monocytogenes was found in samples from two plants, at percentages of 13.3% (n = 128) and 9.6% (n = 114). Samples of raw and pasteurized milk, water, and Minas Frescal cheese were negative for L. monocytogenes, although the pathogen was isolated from the surface of Prato cheese and in brine from one of the plants evaluated. L. monocytogenes was also isolated from different sites of the facilities, mainly in non-food contact surfaces such as drains, floors, and platforms. Serotype 4b was the most predominant in the plants studied. The results of this study indicate the need for control strategies to prevent the dispersion of L. monocytogenes in the environment of cheese manufacturing plants.

Improvement in melting and baking properties of low-fat Mozzarella cheese

Low-fat cheeses dehydrate too quickly when baked in a forced air convection oven, preventing proper melting on a pizza. To overcome this problem, low-fat Mozzarella cheese was developed in which fat is released onto the cheese surface during baking to prevent excessive dehydration. Low-fat Mozzarella cheese curd was made with target fat contents of 15, 30, 45, and 60g/kg using direct acidification of the milk to pH 5.9 before renneting. The 4 portions of cheese curd were comminuted and then mixed with sufficient glucono-δ-lactone and melted butter (45, 30, 15, or 0g/kg, respectively), then pressed into blocks to produce low-fat Mozzarella cheese with about 6% fat and pH 5.2. The cheeses were analyzed after 15, 30, 60, and 120 d of storage at 5°C for melting characteristics, texture, free oil content, dehydration performance, and stretch when baked on a pizza at 250°C for 6min in a convection oven. Cheeses made with added butter had higher stretchability compared with the control cheese. Melting characteristics also improved in contrast to the control cheese, which remained in the form of shreds during baking and lacked proper melting. The cheeses made with added butter had higher free oil content, which correlated (R2≥0.92) to the amount of butterfat added, and less hardness and gumminess compared with the control low fat cheese.

Facultative anaerobic halophilic and alkaliphilic bacteria isolated from a natural smear ecosystem inhibit Listeria growth in early ripening stages

In vitro and in situ anti-listerial properties of 3 strains of Facultative Anaerobic Halophilic and Alkaliphilic (FAHA) species, i.e. Alkalibacterium kapii ALK 6, Marinilactibacillus psychrotolerans ALK 9 and Facklamia tabacinasalis ALK 1, were investigated. The 3 strains were isolated from a smear ecosystem originating from a commercial Raclette type cheese and exhibiting strong anti-listerial activity in situ on cheese surface. In a first step, strains were tested in vitro for production of antimicrobial compounds against Listeria innocua 81000-1 and Listeria ivanovii HPB 28. M. psychrotolerans ALK 9 inhibited both indicator strains in spot-on-the-lawn tests while A. kapii ALK 6 showed no inhibiting effect. F. tabacinasalis ALK 1 exerted an in vitro inhibition on L. ivanovii HPB 28, but induced the formation of dense ball-shaped microcolonies of L. innocua 81000-1 in the soft agar, a typical biofilm microstructure. The extent of the biofilm zone was enhanced when F. tabacinasalis ALK 1 and M. psychrotolerans ALK 9 were tested together. In a second step, different combinations of strains were applied on Raclette cheeses ripened at pilot scale and contaminated with 50 cfu/cm(2)L. innocua at day 7. A control flora of 6 strains, isolated from ecosystem F and corresponding to species commonly found on smear cheeses, was applied on control and test cheeses. In test cheeses, we investigated the impact on Listeria growth of the addition of the 3 FAHA strains, applied as single or mixed cultures. A 1-log inhibition was obtained at day 15 on cheeses treated with FAHA strains applied either as single or mixed cultures. This 1-log inhibition was correlated with the development of FAHA species that reached their maximal count at day 15. This study suggests that the development of FAHA species in early ripening likely contributes to the initial part of the in situ inhibition exerted by the complex cheese surface ecosystem investigated.

Surface growth of geotrichum candidum: effect of the environmental factors on its dynamics

The growth dynamics of Geotrichum candidum was studied on the surface of the skim milk agar with respect to the temperature, pH and water activity/NaCl content. At pH ranging from 5.0 to 7.0, the fungus growth rates were similar, whereas the temperature and water activity represented by salt addition in concentration of 3 % influenced the growth significantly. The effect of incubation temperature on the surface growth rate was modelled with G-Twmodel. Designed model proved to be good predictor of fungus growth at used environmental conditions. As the filamentous fungus under study is commonly present on the surface of various cheeses, the quantitative data found in this work can provide useful information closely related to real fungus growth, e.g. calculation of time required for G. candidum to reach visible 3 mm colony. The predictions showed that, for example at 0 % NaCl content, such colonies were grown for 52.2, 30.7, 18.4, 14.4, 13.9 hat temperatures of 10, 14, 19, 23, 27 °C, respectively.

Effect of biopolymers containing natamycin against Aspergillus niger and Penicillium roquefortii on fresh kashar cheese

The Scientific and Technological Research Council of Turkey (TUBITAK-TOVAG Project No: 104O543)

The arthrobacter arilaitensis Re117 genome sequence reveals its genetic adaptation to the surface of cheese.

Arthrobacter arilaitensis is one of the major bacterial species found at the surface of cheeses, especially in smear-ripened cheeses, where it contributes to the typical colour, flavour and texture properties of the final product. The A. arilaitensis Re117 genome is composed of a 3,859,257 bp chromosome and two plasmids of 50,407 and 8,528 bp. The chromosome shares large regions of synteny with the chromosomes of three environmental Arthrobacter strains for which genome sequences are available: A. aurescens TC1, A. chlorophenolicus A6 and Arthrobacter sp. FB24. In contrast however, 4.92% of the A. arilaitensis chromosome is composed of ISs elements, a portion that is at least 15 fold higher than for the other Arthrobacter strains. Comparative genomic analyses reveal an extensive loss of genes associated with catabolic activities, presumably as a result of adaptation to the properties of the cheese surface habitat. Like the environmental Arthrobacter strains, A. arilaitensis Re117 is well-equipped with enzymes required for the catabolism of major carbon substrates present at cheese surfaces such as fatty acids, amino acids and lactic acid. However, A. arilaitensis has several specificities which seem to be linked to its adaptation to its particular niche. These include the ability to catabolize D-galactonate, a high number of glycine betaine and related osmolyte transporters, two siderophore biosynthesis gene clusters and a high number of Fe3+/siderophore transport systems. In model cheese experiments, addition of small amounts of iron strongly stimulated the growth of A. arilaitensis, indicating that cheese is a highly iron-restricted medium. We suggest that there is a strong selective pressure at the surface of cheese for strains with efficient iron acquisition and salt-tolerance systems together with abilities to catabolize substrates such as lactic acid, lipids and amino acids.

Study of Mucor spp. in semi-hard cheese ripening

A strain of Mucor spp. isolated from a fermented soybean food in central China was smeared on the surface of semi-hard cheese to investigate its role in cheese ripening. Cheese ripening indices showed increase by 3-4 fold upon ripening of cheese for 90days. Electrophoretic studies revealed that caseins in the cheese were degraded gradually during ripening forming several low molecular weight peptides, especially of 14.4kDa. Texture profile analysis and microstructure study of the cheese showed that the proteolytic activity of Mucor led to desirable texture development in ripened cheese. The study indicates that the strain of Mucor used in the experiment has desirable proteolytic capability for use in cheese system.

Genome sequence of Corynebacterium mooreparkense DSM 44702 isolated from the surface of a smear-ripened cheese

Genome sequence of Corynebacterium mooreparkense DSM 44702 isolated from the surface of a smear-ripened cheese

Listeria monocytogenes in Irish Farmhouse cheese processing environments

Sixteen cheesemaking facilities were sampled during the production season at monthly intervals over a two-year period. Thirteen facilities were found to have samples positive for Listeria monocytogenes. Samples were divided into 4 categories; cheese, raw milk, processing environment and external to the processing environment (samples from the farm such as silage, bedding, and pooled water). In order to attempt to identify the source, persistence and putative transfer routes of contamination with the L. monocytogenes isolates, they were differentiated using PFGE and serotyping. Of the 250 isolates, there were 52 different pulsotypes. No pulsotype was found at more than one facility. Two facilities had persistent pulsotypes that were isolated on sampling occasions at least 6 months apart. Of the samples tested, 6.3% of milk, 13.1% of processing environment and 12.3% of samples external to the processing environment, respectively, were positive for L. monocytogenes. Pulsotypes found in raw milk were also found in the processing environment, however, one of the pulsotypes from raw milk was found in cheese on only one occasion. One of the pulsotypes isolated from the environment external to the processing facility was found on the surface of cheese, however, a number of them were found in the processing environment. The results suggest that the farm environment external to the processing environment may in some cases be the source of processing environment contamination with L. monocytogenes.

Anti-listerial potential of food-borne yeasts in red smear cheese

Screening of 175 yeasts in an agar plate co-cultivation assay revealed that five out of 31 species reduced Listeria monocytogenes by 4–5log units, one exceptionally active Pichia norvegensis reduced Listeria by 7log units. To test the anti-listerial activity of this Pichia strain on cheese, Tilsit cheese and smeared acid curd cheese (Harzer) were prepared. The Tilsit cheese surface was inoculated with a 3%-NaCl brine containing Brevibacterium linens, Microbacterium gubbeenense, Corynebacterium casei, Staphylococcus equorum, Debaryomyces hansenii, P. norvegensis and L. monocytogenes. Ripening was done at 13°C and >95% relative humidity. On the Tilsit, but not on the Harzer cheeses, a decrease of listerial cell numbers by 1–2log units was observed. The difference between high inhibition in agar plate co-cultivation versus cheese is probably due to a decreased expression of the unknown inhibitory substance due to lactate, but not by the low pH.

Potent antilisterial cell-free supernatants produced by complex red-smear cheese microbial consortia

The microbial surface ripening consortia of 49 soft cheeses were investigated with respect to their inhibition of Listeria monocytogenes. When L. monocytogenes EGDe (serovar 1/2a) was cultivated in cell-free supernatants obtained from consortia grown for 8h in liquid medium, a strong bactericidal activity was observed in several cases. The cell-free supernatants of 2 of these consortia (I and II) reduced an initial L. monocytogenes inoculum of 5 × 107 cfu/mL to zero after 24h of incubation. No inhibitory substances could be washed off the complex consortia when incubated for a 10-min period. A taxonomical analysis of the antilisterial consortia I and II using Fourier transform infrared spectroscopy yielded a considerable species diversity, with lactic acid bacteria increasing strongly during the 8-h cultivation. Therefore, 23 lactic acid bacteria bacteriocin genes were assayed using specific PCR primers, identifying 3 bacteriocin genes in both microbial communities. However, no transcription of these genes was found on cheese surfaces or in consortia propagated in liquid culture. Individual lactic acid bacteria isolates of consortia I and II displayed no or only weak inhibition of L. monocytogenes on solid medium. The complex cell-free supernatants I and II, in contrast, exhibited an unusually broad inhibitory spectrum, killing L. monocytogenes ssp., Bacillus spp., Staphylococcus aureus, as well as gram-negative bacteria such as Escherichia coli DH5α and Salmonella enterica serovar Typhimurium. Inhibition could not be abolished by heating to 100°C or by proteinase K treatment. Initial purification of an inhibitory substance from consortium I by solid-phase extraction and HPLC indicates the presence of rather small, extremely stable compounds, which, most probably, are not bacteriocins.

From undefined red smear cheese consortia to minimal model communities both exhibiting similar anti-listerial activity on a cheese-like matrix

Starting from one undefined cheese smear consortium exhibiting anti-listerial activity (signal) at 15 °C, 50 yeasts and 39 bacteria were identified by partial rDNA sequencing. Construction of microbial communities was done either by addition or by erosion approach with the aim to obtain minimal communities having similar signal to that of the initial smear. The signal of these microbial communities was monitored in cheese microcosm for 14 days under ripening conditions. In the addition scheme, strains having significant signals were mixed step by step. Five-member communities, obtained by addition of a Gram negative bacterium to two yeasts and two Gram positive bacteria, enhanced the signal dramatically contrary to six-member communities including two Gram negative bacteria. In the erosion approach, a progressive reduction of 89 initial strains was performed. While intermediate communities (89, 44 and 22 members) exhibited a lower signal than initial smear consortium, eleven- and six-member communities gave a signal almost as efficient. It was noteworthy that the final minimal model communities obtained by erosion and addition approaches both had anti-listerial activity while consisting of different strains. In conclusion, some minimal model communities can have higher anti-listerial effectiveness than individual strains or the initial 89 micro-organisms from smear. Thus, microbial interactions are involved in the production and modulation of anti-listerial signals in cheese surface communities.

Evaluation of a chitosan-based edible film as carrier of natamycin to improve the storability of Saloio cheese

The purpose of this study was to evaluate the effects of the application of chitosan coating containing natamycin on the physicochemical and microbial properties of semi-hard cheese. Three cheese groups were prepared: samples without coating, samples coated with chitosan and with chitosan containing 0.50 mg mL −1 of natamycin, whose minimum inhibitory concentration was previously determinated on cheese surface. Microbiological analyses showed that natamycin coated samples presented a decrease on moulds/yeasts of 1.1 log (CFU g −1) compared to control after 27 days of storage. Addition of natamycin also affected O 2 and CO 2 permeability, increasing from 7.12 to 7.68 × 10 −15 g·(Pa s m) −1, and from 10.69 to 64.58 × 10 −14 g·(Pa s m) −1, respectively. The diffusion coefficient values of natamycin from the film to phosphate buffered saline solution and to the cheese were 3.60 × 10 −10 and 1.29 × 10 −12 cm 2 s −1, respectively. This study demonstrated that chitosan-based coating/films can be used as release system containing natamycin to create an additional hurdle for moulds/yeasts in cheese thus contributing to extend its shelf-life.

High biodiversity and potent anti-listerial action of complex red smear cheese microbial ripening consortia

When Listeria monocytogenes EGDe (serovar 1/2a) was cultivated in cell-free supernatants prepared from red smear cheese microbial ripening consortia grown for 8 h in liquid medium, 8 out of 49 supernatants exhibited a bactericidal activity, sometimes even reducing the inoculum of L. monocytogenes from 5 × 107 CFU/ml to zero after 24 h of incubation. Another five consortia displayed a bacteriostatic capacity. No inhibition in supernatants was observed when the complex consortia were incubated for a 10-min period only, indicating that the activity depends on actively growing consortia. Consortia displayed a very high biodiversity (Simpson’s strain diversity index up to 0.97, species diversity up to 0.89). However, biodiversity did not correlate with anti-listerial activity. There was no obvious similarity between the anti-listerial consortia studied, and no general difference in comparison to non-inhibitory communities. The proportion of lactic acid bacteria (LAB) in the consortia ranged between 3 and 45%. Therefore, the presence of 23 different LAB bacteriocin genes was investigated using specific PCR primers, identifying one to five bacteriocin genes in several consortia. In situ transcription of lactococcin G mRNA on the cheese surface was demonstrated by RT-PCR in five samples, but this bacteriocin displayed no anti-listerial activity. Supernatants subjected to thermal and enzymatic treatment suggested the presence of heat-stable, non-proteinaceous molecules as well as heat-labile compounds which are sensitive to proteolytic digestion. Probably, substances other than LAB bacteriocins are responsible for the pronounced antilisterial action of some supernatants.

Influence of the preservative material HOLDBACTM on the growth and proliferation of Listeria on the surfaces of cheeses

Three varieties of cheese were selected for the experiment: Genuine Olomouc-brand soft ripened cheese, Loose-brand acid curd cheese, and Slovak-style string cheese. Their surfaces were inoculated with Listeria cells of a defined density and then treated with the preservative material HOLDBAC<sup>TM</sup> at its optimal concentration. The cheeses were stored at two different temperatures for the periods of 2, 5, 9, and 14 days, after which the effectiveness of the treatment with the preservative material was determined. The results were compared with the effectiveness of the material on the growth and proliferation of Listeria when the application of the preservative material was made before the artificial contamination of the cheese surface with Listeria. The experiment demonstrated only a slightly greater effectiveness of HOLDBAC<sup>TM</sup> when it was applied before the microbial contamination. Complete inhibition of Listeria was not observed when using this approach.

Sampling analytes from cheese products for fast detection using neutral desorption extractive electrospray ionization mass spectrometry

The development of analytical techniques suitable for sensitive, high-throughput, and nondestructive food analysis has been of increasing interest in recent years. In this study, mass-spectral fingerprints of various cheese products were rapidly recorded in the mass range of m/z 50-300 Da without any sample pretreatment, using neutral desorption extractive electrospray ionization mass spectrometry (ND-EESI-MS) in negative ion mode. The results demonstrate that both volatile and nonvolatile analytes on greasy cheese surfaces can be directly sampled by a neutral desorption gas beam. The influence of the neutral desorption gas flow on the analyte signal was systematically investigated. Under optimized experimental conditions, reproducible results were obtained using ND-EESI-MS. Principal component analysis was applied to differentiate a total of 49 individual cheese samples (four different types), which were purchased from three different supermarkets. All samples were successfully classified according to their types; but distributors and sensory properties were not distinguishable from the spectra data. The principal components 2, 3, and 4 scores showed an excellent capacity of distinguishing types of cheese. Molecular markers of interest can be identified using tandem mass spectrometry and matching the data with those from reference compounds. The experimental data show that ND-EESI-MS is able to sensitively and directly detect analytes on greasy surfaces without chemical contamination, providing a convenient method for high-throughput food analysis with a high degree of safety.

Application of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces

To evaluate and optimize the use of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces. The resolution of DHPLC was first evaluated and optimized using a mixture of PCR amplicons of the internal transcribed spacer 2 (ITS2) region of 19 yeast reference strains representing 18 species that are common in the cheese microbiota. Sixteen of the 18 yeast species could be resolved by combining runs at temperatures of 57.5 and 59 degrees C. Then, DHPLC was used to investigate the yeast microbiota of pasteurized Maroilles, Munster and Livarot cheese surfaces by comparing their peak profiles with our reference yeast database and by collecting/sequencing of peak fractions. Debaryomyces hansenii and Geotrichum candidum for Munster and Maroilles cheeses, and Candida catenulata, Candida intermedia and G. candidum for Livarot cheese were identified using the reference database and collecting/sequencing of peak fractions. DHPLC technique was found to have good resolution properties and to be useful for investigating the yeast microbiota of red smear cheese surfaces. This is the first time that DHPLC is applied to study the yeast microbiota of red smear cheese surfaces.

Is microbial diversity an asset for inhibitingListeria monocytogenesin raw milk cheeses?

This study aimed at determining if microbial diversity can be an asset to guarantee the microbial safety of raw milk cheeses. Our results show that microbial consortia from the surface of raw milk cheeses can self-protect against Listeria monocytogenes. Indeed, 10 complex microbial consortia among 34 tested from the surfaces of raw milk Saint-Nectaire cheeses were particularly effective for reducing the growth of L. monocytogenes on cheese surfaces in comparison of a commercial ripening culture, despite the high pH values on the surfaces. One of these consortia (TR15) was selected and propagated on cheese surfaces to create a collection of strains belonging to lactic acid bacteria, Gram-positive and catalase-positive bacteria, Gram-negative bacteria and yeasts. On the surfaces of uncooked cheeses, defined consortia consisting of combinations of several isolates from this collection displayed weaker antagonist activity against L. monocytogenes than the complex consortium TR15. The results from plate counting and analysis by single strand conformation polymorphism (SSCP) converged to show that microbial dynamics in cheeses TR15 differed from that of the defined consortia. TR15 cheeses had the highest levels of cultivable lactobacilli and leuconostocs. Their SSCP profiles were the richest in peaks and were characterised by the presence of Marinilactibacillus psychrotolerans, Carnobacterium mobile, Arthrobacter nicotianae or A. arilaitensis, Arthrobacter ardleyensis or A. bergerei and Brachybactrerium sp. Further investigation will be necessary to gain a better understanding of the microbial interactions involved in inhibiting L. monocytogenes.

Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis

BackgroundSurface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE), a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F) or produced with an old-young smearing process (M).ResultsTTGE revealed nine bacterial species common to both F and M consortia, but consortium F exhibited a higher diversity than consortium M, with thirteen and ten species, respectively. Population dynamics were studied after application of the consortia on fresh-produced Raclette cheeses. TTGE analyses revealed a similar sequential development of the nine species common to both consortia. Beside common cheese surface bacteria (Staphylococcus equorum, Corynebacterium spp., Brevibacterium linens, Microbacterium gubbeenense, Agrococcus casei), the two consortia contained marine lactic acid bacteria (Alkalibacterium kapii, Marinilactibacillus psychrotolerans) that developed early in ripening (day 14 to 20), shortly after the growth of staphylococci (day 7). A decrease of Listeria counts was observed on cheese surface inoculated at day 7 with 0.1-1 × 102 CFU cm-2, when cheeses were smeared with consortium F or M. Listeria counts went below the detection limit of the method between day 14 and 28 and no subsequent regrowth was detected over 60 to 80 ripening days. In contrast, Listeria grew to high counts (105 CFU cm-2) on cheeses smeared with a defined surface culture.ConclusionsThis work reports the first population dynamics study of complex smear ecosystems exhibiting in situ antilisterial activity. TTGE revealed the presence of marine lactic acid bacteria that are likely related to the strong Listeria inhibition, as their early development in the smear occurred simultaneously with a decrease in Listeria cell count.