A multi-sampling approach to evaluate an infrared surface treatment for reducing Listeria monocytogenes contamination on whole Gorgonzola cheese rinds

The microbial ecology of Gorgonzola cheese rind is the focus of many studies because the surface can be contaminated by pathogenic microorganisms. Among food-borne pathogens, particular attention is focused on the behaviour of Listeria monocytogenes that is able to grow at refrigeration temperatures and it could also grow during ripening. The Consortium for the Protection of Gorgonzola Cheese declares the rind not edible but the pathogen may also be transferred during cutting and portioning. Therefore, the decontamination of rinds is important to increasing cheese safety. To achieve this goal, many different strategies have been proposed. In this study, the application of an infrared surface treatment to decontaminate cheese rinds is proposed. The presence of L. monocytogenes, which was artificially inoculated in cheese rinds together with cheese rind microflora, and the cheese rind microflora were monitored before and after the treatment of 32 samples of Gorgonzola cheese rinds.The infrared surface treatment provided good reduction of the rind microflora, and L. monocytogenes was particularly affected by this. The treatment, applied to cheeses at the end of ripening, does not interfere with the ripening process and offers the advantages of short time exposures and easy installation of the equipment in cheese plants. Moreover, this study demonstrated that the sampling method affects the detection of cheese rind microflora. In fact, a non-destructive sampling method, based on a sponge and often used for surface sampling but never before applied to ready to eat food sampling, was compared with a traditional but destructive method, based on rind scraping. Regarding L. monocytogenes, the sponge method allowed to estimate even only 5.71 ± 0.79 log cfu g⁻¹ of cells reduction after the treatment while the higher reduction when considering the rind scraping method was 4.06 ± 3.38 log cfu g⁻¹. The sponge method, combined with the classic scraping one, besides offering the great advantage of not being destructive, allowed to differentiate the effect that the treatment has on the microflora located on the surface from those in deeper layers.     

Microbial benefits and risks of raw milk cheese

Consumer preference for raw milk cheese is continually growing, owing to its more intense and varied flavor than pasteurized milk cheese. Flavor development in raw milk cheese is mainly governed by its naturally existing microbial community, which also contributes to the inhibition of food-borne pathogenic bacterial growth. Lactic acid bacteria, the dominant indigenous microorganisms of raw milk cheese, produce pathogen-inhibiting substances such as bacteriocin, organic acids, and hydrogen peroxide, and it is possible to manufacture cheese with desirable microbiological qualities. Nonetheless, outbreaks of food-borne illnesses have been linked to the consumption of raw milk cheese, and concerns have been raised regarding the microbiological safety of cheese manufactured from raw milk. Consequently, efficient and accurate methods for detecting contaminated bacterial pathogens in raw milk cheese have been promptly developed, including conventional plating, PCR-based technology, and immunoassay-integrated methods. The microbiological risk of the cheese can be reduced by proper ripening processing. However, additionally, hygiene in the environments for milk production and cheesemaking and the post-manufacturing stage needs to be constantly microbiologically monitored.     

Transfer of aflatoxin M1 from milk to ripened cheese in three Italian traditional production methods

Robiola and Primosale, two fresh cheeses, and Maccagno, an hard-type cheese, were produced using milk that was naturally and artificially contaminated with aflatoxin M₁ (AFM₁) at the levels of 10, 50 and 200 ng/l. Concentrations of AFM₁in milk and cheeses were determined by liquid chromatography and fluorimetric detection, coupled with immunoaffinity column extraction. In the Robiola production method, AFM₁ levels in whey ranged between 30% and 65% of the total amount of the toxin present in the milk, while Primosale and Maccagno, that share the same rennet based cheesemaking procedure, showed an higher percentage of AFM₁ partitioning to whey.For each cheese-making method, the concentration of AFM₁ on fresh matter was higher in the cheese compared to the original milk. The fresh cheeses showed a concentration factors of 1.43 and 2.20 for Primosale and Robiola, respectively, whereas the Maccagno cheese showed a value of 6.71. For all the production methods considered, when using milk not exceeding the maximum acceptable level of 0.05 μg AFM₁/kg set by EU, the resulting cheese also complied with current Italian recommendations for AFM₁ contamination (450 ng AFM₁/kg).     

Characterization of the natural microflora of artisanal Mexican Fresco cheese

The objective was to characterize the microflora of Mexican artisanal Fresco cheese, as a first step towards the development of a starter culture, which would allow the making of a safe and uniform product from pasteurized milk. The numbers of several microbial groups (aerobic mesophilic flora, total and fecal coliforms, Salmonella spp., Staphylococcus spp., molds, yeasts and lactic acid bacteria) were investigated. The dominating isolates were characterized as being Lactococcus lactis ssp. lactis, Enterococcus faecium and Lactobacillus casei. The results suggest that an adequate starter in the production of Mexican Fresco cheese may be composed of strains of these species.     

Aflatoxin M1 contamination in white and Lighvan cheese marketed in Rafsanjan,Iran

This study was a screening survey to determine the occurrence of aflatoxin M₁ (AFM₁) in 82 cheese samples composed of white cheese (45 samples) and Lighvan cheese (37 samples) obtained from supermarkets and retail outlets in Rafsanjan city of Iran. The competitive enzyme immunoassay method was used for determination of the toxin in the samples. Aflatoxin M₁ was detected in 39 (47.6%) samples, consisting of 29 (64.4%) white cheese (mean: 135 ng/kg; range: 93.3–309 ng/kg) and 10 (27%) Lighvan cheese samples (mean: 90.8 ng/kg; range: 70.5–203 ng/kg). According to Iranian national standard limit for AFM₁ in cheese (200 ng/kg), 9 samples (20%) of white cheese and 1 sample (2.70%) of Lighvan cheese had levels above the limit. It was concluded that the contamination of the samples with AFM₁ in such a level could be considered as a serious public health problem.     

Identification and characterization of the microbial agent responsible of an alteration in spoiled,Grana Padano cheese during ripening

A peculiar alteration of microbial origin of an extra hard cheese ripened in a factory within the geographic area of production of Grana Padano (GP), Protected Designation of Origin (PDO) cheese, was investigated. The defect appeared after 6–7 months of ripening as whitish spots located in the innermost parts of the cheese wheel and concerned only a few cheeses produced in the spring and summer seasons. The alteration evolved with a degradation of the cheese matrix, followed by a formation of cavities containing an unpleasant smelling serosity, with little accumulation of gas. Notably, the spoiled cheeses did not show evidence of late blowing. According to these characteristics, the study was addressed to the detection of anaerobic sporeforming, proteolytic, butyric clostridia. From three cheese wheels with the defect, samples from both spoiled (S) and non-spoiled (NS) cheese areas were taken. The average number of butyric clostridia spores (BCS) was 2.5 and <-0.5 log MPN g⁻¹ in the S- and NS-areas, respectively. From the positive MPN tubes of both S and NS samples, 24 strains, all proteolytic on milk agar, were isolated. By a species-specific PCR, they were all identified as Clostridium (C.) sporogenes and grouped into four distinct genotypes after RAPD-PCR fingerprinting analysis. One of the four genotypes included only strains isolated from NS-areas whereas the other three were from S-areas. A partial sequencing of the 16S rRNA gene of strains belonging to the four RAPD-PCR clusters confirmed the identification as C. sporogenes. Two strains, one (strain 4990), which represented the dominant genotype from the S-areas and the other (strain 4992), which was the unique genotype isolated from the NS-areas, showed a 5 bp sequence polymorphism within the first 500 bp of the 16S rRNA gene. The two strains showed also distinct High-Resolution Melting profiles, which were in turn different from the type strains of C. sporogenes, C. tyrobutyricum, C. butyricum, and C. beijerinckii, and morphological and motility differences. Metataxonomic analysis showed Clostridium spp. as the most abundant taxon in all the S samples. Overall, these results confirmed the involvement of C. sporogenes in the onset of the defect of the cheeses studied. Genotypic and phenotypic characterization suggested the presence of two different prevailing biotypes of C. sporogenes, corresponding to two separate genetic lines, with notable morphological and taxonomic differences, isolated respectively from spoiled and defect-free zones of the cheeses. A more specific adaptation to the cheese substrate or a better efficiency of the enzymatic processes involved in the Stickland reaction may explain the dominance of strain 4990 in spoiled cheese samples.     

Fate of aflatoxin M1 during production and storage of parmesan cheese

The fate of AFM₁ during production of a long maturing cheese (Parmesan cheese) was assessed. Different levels of AFM₁ contamination and of the fat/casein (F/C) ratio of milk were considered, in order to evaluate if these factors can influence the enrichment factor (EF) of AFM₁ in cheese. For this purpose, 24 cheese-makings were carried out using naturally contaminated milk at 3 different AFM₁ levels and at 2 F/C ratios. AFM₁ analysis was performed by HPLC in raw milk, cream, cauldron milk, liquid cattle rennet, whey, curd and cheese at 3, 9, 16 and 24 months of ageing. The mass balances of the cheese-making processes were close to 100%; in whey, AFM₁ concentration was about 40% less than the concentration in cauldron milk. The EF in curd was between 4.0 and 5.2, with an average value of 4.7 ± 0.4; this factor was not significantly affected by either AFM₁ contamination level or F/C ratio. During maturation, AFM₁ concentration and consequently EF increased from curd to 16 ageing months; successively, AFM₁ slightly decreased at 24 months and consequently the EF. At 3, 9, 16 months of maturation, the EF was significantly higher for cheeses prepared using milk with low F/C than those with high F/C milk; on the contrary, EF was not significantly influenced by the AFM₁ contamination level. In cheeses, EF values were between 4.7 and 6.3; from these results, the maximum admissible level for AFM₁ in Parmesan cheese should be about 0.275 μg kg⁻¹.     

Rheological,physicochemical and authenticity assessment of Minas Frescal cheese

This study was carried out to assess physicochemical (color, moisture, protein and fat content) and rheological properties (fracture stress, fracture strain, degree of solidity and stress decay), as well as to assess the authenticity (using SDS-PAGE) of Minas Frescal cheese in 21 commercial samples acquired in Southern Brazil. Large differences among the samples, which tended to present a yellowish white color were observed. Moisture and protein influenced on the rheological properties and cheeses showed a tendency to being viscous and firm. SDS-PAGE revealed a wide unexpected variability in their proteins profiles and a densitometric casein/β-lactoglobulin ratio allowed to distinguish authentic cheese from adulterated ones. The profiles suggest the irregular presence of whey in the composition of these products, contrary to the enzymatic process legally required. These results emphasize the need for a more effective control, mainly in the selection of raw material, aiming to offer consumers authentic products.     

Modelling the interaction of storage temperature,pH, and water activity on the growth behaviour of Listeria monocytogenes in raw and pasteurised semi-soft rind washed milk cheese during storage following ripening

The objective of this study was to investigate the growth of Listeria monocytogenes in semi-soft rind washed cheese made from raw and pasteurised milk at different storage temperatures (4, 10 and 15 °C) over a 28 day period simulating storage following ripening. Changes in water activity (a_w) and pH in cheeses were also monitored during storage. Response surface models were used to model the interaction of storage temperature and time on a_w, pH and L. monocytogenes population. Growth curves were fitted using Baranyi, modified Gompertz and Logistic models at all storage temperatures for both cheeses, and model parameters were statistically analysed. In raw and pasteurised milk cheeses, all models showed a significant (P < 0.05) increase in the specific growth rate (SGR, Day⁻¹) of L. monocytogenes with an increase in storage temperature. A higher SGR was observed for L. monocytogenes in pasteurised milk cheese (0.18–0.85 Day⁻¹) compared to raw milk cheese (0.05–0.37 Day⁻¹) at all storage temperatures studied. Response surface models indicated an increase in the L. monocytogenes population and pH with an increase in storage temperature. However, a decreasing trend in a_w for both cheese types was observed. The predicted regression model parameters for both the raw and pasteurised milk cheese showed a high correlation coefficient R² > 0.87. Overall, the L. monocytogenes population increased up to 3 log₁₀ cfu⁻g⁻¹ for both cheeses during storage following ripening. The fitted models confirmed different L. monocytogenes growth behaviour between raw and pasteurised milk cheeses, which could support the Food Business Operator in predicting growth during storage following ripening.     

Relevance and analysis of butyric acid producing clostridia in milk and cheese

Via butyric acid fermentation, clostridia – mainly Clostridium tyrobutyricum – are able to transform lactic acid into butyric acid, acetic acid and gas (H₂ and CO₂). The presence of clostridial endospores in milk may lead to severe quality defects in semi-hard and hard cheeses. As a consequence of butyric acid fermentation during ripening, cheeses tend to swell and develop undesired slits, irregular eyes and a rancid taste, thus resulting in high economic losses for producers. Several measures regarding stable, milking and feed hygiene have already been partly implemented to minimise the risk of raw milk contamination with clostridial endospores. Contamination, nevertheless, cannot be avoided completely. Moreover, some of the existing procedures to reduce the bacterial and endospore count in milk (e.g. bactofugation, addition of bacteriocins) are not always applicable or even prohibited for the production of certain cheese types. Therefore, cheese producers may benefit from integrating the determination of the initial count of clostridial endospores in milk into their quality control system of primary materials. This review discusses the role of butyric acid clostridia in the cheese processing environment and methods for the detection and enumeration of cheese-damaging clostridia in milk and cheese.     

Effects of combined organic acid treatments during the cutting process on the natural microflora and quality of chicken drumsticks

Reducing the microbial load on broiler chicken carcasses at each stage of poultry meat processing, is highly important for hygienic meat production. This study was conducted to assess the efficacy of various combined organic acid treatments during the cutting process on the microbial decontamination of chicken drumsticks. Changes in naturally occurring microflora before and after treatment were analyzed through microbiological counting and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE). Results revealed that the most effective treatments obtained through the combination of orthogonal design and sensory evaluation were as follows: 0.5% lactic acid (w/v), 1% citric acid (w/v), and spray-washing for 30 s. Microbiological counting results and PCR-DGGE analysis indicated that the microbial load on the chicken drumsticks decreased significantly after the treatment was administered. The treatment did not affect the physicochemical properties and sensory attributes of the quick-frozen chicken drumsticks during storage. Therefore, the proposed technique could be used to improve the microbial quality of chicken drumsticks. The technique could also be employed in poultry meat production chains.     

Distribution,stability, and protein interactions of Aflatoxin M1 in fresh cheese

Aflatoxin M₁ (AFM₁), a metabolite resulting from the hepatic metabolism of aflatoxin B₁, is a potential carcinogen that can be found in milk, cheese, and others dairy products. In this work, we aimed to elucidate the distribution and fate of AFM₁ in fresh cheese and whey during cheese manufacturing and storage as well as the level of interaction of this toxin with various milk, cheese, and whey proteins. Additionally, we analyzed the in vitro behavior of casein, α-lactalbumin, β-lactoglobulin, bovine serum albumin (BSA), lactoferrin, and mixtures thereof, with a fixed concentration of AFM₁ after covalent crosslinking. Our results show that up to 70% of the AFM₁ levels present in milk spiked with 0.5 and 1.5 μg L⁻¹ are released in whey during fresh cheese manufacturing. The whey and milk proteins with more AFM₁ molecules bound were α-lactalbumin and casein, with 88% and 81%, respectively. We also observed a substantial decrement in AFM₁ concentration during ripening that correlated inversely with plate counts of lactic acid bacteria and directly with the whey-draining process that occurs during fresh cheese maturation or storage. This knowledge may serve as a basis for interventions in the dairy industry aiming to increase the security of cheese and other dairy products.     

The presence,genetic diversity and behaviour of Listeria monocytogenes in blue-veined cheese rinds during the shelf life

Blue-veined cheeses may allow Listeria monocytogenes survival and multiplication due to the biochemical characteristics of the cheese and the growth characteristics of the pathogen. Because of the availability of a considerable number of samples, we wanted this study to take a large view of the microbiota and of the incidence and genetic diversity of L. monocytogenes in blue-veined cheese rinds. Moreover, we wanted to determine if the pathogen present on the rinds at the end of ripening represented a risk to the consumer if the cheese should exceed the domestic storage limit imposed by Commission Regulation (EC) No 2073/2005 for food safety criteria.The rind microflora showed a high level of heterogeneity, and the incidence of L. monocytogenes (never found in the paste samples) was 55%, with an increased presence and concentration associated with a longer ripening time. Amplified Fragment Length Polymorphism (AFLP) was used to characterise L. monocytogenes isolated from blue-veined cheese rinds and highlighted the heterogeneity among the strains, demonstrating its suitability for studying the biodiversity of the pathogen in this environment. L. monocytogenes was able to grow during the shelf life of the cheese and was influenced by the refrigeration temperatures and the physicochemical characteristics of the cheese.     

Starch edible film supporting natamycin and nisin for improving microbiological stability of refrigerated argentinian Port Salut cheese

The effectiveness of natamycin and nisin supported in edible films (NANI) was evaluated, at 7 ± 1 °C, in relation to the improvement of the microbiological stability of Port Salut cheese. This film inhibited the growth of yeasts and moulds and controlled the growth of psychrotrophic bacteria originally present in the Port Salut cheese stored at refrigeration temperature. It also inhibited the development of a mixed culture (Saccharomyces cerevisiae and Listeria innocua) present in the cheese due to a superficial contamination, throughout the entire storage. With respect to a postprocessing contamination affecting the film present in a covered cheese, control film, commercial film and film containing nisin and natamycin (NANI), acted as barriers precluding the mixed culture growth on the cheese surface. But only NANI inhibited the growth of that culture on the film. These results are demonstrating that the film NANI is an extremely effective method to control the population of microorganisms present in both the cheese and the film, enabling to offer the consumer a safer product.     

Survival of Listeria innocua in Minas Traditional Serro cheese during ripening

Cheese produced with raw milk can be a risk to consumer health. It is known that lactic acid bacteria present in raw milk and in natural starters can produce antimicrobial compounds against some foodborne bacteria. This work aimed to evaluate the survival of Listeria innocua in Minas Traditional Serro cheese during cheese ripening. The cheeses were inoculated with 10¹, 10² or 10³ CFU mL⁻¹ of the bacterium and were analyzed for 60 days of ripening at 30 °C. It was observed that the time and the dose of bacteria inoculated affected (p < 0.05) the survival of L. innocua. Even when the lowest dose was inoculated, at the end of the 60 days, approximately 10² CFU mL⁻¹ of L. innocua was detected in the cheese. The lactic acid bacteria present in the milk and in the natural starter were not sufficient to guarantee the absence of L. innocua in Minas Traditional Serro cheese even after 60 days of storage, as is required by Brazilian legislation.     

Survival of microencapsulated Bifidobacterium longum in Cheddar cheese during production and storage

The aim of this study was to evaluate the effect of microencapsulation (ME) in alginate beads on the viability of Bifidobacterium longum 15708 in terms of their tolerance to freezing, storage in a frozen state, cheddar cheese manufacturing and storage as well as to a simulated gastro-intestinal environment. Two ME methods namely i) droplet extrusion method (ADE) and ii) emulsion method, involving two polymers (native (NA) and palmitoylated alginate (PA)) were compared. Results showed that ADE maintained higher viability of B. longum after 24 h freezing at −80 °C with no viability loss as compared to the emulsion process and free cells which lost approximately 0.8 and 1.5 log CFU/mL respectively. However, during a 4 weeks storage period at −80 °C, no significant difference (P > 0.05) was observed in the survival of free and immobilized B. longum, with no loss of viability. Cheddar cheeses supplemented with B. longum culture were prepared and analysed during storage at 4 °C. After 21 days of storage, Cheddar cheese containing encapsulated B. longum in NA and PA polymers produced with the emulsion process showed a good survival with 2 log CFU/mL reduction after 21 days, as compared to ADE-encapsulated B. longum and free cells with 3 and 4 log CFU/mL reductions respectively. The immobilized bacteria in both polymers were also more resistant than free cells to simulated gastric and intestinal environments by a factor of 30.     

Cutting procedures might be responsible for Listeria monocytogenes contamination of foods: The case of Gorgonzola cheese

Commission Regulation (EC) No. 2073/2005 established, as a food safety criterion for tolerable levels of Listeria monocytogenes in ready-to-eat foods which do not support the growth of the pathogen or with shelf life below 5 days, a maximum of 100 cfu g⁻¹. Blue-veined cheeses are among these foods because their rinds can be contaminated, and the pathogen can be transferred to the paste during slicing. The aim of this research was to investigate whether cutting procedures could be responsible for cheese paste contamination. Considering that the Commission Regulation limit is allowed when the pathogen does not grow during the shelf life, we also wanted to verify whether, in the case of positive dragging, L. monocytogenes was able to grow on cut slices beyond the limit imposed, thereby becoming a risk for consumers during storage at 4 °C. Gorgonzola cheese was chosen for this investigation. The cutting simulation on artificially inoculated wheel rinds indicated that greater rind contamination corresponded to a higher percentage of contaminated paste samples. The growth of L. monocytogenes transferred to cut slices was variable relative to the physicochemical characteristics of the cheese, to the contamination level and to the time of storage. In particular, the sweet typology was able to support the growth of L. monocytogenes in the shelf life conditions considered and the quick overcoming of the limit imposed by food safety criteria would not ensure the safety for consumption.     

Presence of aflatoxin M1 in white and hard cheese samples from Serbia

The level of aflatoxin M1 (AFM1) in 54 samples of white and hard type of cheese produced in Serbia was investigated using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Two methodologies were developed for sample extraction, separately for white and hard type of cheese. The first one involved sample extraction with mixture of acetonitrile/water (86:14, v/v) for white cheese without sample clean-up step. The second method consisted of a sample extraction with dichloromethane/acetone for hard cheese followed by solid-phase extraction (SPE) clean-up by SPE C₁₈ cartridges. The average recoveries of AFM1 from spiked samples at levels of 0.25–0.50 μg/kg, were 73–111% and the precision (RSD) ranged from 7 to 9% with the first method, whilst the average recoveries from samples spiked at levels of 0.125–0.50 μg/kg were 71–80%, with RSD ranged from 4 to 10% for the second method. Due to different matrix effects, the quantification limits were 0.125 and 0.02 μg/kg for white and hard type of cheeses, respectively.Seven samples (13%) exceeded the maximum acceptable level of 0.25 μg/kg has been established for AFM1 in some European countries like Switzerland, France, Austria and Turkey.