Gram-positive Catalase-positive Cocci Research Articles

Microbial diversity in dry-cured Iberian ham: an approach to the concept of microbial terroir

Microbial terroir has been scarcely studied in the dry-cured ham industry. The microbial population and ochratoxin A (OTA) contamination in two Spanish facilities located in a specific geographical region (Salamanca) with differences in the environmental conditions used for the ham processing were evaluated for establishing the microbial terroir. Differences between the levels of Gram-positive catalase-positive cocci (GCC+), lactic acid bacteria (LAB), yeasts and moulds were found at the processing stage (post-salting, drying and cellar) and facility levels. Such variations could be responsible for the development of different flavour profiles, which indicate their potential functionality for determining the microbial terroir in the artisan dry-cured Iberian ham industry, despite the fact that this work constitutes a preliminary study. Furthermore, it has been demonstrated that the differences in the microbial population throughout the processing of both sampled facilities could directly impact on one of the main concerns of the meat industry from the food safety point of view consisting of the accumulation of OTA, since differences in its levels were detected between both checked facilities. These findings could lead to the enforcement of the terroir concept in artisan dry-cured Iberian ham elaboration, resulting in a value enhancement that might be reflected in economic and social positive results for the territory. Nevertheless, microbial terroir in the dry-cured Iberian ham must be further explored by the increase of the number of studied facilities.

Metabarcoding analysis reveals a differential bacterial community profile associated with ‘Torta del Casar’ and ‘Queso de la Serena’ PDO cheeses

A detailed understanding of the microbial ecology of artisanal cheeses is a crucial factor for implementation practices that can help to update their quality. In this study, the bacterial communities of Protected Designation of Origin (PDO) ‘Queso de la Serena’ and ‘Torta del Casar’ cheeses were characterised by 16S rRNA gene metabarcoding sequencing over 60 days of ripening at three dairies for each cheese variety. Both PDO cheeses presented a distinctive and unique bacterial community, suggesting that the manufacturing practices of each PDO shaped the microbiota. Lactococcus lactis and, to a lesser extent, Leuconostoc mesenteroides were the key lactic acid bacteria (LAB) species that drove the ripening process of PDO ‘Torta del Casar’, whereas in PDO ‘Queso de la Serena’, Leu. mesenteroides was the main LAB species, followed by Lactococcus raffinolactis and Lc. lactis, depending on the dairy. Furthermore, a wide range of non-starter LAB, Gram-negative bacteria and Gram-positive catalase-positive cocci were found, with a variable presence and abundance between dairies. In this regard, the dairy was a strong contributing factor to the variability in the composition of the bacterial community in each cheese type. In contrast, the impact of the ripening process was limited. Overall, this study enhances the understanding of the bacterial dynamics that occur during the ripening of these traditional PDO cheeses, highlighting the need to improve the standardisation of the raw materials and manufacturing practices to minimise inter-dairy variability, ensuring final quality and their unique sensory traits.

Bacterial species diversity of traditionally ripened sheep legs from the Faroe Islands (skerpikjøt)

Skerpikjøt is a traditionally ripened sheep leg product from the Faroe Islands, constituting a relatively underexplored microbial ecosystem. The objective of this study is to achieve a deeper understanding of the microbial composition of this artisanal product. Nine ripened hind legs, obtained from three different producers, were assessed regarding their bacterial communities and contents of biogenic amines, including both surface and core samples. Biogenic amine concentrations were generally low, although one sample had a somewhat elevated concentration of cadaverine. Bacterial diversity was investigated by culture-dependent and culture-independent techniques. Gram-positive catalase-positive cocci (GCC) constituted the most abundant group. Within this group, Staphylococcus equorum was the most prevailing species, followed by Kocuria sp., Mammaliicoccus vitulinus, and Staphylococcus saprophyticus. Lactic acid bacteria prevailed in only one sample and were mainly represented by Latilactobacillus curvatus. Enterobacterial communities were characterised by the prevalence of Serratia proteamaculans. Despite the majority of GCC, Clostridium putrefaciens was the most abundant bacterial species in some core samples. Taken together, the culture-dependent and culture-independent identification methods gave complementary results.

Selection and Evaluation of Staphylococcus xylosus as a Biocontrol Agent against Toxigenic Moulds in a Dry-Cured Ham Model System.

Toxigenic moulds can develop on the surface of dry-cured meat products during ripening due to their ecological conditions, which constitutes a risk for consumers. A promising strategy to control this hazard is the use of antifungal microorganisms usually found in these foods. However, to date, the effectiveness of gram-positive catalase-positive cocci (GCC+) has not been explored. The aim of this work was to select GCC+ isolates with antifungal activity to study its effectiveness in a dry-cured ham model system at the environmental conditions reached during the ripening. Forty-five strains of GCC+ were evaluated and the isolate Staphylococcus xylosus Sx8 was selected to assess its efficacy at two different concentrations (106 and 104 cfu/mL) against Penicillium nordicum, Aspergillus flavus, Aspergillus parasiticus, and Penicillium griseofulvum at 15, 20, and 25 °C. The results showed that the inoculation of 106 cfu/mL of S. xylosus completely inhibited the growth of most fungi. In addition, in the presence of this strain at 104 cfu/mL, a significant reduction in fungal growth and mycotoxins production was observed at the three temperatures studied. In conclusion, S. xylosus Sx8 possesses great potential as a biological agent to control toxigenic moulds in dry-cured meat products.

Role of Starter Cultures on the Safety of Fermented Meat Products.

Starters are microbial cultures used to promote and conduct the fermentation of meat products. Bacteria, particularly lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS), as well as yeasts and molds, may be used as starters. They can increase the safety of fermented meat products by means of rapid matrix acidification or due to the production of antimicrobial substances, such as bacteriocins. Besides, starters may help to standardize product properties and shorten ripening times. Safety of fermented meat products may be jeopardized by microbiological, namely foodborne pathogens (Salmonella spp., Listeria spp., etc), and chemical hazards, particularly biogenic amines, nitrosamines, polycyclic aromatic hydrocarbons (PAH), and mycotoxins. Biogenic amines (BA) are potentially unsafe nitrogenous compounds that result from the decarboxylation of some amino acids. Some microorganisms may be responsible for their formation. Starters can cause a fast pH decrease, inhibiting the development of microorganisms with amino acid decarboxylative ability, thus preventing the accumulation of BA in fermented meat products. Besides, starters can compete with the autochthonous, non-starter microbiota throughout ripening and storage, thus reducing BA production. Some strains of Lactobacillus sakei and Lactobacillus plantarum have been shown to reduce the formation/accumulation of BA. On the other hand, Staphylococcus xylosus and Debaryomyces hansenii strains have been reported to degrade BA in food. PAH are organic compounds containing multiple aromatic rings and produced by the incomplete combustion of organic matter, such as the wood used for smoking meat. Mixed starters containing Lactobacillus spp., Gram-positive catalase-positive cocci and yeasts have been used in the manufacturing of traditional meat sausages. However, the effect of starters on reducing the accumulation of PAH is poorly understood. Starters may also be engaged in competitive exclusion, outcompeting the spoiling or deteriorating autochthonous microbiota. For example, Pediococcus acidilactici has been shown to inhibit Listeria monocytogenes in meat products. Additionally, the role of molds, such as Penicillium nalgiovense, in the competitive exclusion of undesired filamentous fungi, has also been demonstrated. Most of these undesired fungi produce mycotoxins, secondary metabolites capable of causing disease. The current review addresses the role of starters on the microbiological and chemical safety of fermented meat products.

Selection of native bacterial starter culture in the production of fermented meat sausages: Application potential, safety aspects, and emerging technologies.

The development of standardized and safe food products with the typical characteristics of each region is highly desirable and can be obtained by using native starter cultures that influence the flavor, texture, and color of fermented foods. Therefore, scientists have been employing various techniques for screening and characterizing native bacteria (lactic acid bacteria and Gram-positive catalase-positive cocci) for application in fermented meat sausage. The present review outlines in vitro assays that evaluate the potential application and safety aspects of native isolates and introduces emerging omics technologies applied to the microbiology of fermented meat sausage. Results from current research are presented, and the strengths and limitations of each assay are provided, with references indicating where further details can be obtained. In choosing the most appropriate in vitro method, it is necessary to consider the available analytical infrastructure, the sensitivity and selectivity of the assay, the time it takes to get the results, the ease of the assay, and the costs involved.

Technological implications of reducing nitrate and nitrite levels in dry-fermented sausages: Typical microbiota, residual nitrate and nitrite and volatile profile

Despite the number of studies that have focused on alternatives to nitrite in meat products, it has not been found yet a single compound that performs all its functions. Therefore, nitrate and nitrite are still common additives in the meat industry. However, to reduce nitrosamine formation, both ingoing amounts and residual levels must be controlled. Dry-fermented sausages were prepared with the maximum amounts of nitrate and nitrite allowed by the European Union, 25% and 50% reduction, and no nitrate/nitrite. The concentration of these additives significantly affected Gram-positive catalase-positive cocci, which numbers were 1 and 2 log cfu/g higher in the 50% reduction and control batches, respectively. A higher amount of volatiles derived from amino acid degradation and carbohydrate fermentation was detected, related to the microbiological changes. Enterobacteriaceae increased during fermentation at lower nitrate/nitrite concentrations. A relation was found between ingoing and residual nitrite, which was 3.5 fold higher when the maximum amount was used in comparison to the 50% reduction.

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 (9.14logCFU/g) and LBP14 (8.96logCFU/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.

Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

This study was carried out for the characterization and discrimination of the indigenous Gram positive, catalase-positive cocci (GCC) population in sucuk, a traditional Turkish dry-fermented sausage. Sucuk samples, produced by the traditional method without starter culture were collected from 8 local producers in Kayseri/Turkey and a total of 116 GCC isolates were identified by using different molecular techniques. Two different molecular fingerprinting methods; namely, randomly amplified polymorphic DNA-PCR (RAPD-PCR) and repetitive extragenic palindrome-PCR (rep-PCR), were used for the clustering of isolates and identification at species level was carried out by full length sequencing of 16S rDNA. Combining the results obtained from molecular fingerprinting and 16S rDNA sequencing showed that the dominant GCC species isolated from the sucuk samples was Staphylococcus saprophyticus followed by Staphylococcus succinus and Staphylococcus equorum belonging to the Staphylococcus genus. Real-time PCR DNA melting curve analysis and high-resolution melting (HRM) analysis targeting the V1 + V3 regions of 16S rDNA were also applied for the discrimination of isolates belonging to different species. It was observed statistically different Tm values and species-specific HRM profiles for all except 2 species (S. saprophyticus and Staphylococcus xylosus) that have high 16S rDNA sequence similarity. The combination of rep-PCR and/or PCR-RAPD with 16S rRNA gene sequencing was an efficient approach for the characterization and identification of the GCC population in spontaneously fermented sucuk. On the other hand, intercalating dye assays were found to be a simple and very promising technique for the differentiation of the GCC population at species level.

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.

Probiotic strains Lactobacillus plantarum 299V and Lactobacillus rhamnosus GG as starter cultures for fermented sausages

Probiotic food products are a fast growing area. Although probiotic strains are currently used in dairy products, their commercial application in fermented meat products is not yet common. The aim of this study was to assess the competitiveness of two probiotic Lactobacillus strains (Lactobacillus plantarum 299V and Lactobacillus rhamnosus GG) during the manufacture of Spanish fermented sausages and their effect on the hygienic and sensory qualities of the final products. The inoculated strains were successfully monitored by Randomly Amplified Polymorphic DNA (RAPD)-PCR. Both strains prevented the growth of Enterobacteriaceae throughout the entire ripening process. L. rhamnosus GG and L. plantarum 299V at high inoculum (ca. 107 CFU/g) produced a sharp decrease of pH values and low growth of Gram-positive Catalase-positive Cocci (GCC+), leading to a negative effect on the sensory attributes evaluated. Nevertheless, L. plantarum 299V inoculated at 105 CFU/g achieved and maintained high counts until the end of ripening and storage (ca.108 CFU/g), co-dominating (60%) with the endogenous microbiota, producing functional sausages with a satisfactory overall sensory quality. No major differences in physico-chemical parameters or sensory attributes were recorded when compared to spontaneously fermented sausages, thus adding further value to this type of meat product as a probiotic vehicle.

The microbial flora in the nasal septum area prone to perforation

To explore the colonizing bacterial flora of the nasal septum area, that is mostly afflicted by perforations, 101 healthy police students had swab samples taken from that location. The described culture strategy recovered positive cultures from 95% of the test subjects and from 60% with more than one organism. In total, 191 bacterial isolates were classified according to colony morphology, Gram-stain and a panel of standard laboratory techniques. A part of the bacteria was identified to species-level by biochemical methods and by sequencing of the 16S rRNA gene. The predominant finding was Gram-positive irregular rods - 65 presumptive Corynebacterium isolates, both lipophilic and non-lipophilic, and 37 anaerobic Propionibacterium isolates. The second largest bacterial group was Gram-positive catalase-positive cocci, of which 13 isolates were identified as Staphylococcus aureus and 53 as coagulase-negative staphylococci. The few potential airway pathogens included Streptococcus pneumonia (n = 1) and Moraxella catarrhalis (n = 3) isolates. The bacterial flora colonizing the nasal septum mainly consists of Gram-positive bacteria. Although of low virulence, the microbial flora may impact on occlusion treatment of nasal septum perforations with silicone obturators.

Diagnosis of intramammary infection in samples yielding negative results or minor pathogens in conventional bacterial culturing

Up to half of quarter milk samples submitted for mastitis diagnosis are culture-negative results or lead to identification of coagulase-negative staphylococci or Corynebacterium bovis in conventional culturing, the so-called minor pathogens. The interpretation and usefulness of these results in terms of udder and animal health management is limited, even though the amount of resources spent is relatively high. This work aimed to test two methods of analysis of milk samples with the goal of increasing detection of intramammary pathogens. In the first study, 783 milk samples were processed in duplicate: before and after freezing at -20°C for 24 h, using standard bacteriological techniques. There was a significant difference between the two methods with samples frozen for 24 h yielding significantly fewer Gram-positive catalase-positive cocci, Gram-negative bacilli, Gram-positive bacilli and significantly more samples leading to no growth, than samples before freezing. The number of samples yielding Gram-positive catalase-negative cocci was not significantly affected by freezing. In the second study, a real-time PCR-based test was performed on milk samples with an individual quarter somatic cell count above 500,000 cells/ml that were either negative (n=51 samples) or that led to the isolation of minor pathogens in culturing: Corynebacterium bovis (n=79 samples) or non-aureus staphylococci (NAS, n=32). A mastitis pathogen, beyond the result obtained with standard bacteriology, was detected on 47% of the no-growth samples, on 35% of the samples from which C. bovis had been isolated and on 25% of the samples from which NAS had been isolated. The most commonly detected major pathogen was Escherichia coli, followed by Streptococcus uberis, Arcanobacterium pyogenes/Peptoniphilus indolicus and Streptococcus dysgalactiae. These results suggest that simply freezing milk samples for 24 h does not increase the detection of intramammary bacteria in milk samples and therefore should not be recommended. However, use of the real-time PCR-based test may be useful in diagnosing intramammary infections when milk samples with high somatic cell counts are culture-negative or when culturing results in the detection of minor pathogens.

Characterization by Volatile Compounds of Microbial Deep Spoilage in Iberian Dry‐Cured Ham

In the present study, volatile compounds of spoiled dry-cured Iberian ham with deep spoilage or "bone taint" were analyzed and correlated with level of spoilage and the microorganisms detected. Volatile compounds extracted by a solid phase micro-extraction technique were assayed by gas chromatography/mass spectrometry. The spoiled hams were evaluated sensorially, and the correlations among volatile compounds, spoilage level, and microbial counts were studied. The spoiled hams had higher concentrations of hydrocarbons, alcohols, acids, esters, pyrazines, sulfur compounds, and other minor volatile compounds than unspoiled hams. The sensorial analysis showed that the spoilage level of hams correlated with several volatile compounds, most of them associated with Gram-positive catalase positive cocci and Enterobacteriaceae counts. Cyclic compounds such as cyclohexanone, some ethers, and pyrazines should be considered as indicators to monitor incipient microbial deep spoilage in the elaboration of this meat product.

Volatile profile and microbiological characterization of hollow defect in dry-cured ham

This paper describes the physicochemical and microbiological parameters and volatile profile of the muscles surrounding the coxofemoral joint which are affected with hollow defect when compared to the part of muscles nearby which did not show this defect. Differences of the same parameters between both areas in dry-cured hams without this defect were also analyzed. Gram-positive catalase-positive cocci were the predominant flora in the muscles affected with hollow defect, with the main species being Staphyloccocus equorum, Staphylococcus nepalensis and Staphylococcus xylosus. Lactic acid bacteria, Listeria monocytogenes and Staphylococcus aureus were <2 log CFU g(-1) and Salmonella spp. was not detected. Volatile profile of samples affected with hollow defect had higher amounts of compounds such as esters, pyrazines, sulfurs and furans. There was a positive correlation between the amount of ester compounds and the hollow defect intensity. Moisture content was lower and pH values were higher in affected areas than in unaffected areas.

Identification of Lactic Acid Bacteria and Gram‐Positive Catalase‐Positive Cocci Isolated from Naturally Fermented Sausage (Sucuk)

The aim of the study was to identify lactic acid bacteria and Gram-positive catalase-positive cocci isolated from Turkish dry fermented sausage (sucuk) produced by 7 different manufacturers without using starter culture. A total of 129 isolates of lactic acid bacteria were identified phenotypically. Lactobacillus plantarum was the dominant species (45.7%) followed by L. curvatus (10.9%) and L. fermentum (9.3%). Pediococcus isolates were identified as P. pentosaceus and P. acidilactici. All the isolates of gram-positive and catalase-positive cocci (123 isolates) were classified as Staphylococcus except for 1 isolate assigned to Kocuria rosea. The species isolated most often were S. xylosus (41.5%) and S. saprophyticus (28.5%). Four isolates were identified as S. equorum (3.3%), 1 isolate was assigned to S. carnosus (0.8%).

Effect of the inoculation of a starter culture and vacuum packaging during the resting stage on sensory traits of dry-cured ham

The effects of the inoculation of a mixed starter culture and vacuum packaging (during resting stage) on odour, appearance, texture and flavour of dry-cured ham were studied. After salting, half of the 36 processed hams were inoculated with a commercial starter culture containing lactic-acid bacteria, Gram-positive catalase-positive cocci and yeasts. Nine hams per group (inoculated and non-inoculated) remained vacuum-packaged during resting. External odour during the process, as well as appearance of the cut surface, texture and flavour on semimembranosus and biceps femoris of the final product were assessed. Vacuum packaging during resting caused an increase in white film and feedstuff flavour, as well as a decrease in aged flavour, hardness, fibrousness and overall liking. The use of the starter culture brought about an increase in feedstuff flavour, a decrease in sweetness, aged flavour, nutty flavour and overall liking and, only in vacuum-packaged hams, the development of a floral flavour, but had no significant effect on texture descriptors. The starter culture studied is considered inappropriate for the production of traditional Spanish dry-cured ham regardless of the type of resting used.

Effect of the inoculation of a starter culture and vacuum packaging (during resting stage) on the appearance and some microbiological and physicochemical parameters of dry-cured ham

The effect of the inoculation of a starter culture and vacuum packaging (during the resting stage) on dry-cured ham appearance, microbiological and physicochemical parameters was studied. Half of the 36 processed hams were inoculated, after salting, with a commercial starter culture containing lactic-acid bacteria, Gram-positive catalase-positive cocci and yeasts. 18 hams per group (inoculated and non-inoculated) remained vacuum packaged during resting. Microbiological analyses were carried out on the lean surface during processing, on subcutaneous fat tissue at the drying stage and on lean tissue in the finished product. Appearance was evaluated during processing. Physicochemical analyses (NaCl, H 2O, proteolysis index, a w) were done on Semimembranosus and Biceps femoris in the final product. Inoculation caused a reduction of mould growth and oil drip. Vacuum packaging induced increased proteolysis and increases in all microbial counts and a reduction of oil drip, mould growth and weight loss during processing.

Molecular, technological and safety characterization of Gram-positive catalase-positive cocci from slightly fermented sausages

The population of Gram-positive catalase-positive cocci from slightly fermented sausages was characterized at species and strain level by molecular techniques and some technological and hygienic aspects were also considered. Staphylococcus xylosus was the predominant species (80.8%) followed by Staphylococcus warneri (8.3%), Staphylococcus epidermidis (5.8%) Staphylococcus carnosus (4.6%), and Kocuria varians (0.4%). Proteolytic activity was observed in 23% of the isolates. The species with the highest percentage of proteolytic strains was S. warneri. Lipolytic activity was found in 45.8% of the isolates and S. xylosus was the species with the highest percentage of lipolytic isolates. Biogenic amine production was not widely distributed (only 14.6% of the isolates). Tyramine was the most intense amine produced, although by only 4.6% of the isolates. Phenylethylamine was more frequently detected (10.8% of isolates) but at lower levels. Some strains also produced putrescine (3.3%), cadaverine (2.9%), histamine (1.3%) and tryptamine (0.4%). All isolates were susceptible to linezolid and vancomicin and over 70% were resistant to penicillin G, ampicilin and sulphonamides. Most of the mecA + strains (only 4.6% of isolates) also displayed resistance to multiple antibiotics. A reduced enterotoxigenic potential was found. Only 3.3% of isolates showed staphylococcal enterotoxins genes, all identified as entC gene. The combination of RAPD-PCR and plasmid profiling allowed the discrimination of 208 different profiles among the 240 Gram-positive catalase-positive cocci characterized, indicating a great genetic variability.