Coagulase-negative Cocci Research Articles

Tasting of traditional Polish fermented cucumbers: Microbiology, morpho-textural features, and volatilome

In the present study, naturally fermented and unpasteurized cucumbers (Cucumis sativus L.) collected from 4 producers located in different regions of Poland were studied. The fermented cucumbers were characterized by significant nutritional features in terms of polyphenols content and antioxidant activity. Microbiological analyses revealed active bacterial populations of lactococci, thermophilic cocci, lactobacilli, and coagulase-negative cocci. The microbiological characterization of cucumber and brine samples through metataxonomic analysis allowed the dominant species to be detected, being Lactococcus and Streptococcus in cucumbers, and Lactiplantibacillus, Leuconostoc, Pediococcus, Secundilactobacillus, and Lentilactobacillus in brine. The isolation activity offered a clear picture of the main active lactic acid bacteria at the end of fermentation, being Pediococcus parvulus and Lactiplantibacillus plantarum group. All the studied isolates showed a good attitude in fermenting a cucumber-based broth, thus suggesting their potential application as starter or adjunct cultures for guided cucumber fermentation. Moreover, for the same isolates, strong aminopeptidase activity (due to leucine arylamidase and valine arylamidase) was observed, with potential effect on the definition of the final sensory traits of the product. Only a few isolates showed the ability to produce exopolysaccharides in synthetic medium. Of note, the presence of the hdcA gene in some Pediococcus ethanolidurans isolates also confirmed the need for a thorough characterization of starter candidates to avoid undesired adverse effects on consumer’s health. No isolate showed the production of bacteriocins against Listeria innocua used as surrogate for Listeria monocytogenes. Based on the results of Headspace Solid-Phase Microextraction-Gas Chromatography/Mass Spectrometry analysis, a rich and complex volatilome, composed by more than 80 VOCs, was recognized and characterized. In more detail, the detected compounds belonged to 9 main classes, being oxygenated terpenes, alcohols, terpenes, ketones, acids, aldehydes, esters, sulfur, and sesquiterpenes.

Spotlight on autochthonous microbiota, morpho-textural characteristics, and volatilome of a traditional Polish cold-smoked raw sausage

The aim of the present study was to obtain information on the bacterial diversity of traditional Polish cold-smoked raw sausages (Kiełbasa polska wędzona) manufactured by two artisanal producers using different selective growth media and a metataxonomic analysis. Physico-chemical and morpho-textural characteristics were also carried out, together with Microextraction-Gas Chromatography/Mass Spectrometry (HS-SPMEGC/MS) to study the volatile organic compounds (VOCs). The results overall obtained allowed a picture of the microbiota, the morpho-textural characteristics, and the volatilome of traditional Polish cold-smoked raw sausages (Kiełbasa polska wędzona) to be drawn for the first time. In more detail, viable counting revealed active populations of presumptive lactobacilli, enterococci, coagulase-negative cocci, and a few spoilage microorganisms typically occurring in raw meat products. The metataxonomic analysis revealed the dominance of Latilactobacillus sakei occurring with a relative frequency between 77% and 89%. Pediococcus pentosaceus, Weissella hellenica, and Leuconostoc carnosum were detected among the minority taxa. In the sausages herein studied, no histamine levels of concern were detected. The Principal Component Analysis (PCA) performed on the Amplicon Sequence Variants (ASVs) did not show significant differences in the microbiota composition among producers. The HS-SPMEGC/MS analysis allowed the detection and identification of more than 90 volatile components belonging to ten main classes, namely: aldehydes, ketones, esters and acetates, acids, alcohols, phenols, furans, sulphur compounds, terpenoids, and benzene derivatives. The detected VOCs originated from spices, smoke, and microbial metabolism. The PCA of volatile compounds allowed differences between the sausage samples of the two producers to be identified.

Justification and microbiota compositions development for the fermentation of raw meat

In the production of fermented meat products, microorganisms of various taxonomic groups play an extremely important role, namely in the formation of specific taste, aroma, colour, and consistency. Both fermentative and spontaneous microflora take part in the components' transformation of meat raw materials during the maturation of such products, and the course of this process depends on the metabolic activity of the strains. In accordance, this article's purpose is to select microbiota compositions (lactic acid bacteria and coagulase-negative cocci) for the fermentation of meat raw materials. So, as a result of the research, 4 compositions were selected, two of which are lactic acid bacteria with micrococci (No. 2, 3) and two lactic acid bacteria with staphylococci (No. 1, 6). They were characterized by the high productivity of each of the components of the leavening composition, in particular, it was established that the number of MKB increased – by 4.3-6.5 times, and micrococci and staphylococci – by 7.7-28.6 times, respectively. For these compositions, mutual stimulation of the components was observed, contributing to the active microorganisms' development and their biochemical activity. Fermentation compositions No. 1, 6, 4, and 6 had the highest nitrite-reducing activity, and a high level of proteolysis characterized compositions No. 1, 2, 3, and 4. According to the results of determining the antagonistic activity against opportunistic and pathogenic microorganisms, it was established that the investigated compositions exhibit antagonistic activity against both gram-negative and gram-positive microorganisms.

Microbiological, morpho-textural, and volatile characterization of Portuguese Queijo de Nisa PDO cheese

Queijo de Nisa PDO (Protected Designation of Origin) is a semi-hard cheese obtained from raw ewe's milk clotted with cardoon (Cynara cardunculus, L.) rennet. The aim of the present study was to characterize the bacterial communities naturally occurring in Queijo de Nisa PDO cheese samples through viable counting and metataxonomic analysis. Moreover, physico-chemical and morpho-textural analyses were also performed, together with the analysis of volatile organic compounds (VOCs) through solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC–MS). In the analyzed samples, pH values were comprised between 4.84 and 5.74. As for lactic acid, the concentration varied between 0.83 and 2.10 g 100 g-1. Water activity varied between 0.942 and 0.960. Lightness of the samples ranged from 107.82 to 119.16, whereas hardness ranged between 34.45 N and 126.05 N. As for microbiological analyses, lactococci and lactobacilli counts were up to 9.01 Log CFU g-1. Coagulase-negative cocci and enterococci counts were up to 7 Log CFU g-1. Metataxonomic analysis revealed that Lactococcus lactis and Leuconostoc mesenteroides occurred at high frequency in all the analyzed samples. Moreover, Lactiplantibacillus plantarum, Lactococcus piscium, and Lacticaseibacillus zeae were also detected. The most represented VOCs were carboxylic acids, carbonyl compounds, alcohols, and esters. In the analyzed cheese samples, significant relationships between bacteria and VOCs were also evidenced. Of note, research on the microbiological and chemical features, as well as on morpho-textural characteristics, of Queijo de Nisa PDO cheese can increase the knowledge on the interactions between raw materials, environment, and autochthonous microorganisms, thus enabling protection of biodiversity and tradition.

Safety and technological issues of dry fermented sausages produced without nitrate and nitrite

The aim of this work was to investigate the possibility to industrially produce fermented sausages without the addition of nitrate and nitrite. Indeed, despite their antimicrobial effect and multiple technological roles, an increasing pressure for their removal has recently raised. To achieve this goal while maintaining an acceptable final product quality, we deeply modified the whole process, that was carried out at 10–15 °C (i.e., temperatures lower than traditional Mediterranean products) and by using bioprotective starter cultures at high concentrations (7 log CFU/g) to lead the fermentation. Different glucose amounts (0.2 or 0.4 % w/w) were also tested to optimize the process. The results showed no significant differences between the control (with nitrate/nitrite) and the sausages without preservatives in terms of aw (value range 0.908–0.914), weight loss (about 38% in all samples), lactic acid bacteria (value range 8.1–8.3 log CFU/g) and coagulase negative cocci (value range 6.8–7.1 log CFU/g). The amount of sugar affected the final characteristics of sausages. Indeed, in the absence of curing salts, lower sugar concentration resulted in better textural features (reduced hardness and gumminess) and lower oxidation (TBARS values 0.80 vs. 1.10 mg MDA/kg of meat product in samples with 0.2% or 0.4% of glucose, respectively). Finally, challenge tests evidenced the inability of selected strains of Listeria innocua, Salmonella enterica sub. enterica and Clostridium botulinum to grow, under the adopted conditions, in fermented sausages. This research highlighted that nitrate/nitrite removal from these meat products requires accurate technological changes to guarantee the final quality.

Microbial diversity, morpho-textural characterization, and volatilome profile of the Portuguese thistle-curdled cheese Queijo da Beira Baixa PDO

The aim of the present study was to characterize the bacterial and fungal communities naturally occurring in Queijo da Beira Baixa PDO cheese samples produced in Castelo Branco district (Beira Baixa Region, Portugal) through viable counts and metataxonomic analyses. Physico-chemical and morpho-textural analyses were also carried out, together with the analysis of volatile organic compounds (VOCs). In the analyzed samples, pH values ranged between 4.72 ± 0.15 and 5.85 ± 0.02, with values of lactic acid content comprised between 0.64 ± 0.00 and 1.95 ± 0.16 g 100 g−1. Specific volume of cheese ranged from 1.09 ± 0.08 to 1.32 ± 0.02 g mL−1. Texture profile analysis showed hardness ranging between 38.3 ± 9.6 N and 68.55 ± 7.5 N. As for lactic acid bacteria, presumptive lactococci, thermophilic cocci, and lactobacilli counts up to 9 Log cfu g−1. Coagulase-negative cocci showed counts up to 7 Log cfu g−1. Enterococci counts were up to 6 Log cfu g−1. Finally, counts of eumycetes showed values up to 4 Log cfu g−1. The results of metataxonomic analysis of bacteria showed the dominance of Lactococcus lactis in all the samples. Moreover, other taxa were detected, including Lactiplantibacillus plantarum, Loigolactobacillus coryniformis, Lactococcus piscium, Streptococcus thermophilus, and Lacticaseibacillus zeae. Mycobiota was characterized by the presence of Candida sake, Ustilago, Cladosporium variabile, Starmerella, Debaryomyces hansenii, and Pichia kluyveri. In the analyzed Queijo da Beira Baixa PDO cheese samples, carboxylic acids represented the most detected VOCs, followed by esters, carbonyl compounds, and alcohols.

Unfolding microbiota and volatile organic compounds of Portuguese Painho de Porco Preto fermented sausages

In the present study, bacterial and fungal diversity, as well as volatile profiles, of ready-to-eat Portuguese Painho de Porco Preto fermented sausages manufactured by two artisanal producers in the districts of Beja (producer A) and Evora (producer B) were studied. To this end, different selective growth media and a metataxonomic analysis were combined with Headspace Solid-Phase Microextraction-Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS) analysis. The results of the microbiological viable counts revealed active microbial populations of lactic acid bacteria (up to 8 Log cfu g−1), coagulase negative cocci (up to 6 Log cfu g−1), and eumycetes (up to 6 Log cfu g−1). Bacterial populations were characterized by high relative frequencies of Latilactobacillus sakei (up to 72%), together with Weissella and Staphylococcus equorum. The mycobiota was mainly dominated by Debaryomyces hansenii (up to 55% of the relative frequency) and Kurtzmaniella zeylanoides (up to 24% of the relative frequency). Unexpected species as Wickerhamomyces subpelliculosus and Zygosaccharomyces rouxii were also detected. HS-SPME-GC/MS analysis allowed to identify a complex volatile profile, showing 159 volatile organic compounds (VOCs). VOCs belonged to twelve classes, such as aldehydes, ketones and lactones, esters and acetates, alcohols, terpenoids, sulfur compounds, aliphatic hydrocarbons, aromatic hydrocarbons, nitrogen compounds, acids, furans and pyrans, and phenols. The analysis of VOCs composition provided evidence that samples from the two producers (A and B) were different, as confirmed by the Principal Component Analysis. Hence, it is likely that the raw materials used, as well as variations related with the empirical practice of the butchers, strongly influenced the final product. The results obtained in the present study represent a further advancement in the knowledge on the biodiversity and VOCs composition of Portuguese fermented sausages. To better understand the interactions occurring between the autochthonous microorganisms and the meat batter in the Painho de Porco Preto fermented sausage, microbial and VOCs dynamics must be further deepened throughout the production process.

Unravelling microbial populations and volatile organic compounds of artisan fermented liver sausages manufactured in Central Italy

The aim of the present study was to obtain information on the occurrence of bacteria and eumycetes in ready-to-eat fermented liver sausages manufactured by 20 artisan producers located in the Marche Region (Italy). To this end, culture-dependent analyses and metataxonomic sequencing were carried out. Physico-chemical parameters and volatilome of the fermented liver sausages were also studied. Finally, the presence of hepatitis E virus (HEV) was also assessed via real-time-RT-(q)PCR assays. Active microbial populations mainly represented by lactic acid bacteria, enterococci, coagulase-negative cocci, and eumycetes were detected. Enterobacteriaceae, Pseudomonadaceae, and sulfite-reducing anaerobes were not detected in most of the samples. Latilactobacillus sakei dominated in all the analyzed samples, reaching abundances up to 80%. Staphylococcus xylosus and Staphylococcus equorum were also detected. Among minority bacterial taxa, Weissella spp., Leuconostoc spp., Macrococcus caseolyticus, Brochothrix thermosphacta, Staphylococcus succinus, Lactobacillus coryniformis, Lactiplantibacillus plantarum, Lactococcus garviae, Psychrobacter spp., and Carnobacterium viridans were detected. The mycobiota was mainly composed by Debaryomyces hansenii that was present in all samples at the highest frequency. Among minority fungal taxa, Aspergillus spp., Penicillium spp., Kurtzmaniella zeylanoides, Candida spp., Yamadazyma spp., Scopulariopsis spp., Yarrowia spp., and Starmerella spp. were detected. Interestingly, associations between some taxa and some physico-chemical parameters were also discovered. The absence of HEV in all the samples attested a high level of safety. Finally, most of the VOCs detected in the analyzed fermented liver sausages belonged to six classes as: terpenoids, aldehydes, ketones, alcohols, esters, and acids. Nitrogen compounds, sulfur compounds, phenols, hydrocarbons, lactones, furans, and aromatic hydrocarbons were also identified. Several significant relationships were observed between mycobiota and VOCs.

Technological and Safety Characterization of Kocuria rhizophila Isolates From Traditional Ethnic Dry-Cured Ham of Nuodeng, Southwest China.

Nuodeng ham is known for its unique processing techniques and flavor. In the present study, proteolytic microorganisms from cured artisanal Nuodeng ham were investigated in order to identify and select potential starter cultures for its faster and safer fermentation. Eight isolates, accounting for 57% of proteolytic microorganisms, were found to be related to Kocuria rhizophila. Relevant properties of K. rhizophila as potential starter culture were evaluated in vitro for the first time. Intra-species diversities were found in phylogenetic and physiological properties of K. rhizophila isolates. Nevertheless, desirable attributes, such as halo-tolerance, nitrate reductase and protease activity, as well as the absence of antimicrobial resistance and amino acid decarboxylase activity, were observed in selected isolates. Moreover, genome analysis of isolates K24 and K45 confirmed their lack of typical genes for virulence, antimicrobial resistance and amino acid decarboxylase. K. rhizophila may thus represent a novel starter candidate of coagulase-negative cocci group and contribute to color and flavor development of fermented meats.

Mediterranean Spontaneously Fermented Sausages: Spotlight on Microbiological and Quality Features to Exploit Their Bacterial Biodiversity.

The wide array of spontaneously fermented sausages of the Mediterranean area can represent a reservoir of microbial biodiversity and can be an important source of new technological and functional strains able to preserve product properties, counteracting the impoverishment of their organoleptic typical features due to the introduction of commercial starter cultures. We analysed 15 artisanal salamis from Italy, Spain, Croatia and Slovenia to evaluate the microbiota composition, through culture-dependent and culture-independent techniques (i.e., metagenomic analysis), chemical–physical features, biogenic amines and aroma profile. The final pH varied according to origin and procedures (e.g., higher pH in Italian samples due to long ripening and mold growth). Lactic acid bacteria (LAB) and coagulase-negative cocci (CNC) were the dominant population, with highest LAB counts in Croatian and Italian samples. Metagenomic analysis showed high variability in qualitative and quantitative microbial composition: among LAB, Latilactobacillus sakei was the dominant species, but Companilactobacillus spp. was present in high amounts (45–55% of the total ASVs) in some Spanish sausages. Among staphylococci, S. epidermidis, S. equorum, S. saprophyticus, S. succinus and S. xylosus were detected. As far as biogenic amines, tyramine was always present, while histamine was found only in two Spanish samples. These results can valorize the bacterial genetic heritage present in Mediterranean products, to find new candidates of autochthonous starter cultures or bioprotective agents.

The Effect of Catabolic Transformations of Proteins and Fats on the Quality and Nutritional Value of Raw Ripened Products from Zlotnicka Spotted and Zlotnicka White Meat

Abstract The aim of the study was to compare the advancement of the ripening as well as catabolic changes in proteins and fats of Zlotnicka Spotted (ZS) and Zlotnicka White (ZW) meat and their impact on the quality and nutritional value of ready-to-eat products. The meat of the breeds ZS and ZW differed not only in the basic chemical composition but also in the susceptibility to catabolic transformations of proteins and lipids, which translated into a separate technological and nutritional quality as well as the profile of volatile odor compounds. Loins due to their compact histological structure, low pH (5.4) and decreased water activity (0.92–0.93) were characterized by a lower number of coagulase-negative cocci (3.3 log cfu/g) compared to hams. The products of both breeds differed in the content of selected neutral glucogenic amino acids with a pI in the range of 5.6–6.1 mainly. The content of biogenic amines was therefore completely dependent on the metabolic potential of acidifying bacteria. Larger number of lactic acid bacilli (7.5–7.7 log cfu/g) and lactic acid cocci (7.9–8.3 log cfu/g), as well as a higher content of saturated (55.2–53.7%) and polyunsaturated fatty acids (6.4–7.0%) shaped the final pH of hams (5.3). Presence of aldehydes, ketones and alcohols indicated existing fat oxidation despite the small values of the TBA index of hams (1.1 mg/kg) and loins (0.4–0.6 mg/kg). The volatile compounds that differentiated products of ZS and ZW formed by the oxidation and microbial activity, were, primarily: octanal, 1-hydroxypropan- 2-one, 3-methylpentan-2-one, propane-1,2-diol, 2,5-dimethylfuran and 3-hydroxybutan- 2-one, butane-2,3-dione, butane-1,2-diol, respectively.

Microbial communities and volatile profile of Queijo de Azeitão PDO cheese, a traditional Mediterranean thistle-curdled cheese from Portugal

The production of ovine or caprine milk cheeses with thistle rennet is a common practice in the Mediterranean basin. The aim of the present study was to obtain information on bacteria and yeast communities harboured by Queijo de Azeitão PDO cheese through viable counting and, for the first time, via metataxonomic analysis. Moreover, solid phase microextraction (SPME) technique was applied to characterize Queijo de Azeitão PDO cheese volatile compounds. Nine cheese samples were collected from three different artisan producers located in Portugal. The results of physico-chemical analyses showed significant differences between producers, with mean values ranging from 5.40 ± 0.25 (Producer 1) to 6.00 ± 0.22 (Producer 2). As for TTA, Producer 1 showed the highest mean value attesting at 18.04 ± 6.57 mL of 0.1 M NaOH used to reach pH 8.3. Regarding lactic acid concentration, Producer 1 showed the highest mean value attesting at 0.488 ± 0.106 g 100 g−1, whereas, for acetic acid, no significant differences were evidenced among producers with values comprised between 0.141 ± 0.021 g 100 g−1 and 0.245 ± 0.016 g 100 g−1. No significant differences were observed between overall mean values of the three producers for viable counts of presumptive lactococci, thermophilic cocci, presumptive lactobacilli, thermophilic lactobacilli and total mesophilic aerobes with values in the order of 7–8 log cfu g−1. Moreover, no significant differences were evidenced for viable counts of coagulase-negative cocci, enterococci, Enterobacteriaceae and Pseudomonadaceae. As for eumycetes, cheeses from Producer 1 showed the lowest mean value (2.78 ± 2.42 log cfu g−1) in respect with values detected in cheeses from Producer 2 and 3. Concerning microbiota and mycobiota of the analyzed cheeses, the alpha diversity index did not show any significant difference between the three producers in terms of composition and complexity of the microbial population. A simple composition was apparently shared by the three producers, whose cheese manufactures were dominated by the presence of Leuconostoc mesenteroides (37% of the relative frequency in average), Lactococcus lactis (29%), Lacticaseibacillus zeae (4.7%), Lentilactobacillus kefiri (4.4%), Serratia spp. (3.5%), Lactiplantibacillus plantarum (2.7%), and Latilactobacillus sakei (2.5%). The mycobiota composition showed the neat dominance of Yarrowia lipolytica (46.7% of the relative frequency in average), followed by Candida ethanolica (13.6%), Kurtzmaniella zeylanoides (9.4%), Geotrichum candidum (8.8%), Galactomyces geotrichum (8.7%), Kluyveromyces lactis (3.5%), and Geotrichum silvicola (2.7%). The volatile profile analysis allowed 24 different compounds to be identified: 7 acids, 7 esters, 4 alcohols, 3 ketones, 2 aromatic hydrocarbons, and 1 aldehyde. The most represented volatile organic compounds (VOCs) were 2-butanone, butanoic acid and hexanoic acid. A positive correlation between Len. kefiri and hexanoic acid and isopentyl isobutyrate was observed (P < 0.05), whereas Y. lipolytica displayed the highest number of positive correlations with 3-methyl-butanal, 2-pentanone and 2-pentanol (P < 0.05). To the authors' knowledge, this is the very first detection of Len. kefiri in a raw ewe’s milk cheese coagulated with vegetable rennet.

Portuguese cacholeira blood sausage: A first taste of its microbiota and volatile organic compounds

Among typical Portuguese sausages, the cacholeira blood sausage undoubtedly represents one of the most popular preparations. To the authors’ knowledge, a lack of information on both the microbiota and the volatile organic compounds (VOCs) of this blood-containing sausage emerges from the available scientific literature. This study represents the first characterization of physico-chemical, microbiological and volatile traits of Portuguese cacholeira blood sausage. To this end, ready-to-eat cacholeira blood sausages were collected from two production batches manufactured in summer (batch 1) and autumn (batch 2). Viable counts showed active microbial communities mainly composed by lactic acid bacteria, coagulase negative cocci, enterococci and eumycetes. The metataxonomic approach showed a simple bacterial composition, which was dominated by Lactobacillus sakei in both the analyzed batches (1 and 2) considered. Carnobacterium, Enterococcus, Kluyvera, Lactococcus and Serratia were found as minor genera. The mycobiota varied according to the production season. Batch 1 was dominated by Starmerella apicola, Debaryomyces hansenii and Candida tropicalis, whereas batch 2 was dominated by D. hansenii. Moreover, Aspergillus spp., Kurtzmaniella zeylanoides, Saccharomyces cerevisiae, Kurtzmaniella santamariae, Brettanomyces bruxellensis and Pichia kluyveri were detected in both the batches as minority species. Seventy-two volatile compounds were identified, including esters, phenols, terpenoids, acids, alcohols, ketones, aldehydes, lactones, furans, sulphur and nitrogen compounds. Significant differences were seen in the amount of some compounds, as a feasible consequence of differences in the raw materials, artisan production and seasonality.

Low-Fat and High-Quality Fermented Sausages.

The present study, considering for the first time microbiological concerns due to the use of lemon albedo as a fat replacer, aimed at the selection of an anti-Listeria strain to be used as protective culture in low-fat southern Italian fermented sausages. In fact, these kinds of products require appropriate bio-protective strategies to avoid risks due to Listeria monocytogenes. Sixty-seven Lactiplantibacillus plantarum strains isolated from diverse sources were screened for their antimicrobial activity and their interaction with starter strains (Latilactobacillus sakei 152 and Staphylococcus xylosus MVS9). Lactiplantibacillus plantarum Lpls100, highlighting both listericidal activity and the ability to promote Staphylococcus xylosus MVS9 growth, was used as a protective strain in low-fat fermented sausages prepared with lemon albedo as a fat replacer. The effect of the albedo and the protective strain on the fermentation process and the final quality was ascertained. Results highlighted that the use of the albedo did not affect the growth of starter strains and enhanced some quality features, such as fatty acid profiles and certain sensory attributes. However, the albedo also produced a slow decrease in water activity, compromising the microbial quality. The anti-Listeria strain, enhancing coagulase negative cocci growth and exerting antimicrobial activity, avoided the inconveniences caused by the use of the albedo. Moreover, the anti-Listeria effectiveness was assessed through a challenge test using a Listeria cocktail. The study revealed that Lactiplantibacillus plantarum Lpls100, regardless of the presence of the albedo, assures a prompt inhibition of Listeria spp. Therefore, its use could be an important contribution to the quality of low-fat fermented sausages.

Staphylococcus saprophyticus Proteomic Analyses Elucidate Differences in the Protein Repertories among Clinical Strains Related to Virulence and Persistence.

Staphylococcus saprophyticus is a Gram-positive and coagulase negative cocci that composes the skin microbiota and can act as an opportunistic agent causing urinary tract infections, being more frequent in sexually active young women. The ability of a pathogen to cause infection in the host is associated to its ability to adhere to host cells and to survive host immune defenses. In this work, we presented the comparative proteomic profile of three S. saprophyticus strains. It was possible to characterize differences in the proteome content, specially related to expression of virulence factors. We compiled this data and previous data and we detected one strain (9325) possessing higher production and secretion of proteins related to virulence. Our results show that phenotypic, genotypic, and proteomic differences reflect in the ability to survive during interaction with host cells, since the 9325 strain presented a higher survival rate after macrophage interaction. In counterpart, the 7108 strain that possesses lower content of proteins related to virulence presented higher ability to form biofilm suggesting that this strain can be better adapted to persist in the host and in the environment. Our work describes, for the first time, proteomic flexibility among S. saprophyticus strains, reflecting in virulence and persistence.

Molecular characterization of multi-drug resistant coagulase negative cocci in non-hospital environment

Antibiotic resistance crisis occasioned by sporadic appearance of multi-drug resistance (MDR) in human pathogens to clinically applied antimicrobials is a serious threat to global health. In this study, we investigated the drug resistant phenotype of Gram-positive cocci isolates from environment. Staphylococcus capitis and Staphylococcus haemolyticus colonies were isolated on mannitol-salt agar plates supplemented with tetracycline. Antibiotic susceptibility profile of the isolates via minimum inhibitory concentration (MIC) determination was examined. Isolates showed decreased sensitivity to clinically applied antimicrobial agents: tetracycline, kanamycin, erythromycin, norfloxacin, teicoplanin, and ampicillin. Genomic analysis demonstrated the presence of multiple antibiotic resistant genes in these bacteria, suggesting the origin of the multiple antimicrobials resistant phenotype. Tetracycline resistance of these isolates was transduced to Staphylococcus aureus-RN4220 strain. These findings indicate the presence of multiple antimicrobials resistant S. capitis and S. haemolyticus strain in a non-hospital setting. Moreover, the presence of plethora of genes responsible for MDR suggest that these strains could present potential threat to human health by serving as reservoir for lateral transference of antimicrobial resistance conferring foreign genetic elements to other clinically relevant pathogens.

Physicochemical, microbiological, and sensory characterization of ‘Nduja, a typical sausage from Calabria, southern Italy

AbstractPhysicochemical parameters, microbiota, and sensory profile of 13 samples of ‘Nduja, a typical sausage from Calabria, Southern Italy, were determined. The chemical composition is characterized by a wide variability, confirming the differences between the producers' recipes. A common feature is the presence of fibers and sugars due to the addition of pepper (about 25%). The presence of sugars in the ripened product confirms that the fermentations were incomplete, as evidenced by the low counts of lactic acid bacteria and coagulase negative cocci. No spoilage bacteria were found. It has been verified that the pepper limit microbial growth, lowering pH and Aw in the mixture, so that Nduja may be considered a meat preserve, not a fermented sausage. Total biogenic amines content was &lt;200 mg/kg, mainly cadaverine, histamine, and tyramine. Finally, as regards the sensory aspect, a trained panel identified 12 specific descriptors.Practical applicationsAlthough ‘Nduja is produced artisanally and in a circumscribed area, having followed the migratory flows of Calabrian people it is known and diffused worldwide. Despite this diffusion, however, studies of the chemical composition, the microbiological, and the sensory characteristics are poor. This study provides information about the chemical and microbiological composition of different samples of ‘Nduja. The safety aspects, due to the massive addition of peppers, have also been verified. These traits are useful for producers who must guarantee a safe product to consumers. This paper is the first to show the sensory aspects of ‘Nduja in detail, creating a specific sensory profile with proper descriptors. All this information could help in drafting a protocol for Community Protected Geographical Indication when required.

Microbial dynamics of model Fabriano-like fermented sausages as affected by starter cultures, nitrates and nitrites

The present study promotes the valorization of Fabriano-like fermented sausages, which are central-Italy salami with an origin that dates to the early 17th century, for the possible future selection of autochthonous starter cultures to be used with respect to local traditions. To the best of the authors' knowledge, this study represents the first attempt to define the microbial dynamics in Fabriano-like fermented sausage and the effect of nitrates/nitrites and starter cultures on its natural bacterial biota. Culture and RNA-based techniques (RT-PCR-DGGE and Illumina sequencing) were used to assess the microbial ecology of model Fabriano-like fermented sausages together with the impact of starter cultures and different nitrate and nitrite concentrations. The meat batter was used to produce two batches of fermented sausages that were prepared as follows: i) without commercial starters or ii) with the use of starter cultures composed of Pediococcus pentosaceus and Staphylococcus xylosus. Each batch was further divided into three different batches with the addition of 0/0 mg kg−1 nitrate/nitrite, 75/60 mg kg−1 nitrate/nitrite and 150/125 mg kg−1 nitrate/nitrite to the first, second and third batch, respectively. The samples, which were produced in triplicate, were analyzed on the day of production and after 7, 21, and 42 days of ripening. Enterobacteriaceae counts were always higher in model Fabriano-like sausages produced without the use of starter cultures at all of the sampling times irrespective of the tested nitrate/nitrite concentrations. Lactobacilli counts were positively influenced by the starters, although this influence was not evident over time; moreover, the effect of nitrates and nitrites on the counts of lactobacilli differed over time. As a general trend, coagulase-negative cocci counts were apparently not influenced by the tested nitrate/nitrite concentrations. Regarding the effect of nitrates/nitrites on the microbial diversity revealed by RT-PCR-DGGE, the higher the concentration, the lower the presence of some genera/species such as Pseudomonas spp., Serratia liquefaciens and Staphylococcus spp. However, Illumina sequencing detected Pseudomonas spp. as a minority species after 7, 21 and 42 days of ripening irrespective of the nitrate/nitrite concentration. The presence of Staphylococcus species was highlighted by both RT-PCR-DGGE and Illumina sequencing at all of the stages of ripening, although its presence was massively detected in fermented sausages produced without the use of nitrates/nitrites at the end of ripening. Overall, the data collected clearly highlighted the dominance of Lactobacillus sakei in all of the fermented sausages during ripening (from day 7 to day 42) and irrespective of the nitrate/nitrite concentration and added starter cultures. Moreover, Pediococcus spp. was principally detected in model Fabriano-like fermented sausage with added starter cultures irrespective of the nitrate/nitrite concentration.

Different Lactobacillus populations dominate in “Chorizo de León” manufacturing performed in different production plants

“Chorizo de Léon” is a high-value Spanish dry fermented sausage traditionally manufactured without the use of starter cultures, owing to the activity of a house-specific autochthonous microbiota that naturally contaminates the meat from the environment, the equipment and the raw materials. Lactic acid bacteria (particularly Lactobacillus) and coagulase-negative cocci (mainly Staphylococcus) have been reported as the most important bacterial groups regarding the organoleptic and safety properties of the dry fermented sausages. In this study, samples from raw minced meat to final products were taken from five different producers and the microbial diversity was investigated by high-throughput sequencing of 16S rRNA gene amplicons. The diverse microbial composition observed during the first stages of “Chorizo de Léon” evolved during ripening to a microbiota mainly composed by Lactobacillus in the final product. Oligotyping performed on 16S rRNA gene sequences of Lactobacillus and Staphylococcus populations revealed sub-genus level diversity within the different manufacturers, likely responsible of the characteristic organoleptic properties of the products from different companies.

Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR

The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media.For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads<5.0 Logcfug−1. Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products.