Technological and Safety Characterization of Coagulase-Negative Staphylococci Isolated from Sardinian Fermented Sausage Made by Ovine Meat.

Ripened sheep sausages are widely consumed in Italy, particularly in Sardinia. Despite their driving role in flavor and color development, coagulase-negative staphylococci in these products have been rarely investigated. A total of 70 CoNS cultures isolated from Sardinian sheep sausages were characterized by rep-PCR and M13-RAPD typing and identified by 16S rDNA sequencing. S. xylosus and S. equorum accounted for more than 70% of the total isolates, whilst S. pasteuri (8.5%), S. succinus (2.8%), and S. haemolyticus (2.8%) were less represented. The genes encoding the synthesis of putrescine, tyramine, cadaverine, and histamine were evaluated by PCR. None of the strains hosted genes for decarboxylases, except one S. pasteuri strain that was potentially a tyramine-producer. Antibiotic resistance was evaluated, along with nitrate reductase, lipolytic, and proteolytic activity, in a pool of selected cultures. Resistance to the primary antibiotics was rather widespread. S. xylosus, S. equorum, and S. pasteuri strains were all resistant to amoxicillin and kanamycin. S. equorum strains were sensitive to all tested antibiotics. S. xylosus strains were all resistant to penicillin B. Conversely, all S. pasteuri strains were resistant to both ampicillin and penicillin B, and four out of five strains exhibited tetracycline resistance. The high variability in the production of sheep sausages makes the search for adjunct cultures of crucial relevance. According to this perspective, the characterization of the autochthonous CSN population represents the first step to approach a starter selection.

Technological Parameters, Anti-Listeria Activity, Biogenic Amines Formation and Degradation Ability of L. plantarum Strains Isolated from Sheep-Fermented Sausage.

The aim of this work was to identify and characterize, from a technological and safety point of view, the lactic acid bacteria (LAB) isolated from traditional sheep-fermented sausage. First, LABs were identified then were screened for some technological parameters such as acidifying and growth ability, proteolytic and lipolytic activity and for antimicrobial activity. Finally, biogenic amine production and degradation abilities were also evaluated. This research reveals the predominance of Lactiplantibacillus (L.) plantarum on LAB community. Almost all L. plantarum strains were active against Listeria monocytogenes strains (inhibition zone diameters > 1 cm). None of the tested strains were positive in histidine (hdcA), lysine (ldc) and tyrosine (tyrdc) decarboxylase genes and only one (L. plantarum PT9-2) was positive to the agmatine deiminase (agdi) gene. Furthermore, given the positive results of the sufl (multi-copper oxidase) gene detection, all strains showed a potential degradation ability of biogenic amines.

In Vitro Activity of Several Essential Oils Extracted from Aromatic Plants against Ascosphaera apis.

The use of natural substances such as essentials oils against bee pathogens is of great interest as an alternative to traditional methods based on synthetic compounds like antibiotics and fungicides, in order to minimize the risk of having toxic residues in hive products and to prevent the development of resistance phenomena. This study evaluated the inhibitory, fungicidal and sporicidal activity of ten essential oils extracted from aromatic plants against Ascosphaera apis, the etiological agent of chalkbrood, an invasive honey bee mycosis. The most effective essential oils were Thymus herba-barona, Thymus capitatus and Cinnamomum zeylanicum, which showed values of minimum fungicidal concentration and minimum sporicidal concentration ranging from 200 to 400 ppm. Carvacrol was the main component of Thymus capitatus and Thymus herba-barona oils, whereas cinnamic aldehyde prevailed in Cinnamomum zeylanicum oil. Further in-apiary studies will allow the evaluation of side effects on bees and residues in hive products.

Mobility and potential bioavailability of antimony in contaminated soils: Short-term impact on microbial community and soil biochemical functioning.

Antimony (Sb) and its compounds are emerging priority pollutants which pose a serious threat to the environment. The aim of this study was to evaluate the short-term fate of antimonate added to different soils (S1 and S2) with respect to its mobility and impact on soil microbial communities and soil biochemical functioning. To this end, S1 (sandy clay loam, pH 8.2) and S2 (loamy coarse sand, pH 4.9) soils were spiked with 100 and 1000 mg Sb(V) kg-1 soil and left in contact for three months. Sequential extractions carried out after this contact time indicated a higher percentage of labile antimony in the Sb-spiked S1 soils than S2 (e.g. ~13 and 4% in S1 and S2 treated with 1000 mg Sb(V) kg-1 respectively), while the opposite was found for residual (hardly bioavailable) Sb. Also, a reduced number of culturable heterotrophic bacteria was recorded in Sb-spiked S1 soil (compared to the unpolluted S1), while an increased one was found in S2. Heterotrophic fungi followed the opposite trend. Actinomycetes and heat-resistant aerobic bacterial spores showed a variable trend depending on the soil type and Sb(V) treatment. The Biolog community level physiological profile indicated a reduced metabolic activity potential of microbial communities from the Sb-spiked S1 soils (e.g. <50% for Sb-1000 compared to the unpolluted S1), while an increase was recorded for those extracted from the Sb-spiked S2 soils (e.g. >2-fold for Sb-1000). The soil dehydrogenase activity followed the same trend. High-throughput 16S rRNA amplicon sequencing analysis revealed that Sb did not influence the bacterial α-diversity in both soils, while significantly affected the composition of the respective soil bacterial communities. Several phyla (e.g. Nitrosospira Nitrososphaeraceae, Adheribacter) were found positively correlated with the concentration of water-soluble Sb in soil. Overall, the results obtained suggest that the risk assessment in soils polluted with antimony should be a priority especially for alkaline soils where the high mobility of the anionic Sb(OH)6- species can pose, at least in the short-term, a serious threat for soil microbial abundance, diversity and functionality, soil fertility and eventually human health.

Microbiological characterization of Gioddu, an Italian fermented milk.

Gioddu, also known as "Miciuratu", "Mezzoraddu" or "Latte ischidu" (literally meaning acidulous milk), is the sole variety of traditional Italian fermented milk. The aim of the present study was to elucidate the microbiota and the mycobiota occurring in artisan Gioddu sampled from three Sardinian producers by combining the results of viable counting on selective culture media and high-throughput sequencing. Physico-chemical parameters were also measured. The overall low pH values (3.80-4.22) recorded in the analyzed Gioddu samples attested the strong acidifying activity carried out by lactic acid bacteria during fermentation. Viable counts revealed the presence of presumptive lactococci, presumptive lactobacilli and non-Saccharomyces yeasts. A complex (kefir-like) microbiota of bacteria and yeasts was unveiled through sequencing. In more detail, Lactobacillus delbrueckii was found to dominate in Gioddu together with Streptococcus thermophilus, thus suggesting the establishment of a yogurt-like protocooperation. Unexpectedly, in all the three analyzed batches from two out of the three producers Lactobacillus kefiri was also detected, thus representing an absolute novelty, which suggests the presence of bioactive compounds (e.g. exopolysaccharides) similar to those characterizing milk kefir beverage. Mycobiota population, studied for the very first time in Gioddu, revealed a more complex composition, with Kluyveromyces marxianus, Galactomyces candidum and Geotrichum galactomyces constituting the core species. Further research is needed to disclose the eventual occurence in Gioddu of probiotic cultures and bioactive compounds (e.g. exopolysaccharides, angiotensin-converting enzyme inhibitory peptides and antimicrobial compounds) with potential health-benefits for the consumers.

Bacterial and Fungal Communities of Gioddu as Revealed by PCR-DGGE Analysis.

Gioddu is the sole variety of fermented milk originating in Italy. Despite the long history of consumption, Gioddu still represents an undisclosed source of microbial diversity. The present study was aimed to get an insight into the bacterial and fungal diversity of Gioddu samples collected from two artisan producers located in Sardinia. To this end 3 batches of Gioddu were collected from each producer and subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analyses. Gioddu was produced with sheep milk in accordance with the local tradition. Regarding the bacterial population, a low biodiversity emerged. In more detail, the sole species Lactobacillus delbrueckii subsp. bulgaricus was detected in all the samples, irrespective of the producer or the batch. A more ample microbial diversity was highlighted for the fungal population that included closest relatives to Pichia cactophila, Kluyveromyces marxianus and Galactomyces candidum. Based on the results, the detected bacterial and fungal species generally clustered in accordance with the producer, irrespective of the batch considered. It is noteworthy that, Gioddu revealed several microbiological similarities with kefir beverage, thus suggesting, by analogy, potential health benefits related to its consumption. More research is needed to better clarify the microbiota composition of Gioddu by using more powerful metagenomic techniques.

Influence of autochthonous lactic acid bacteria and enzymatic yeast extracts on the microbiological, biochemical and sensorial properties of Lben generic products.

In this study we identified Lactococcus lactis subsp. lactis, Lc. lactis subsp. lactis biovar diacetylactis, Kluyveromices lactis and Saccharomyces cerevisiae as the dominant microorganisms of traditional Moroccan acid-alcoholic fermented milk named Lben. The low pH (3·8±0·3), lactose (16·8±3·4 mg/l) and lactic acid (8·16±0·6 mg/l) content indicated that a strong fermentation occurred in the traditional product which was also characterised by the substantial presence of ethanol and typical volatile carbonyl compounds (i.e., acetoin, diacetyl and acetaldehyde). Microbiological analyses of experimental Lben manufactured with selected strains (isolated from the traditional product) of Lc. lactis subsp. lactis and Lc. lactis subsp. lactis biovar. diacetylactis alone (batch A) and in combination with enzymatic extract of a K. lactis strain (batch B) indicated a good effectiveness of the starters employed (∼1010 CFU/g of lactococci after 8 h of incubation) and a significant effect of the yeast enzyme extract on lactococci viability. Despite slight changes in the physicochemical characteristics of the two Lben during the 15 d storage period, volatile compounds (i.e. ethanol, acetaldehyde, diacetyl and acetoin) were consistently higher in batch B. Moreover, sensorial analysis performed after 15 d of storage, highlighted higher odour and flavour intensity, vegetable odour and viscosity in batch B while batch A displayed higher astringency.

Antimicrobial activity against beneficial microorganisms and chemical composition of essential oil of Mentha suaveolens ssp. insularis grown in Sardinia.

UNLABELLED: The aim of this work was to determine the chemical constituents and in vitro antimicrobial activity of the essential oil (EO) of the aerial parts of Mentha sueveolens spp. insularis grown in Sardinia (Italy) against probiotic and starter microorganisms. The gas chromatography-mass spectrometry (GC-MS) analysis allowed to identified 34 compounds, most of oxygenated monoterpene compounds (82.5%) and among them, pulegone was found as major compound (46.5%). The agar diffusion test carried out employing the EO of Mentha suaveolens spp. insularis showed a low antibacterial activity, in particular no action was noticed for probiotic bacteria belonging to lactic acid bacteria groups, whereas almost all yeasts strains tested were inhibited. The automated microtitter dilution assay showed a clear effect at increasing concentration of EO on the specific growth rate (µ) and extension of the lag phase (λ) only for S. xylosus SA23 among bacteria and for Saccharomyces cerevisiae, Tetrapisispora phaffii CBS 4417, Metschnikowia pulcherrima, and Candida zemplinina among yeasts. Results obtained in this work allow us to broaden the knowledge on the effect of EOs on probiotic and food-related microorganisms. PRACTICAL APPLICATION: Mentha suaveolens spp. insularis may be used in combination with probiotic bacteria into the food matrix or encapsulated in coating and edible films for food preservation.

Influence of selected lab cultures on the evolution of free amino acids, free fatty acids and Fiore Sardo cheese microflora during the ripening.

Fiore Sardo Protected Denomination of Origin is a traditional Sardinian (Italy) hard cheese produced exclusively from whole raw ovine milk and coagulated with lamb rennet paste. Currently, Fiore Sardo is still produced by shepherds at the farmhouse level without the addition of any starter culture and the cheese-making process is characterized by significant waste. The first objective of the present work was to investigate the autochthonous microflora present in milk and Fiore Sardo cheese in order to select lactic acid bacterial (LAB) cultures with suitable cheese-making attributes and, possibly reduce the production waste. Secondly, the ability of selected cultures to guarantee cheese healthiness and quality was tested in experimental cheese-making trials. In this study, we show that the typical lactic microflora of raw ewe's milk and Fiore Sardo cheese is mostly composed of mesophilic LAB such as Lactococcus lactis subsp. lactis, Lactobacillus plantarum and Lactobacillus casei subsp. casei. Moreover, strains belonging to the species were selected for cheese-making attributes and used in experimental cheese-making trials carried out in different farms producing Fiore Sardo. The evolution of the cheese microflora, free amino acids and free fatty acids during the ripening showed that the experimental cheeses were characterized by a balanced ratio of the chemical constituents, by a reduced number of spoilage microorganisms and, remarkably, by the absence of production waste that were significant for the control cheeses.