Prevalence of Histidine Decarboxylase Genes of Gram-Positive Bacteria in Surströmming as Revealed by qPCR.

Histamine is a degradation product of the bacterial decarboxylation of the amino acid histidine; such activity is determined by histidine decarboxylase encoded by a gene cluster, carried by some Gram-positive bacteria, that includes the hdcA gene. In this study, the presence of the hdcA gene in ready-to-eat surströmming samples collected from three producers based in Sweden was directly assessed via qPCR analysis for the very first time. Samples from producer A showed hdcA average gene abundance of 6.67 ± 0.13 Log cells/gene copies g-1; in samples from producer B the average value attested at 5.56 ± 0.06 Log cells/gene copies g-1, whereas for samples of producer C hdcA average gene abundance attested at 5.30 ± 0.08 Log cells/gene copies g-1. ANOVA showed a significantly higher average hdcA gene copy number in samples from producer A, whereas no significant differences were seen between average values of hdcA gene copy numbers detected in samples from producer B and C. The hdcA gene copies detected in the present study could give an estimation of the load of potential histamine-producing bacteria in surströmming.

Discovering microbiota and volatile compounds of surströmming, the traditional Swedish sour herring.

In this study, the microbiota of ready-to-eat surströmming from three Swedish producers were studied using a combined approach. The pH values of the samples ranged between 6.67 ± 0.01 and 6.98 ± 0.01, whereas their aw values were between 0.911 ± 0.001 and 0.940 ± 0.001. The acetic acid concentration was between 0.289 ± 0.009 g/100 g and 0.556 ± 0.036 g/100 g. Very low concentrations of lactic acid were measured. Viable counting revealed the presence of mesophilic aerobes, mesophilic lactobacilli and lactococci as well as halophilic lactobacilli and lactococci, coagulase-negative staphylococci, halophilic aerobes and anaerobes. Negligible counts for Enterobacteriaceae, Pseudomonadaceae and total eumycetes were observed, whereas no sulfite-reducing anaerobes were detected. Listeria monocytogenes and Salmonella spp. were absent in all samples. Multiplex real-time PCR revealed the absence of the bont/A, bont/B, bont/E, bont/F, and 4gyrB (CP) genes, which encode botulinic toxins, in all the samples analyzed. Metagenomic sequencing revealed the presence of a core microbiota dominated by Halanaerobium praevalens, Alkalibacterium gilvum, Carnobacterium spp., Tetragenococcus halophilus, Clostridiisalibacter spp. and Porphyromonadaceae. Psychrobacter celer, Ruminococcaceae, Marinilactibacillus psychrotolerans, Streptococcus infantis and Salinivibrio costicola were detected as minor OTUs. GC-MS analysis of volatile components revealed the massive presence of trimethylamine and sulphur compounds. Moreover, 1,2,4-trithiolane, phenols, ketones, aldehydes, alcohols, esters and long chain aliphatic hydrocarbons were also detected. The data obtained allowed pro-technological bacteria, which are well-adapted to saline environments, to be discovered for the first time. Further analyses are needed to better clarify the extent of the contribution of either the microbiota or autolytic enzymes of the fish flesh in the aroma definition.

A Glimpse into the Microbiota of Marketed Ready-to-Eat Crickets (Acheta domesticus).

The present study was aimed to get an insight into the bacterial biota of ready-to-eat small crickets (Acheta domesticus) already marketed in the European Union. 16S rRNA gene of the DNAs extracted from thirty-two samples of ready-to-eat crickets commercialized by 4 European Union producers located in Austria, Belgium, France and the Netherlands (2 batches per producer) was analyzed by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The species belonging to the genera Hespellia, Ruminococcus and Clostridium were detected in samples from Austria, while those from genera Lysobacter, Staphylococcus and Clostridium were detected in samples from Belgium. Moreover, samples from France were characterized by Staphylococcus, Pseudomonas, and Hydrogenophilus genera. Finally, the genera Staphylococcus, Hydrogenophilus, Clostridium and Ruminococcus were identified in the samples produced in the Netherlands. When insects are intended for commercialization, rearing, processing and handling could affect the presence of the occurring microbial species. Hence, to assure a safe product, the need for a full standardization of production technologies, including feed supply as well as rearing and processing practices, is recommended.

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.

Unveiling hákarl: A study of the microbiota of the traditional Icelandic fermented fish.

Hákarl is produced by curing of the Greenland shark (Somniosus microcephalus) flesh, which before fermentation is toxic due to the high content of trimethylamine (TMA) or trimethylamine N-oxide (TMAO). Despite its long history of consumption, little knowledge is available on the microbial consortia involved in the fermentation of this fish. In the present study, a polyphasic approach based on both culturing and DNA-based techniques was adopted to gain insight into the microbial species present in ready-to-eat hákarl. To this aim, samples of ready-to-eat hákarl were subjected to viable counting on different selective growth media. The DNA directly extracted from the samples was further subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and 16S amplicon-based sequencing. Moreover, the presence of Shiga toxin-producing Escherichia coli (STEC) and Pseudomonas aeruginosa was assessed via qualitative real-time PCR assays. pH values measured in the analyzed samples ranged from between 8.07 ±â€¯0.06 and 8.76 ±â€¯0.00. Viable counts revealed the presence of total mesophilic aerobes, lactic acid bacteria and Pseudomonadaceae. Regarding bacteria, PCR-DGGE analysis highlighted the dominance of close relatives of Tissierella creatinophila. For amplicon sequencing, the main operational taxonomic units (OTUs) shared among the data set were Tissierella, Pseudomonas, Oceanobacillus, Abyssivirga and Lactococcus. The presence of Pseudomonas in the analyzed samples supports the hypothesis of a possible role of this microorganism on the detoxification of shark meat from TMAO or TMA during fermentation. Several minor OTUs (<1%) were also detected, including Alkalibacterium, Staphylococcus, Proteiniclasticum, Acinetobacter, Erysipelothrix, Anaerobacillus, Ochrobactrum, Listeria and Photobacterium. Analysis of the yeast and filamentous fungi community composition by PCR-DGGE revealed the presence of close relatives of Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida zeylanoides, Saccharomyces cerevisiae, Debaryomyces, Torulaspora, Yamadazyma, Sporobolomyces, Alternaria, Cladosporium tenuissimum, Moristroma quercinum and Phoma/Epicoccum, and some of these species probably play key roles in the development of the sensory qualities of the end product. Finally, qualitative real-time PCR assays revealed the absence of STEC and Pseudomonas aeruginosa in all of the analyzed samples.

Insight into the bacterial diversity of fermentation woad dye vats as revealed by PCR-DGGE and pyrosequencing.

The bacterial diversity in fermenting dye vats with woad (Isatis tinctoria L.) prepared and maintained in a functional state for approximately 12 months was examined using a combination of culture-dependent and -independent PCR-DGGE analyses and next-generation sequencing of 16S rRNA amplicons. An extremely complex ecosystem including taxa potentially contributing to both indigo reduction and formation, as well as indigo degradation was found. PCR-DGGE analyses revealed the presence of Paenibacillus lactis, Sporosarcina koreensis, Bacillus licheniformis, and Bacillus thermoamylovorans, while Bacillus thermolactis, Bacillus pumilus and Bacillus megaterium were also identified but with sequence identities lower than 97%. Dominant operational taxonomic units (OTUs) identified by pyrosequencing included Clostridium ultunense, Tissierella spp., Alcaligenes faecalis, Erysipelothrix spp., Enterococcus spp., Virgibacillus spp. and Virgibacillus panthothenicus, while sub-dominant OTUs included clostridia, alkaliphiles, halophiles, bacilli, moderately thermophilic bacteria, lactic acid bacteria, Enterobacteriaceae, aerobes, and even photosynthetic bacteria. Based on the current knowledge of indigo-reducing bacteria, it is considered that indigo-reducing bacteria constituted only a small fraction in the unique microcosm detected in the natural indigo dye vats.

The microbiota of marketed processed edible insects as revealed by high-throughput sequencing.

Entomophagy has been linked to nutritional, economic, social and ecological benefits. However, scientific studies on the potential safety risks in eating edible insects need to be carried out for legislators, markets and consumers. In this context, the microbiota of edible insects deserves to be deeply investigated. The aim of this study was to elucidate the microbial species occurring in some processed marketed edible insects, namely powdered small crickets, whole dried small crickets (Acheta domesticus), whole dried locusts (Locusta migratoria), and whole dried mealworm larvae (Tenebrio molitor), through culture-dependent (classical microbiological analyses) and -independent methods (pyrosequencing). A great bacterial diversity and variation among insects was seen. Relatively low counts of total mesophilic aerobes, Enterobacteriaceae, lactic acid bacteria, Clostridium perfringens spores, yeasts and moulds in all of the studied insect batches were found. Furthermore, the presence of several gut-associated bacteria, some of which may act as opportunistic pathogens in humans, were found through pyrosequencing. Food spoilage bacteria were also identified, as well as Spiroplasma spp. in mealworm larvae, which has been found to be related to neurodegenerative diseases in animals and humans. Although viable pathogens such as Salmonella spp. and Listeria monocytogenes were not detected, the presence of Listeria spp., Staphylococcus spp., Clostridium spp. and Bacillus spp. (with low abundance) was also found through pyrosequencing. The results of this study contribute to the elucidation of the microbiota associated with edible insects and encourage further studies aimed to evaluate the influence of rearing and processing conditions on that microbiota.

The occurrence of spoilage yeasts in cream-filled bakery products.

BACKGROUND: Filling creams can provide an adequate substrate for spoilage yeasts because some yeasts can tolerate the high osmotic stress in these products. To discover the source of spoilage of a cream-filled baked product, end products, raw materials, indoor air and work surfaces were subjected to microbiological and molecular analyses. The efficacy of disinfectants against spoilage yeasts was also assessed. RESULTS: The analyses on end products revealed the presence of the closest relatives to Zygosaccharomyces bailii with counts ranging from 1.40 to 4.72 log cfu g-1 . No spoilage yeasts were found in the indoor air and work surfaces. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis, carried out directly on filling creams collected from unopened cans, showed the presence of bands ascribed to the closest relatives to Z. bailii sensu lato, although with counts < 1 log cfu g-1 . Susceptibility testing of yeast isolates to disinfectants showed a significantly lower effect of 10% alkyl dimethyl benzyl ammonium chloride. Different responses of isolates to the tested disinfectants were seen. CONCLUSION: To guarantee the quality of end products, reliable and sensitive methods must be used. Moreover, hygiene and the application of good manufacturing practices represent the most efficient way for the prevention and minimization of cross-contamination. © 2016 Society of Chemical Industry.

Yeast and mould dynamics in Caciofiore della Sibilla cheese coagulated with an aqueous extract of Carlina acanthifolia All.

Caciofiore della Sibilla is a speciality ewes' milk cheese traditionally manufactured in a foothill area of the Marche region (Central Italy) with a crude extract of fresh young leaves of Carlina acanthifolia All. subsp. acanthifolia as a coagulating agent. The fungal dynamics and diversity of this speciality cheese were investigated throughout the manufacturing and 20-day ripening process, using a combined PCR-DGGE approach. The fungal biota of a control ewes' milk cheese, manufactured with the same batch of milk coagulated with a commercial animal rennet, was also monitored by PCR-DGGE, in order to investigate the contribution of the peculiar vegetable coagulant to the fungal diversity and dynamics of the cheese. Based on the overall results collected, the raw milk and the dairy environment represented the main sources of fungal contamination, with a marginal or null contribution of thistle rennet to the fungal diversity and dynamics of Caciofiore della Sibilla cheese. Copyright © 2016 John Wiley & Sons, Ltd.

Microbial evolution of traditional mountain cheese and characterization of early fermentation cocci for selection of autochtonous dairy starter strains.

The microbial population of Traditional Mountain (TM) cheese was investigated and characterized for the selection of cocci suitable for developing new starter cultures. Samples of milk, curd and cheese at different ripening times were enumerated in selective culture media and 640 colonies were isolated from curd and cheese after 24 h of ripening. The Lactic Acid Bacteria (LAB) isolated from M17 were clustered into 231 biotypes by RAPD-PCR analysis and identified as Lactococcus lactis, Streptococcus thermophilus and Enterococcus faecalis. Forty percent of enterococci showed the in vitro ability to inhibit raw milk resident coliforms, but they were excluded as possible starters due to the presence of associated risk factors. All lactococci and streptococci were tested for their technological properties; 4 Lc. lactis subsp. lactis and 2 Sc. thermophilus which were fast acidifiers and did not produce unpleasant flavours were subjected to the freeze-drying stability test. Lc. lactis subsp. lactis biotype 68 and Sc. thermophilus biotype 93 showed the best technological properties and may be appropriate for cheese production. This work gave evidence of the high biodiversity of TM-cheese autochthonous biotypes which could be used as starter cultures for the improvement of TM-cheese technology.

Bacteria and yeast microbiota in milk kefir grains from different Italian regions.

Kefir grains are a unique symbiotic association of different microrganisms, mainly lactic acid bacteria, yeasts and occasionally acetic acid bacteria, cohabiting in a natural polysaccharide and a protein matrix. The microbial composition of kefir grains can be considered as extremely variable since it is strongly influenced by the geographical origin of the grains and by the sub-culturing method used. The aim of this study was to elucidate the bacteria and yeast species occurring in milk kefir grains collected in some Italian regions by combining the results of scanning electron microscopy analysis, viable counts on selective culture media, PCR-DGGE and pyrosequencing. The main bacterial species found was Lactobacillus kefiranofaciens while Dekkera anomala was the predominant yeast. The presence of sub-dominant species ascribed to Streptococcus thermophilus, Lactococcus lactis and Acetobacter genera was also highlighted. In addition, Lc. lactis, Enterococcus sp., Bacillus sp., Acetobacter fabarum, Acetobacter lovaniensis and Acetobacter orientalis were identified as part of the cultivable community. This work further confirms both the importance of combining culture-independent and culture-dependent approaches to study microbial diversity in food and how the combination of multiple 16S rRNA gene targets strengthens taxonomic identification using sequence-based identification approaches.

Evaluation of HACCP system implementation on the quality of mixed fresh-cut salad prepared in a university canteen: a case study.

The increasing awareness that foods can represent vehicles for health risk factors has caused scientists and public authorities to multiply their efforts to reduce these risks to within acceptable limits. Nevertheless, some challenging issues still remain unsolved and new ones have recently emerged, such as the increase in outbreaks of foodborne diseases originating from the consumption of meals at catering facilities. The study described in this article was aimed at evaluating the microbiological quality of mixed fresh-cut salads at an Italian university canteen operating in conformity with the hazard analysis and critical control point (HACCP) system. The effectiveness of the preventive and corrective measures taken was also assessed with respect to the frequency of unsatisfactory salad samples. During the investigation, E. coli, Salmonella spp., and Listeria monocytogenes were never detected. By contrast, a high number of samples exceeded the mandatory or suggested limits for food processing hygiene (in terms of mesophilic aerobes, coliforms, Staphylococcus aureus, Bacillus cereus, and sulfite-reducing clostridia counts). Despite the introduction of a series of preventive and corrective actions, the results were only partially satisfactory; this was most likely due to the impossibility of having available an adequate level of human resources that are indispensable to correctly putting the HACCP procedures into daily practice.

Evaluation of the quality of foods for special diets produced in a school catering facility within a HACCP-based approach: a case study.

A study was carried out to verify the appropriateness of the Hazard Analysis and Critical Control Point (HACCP) plan adopted in a school catering facility. To that end, the microbiological quality of foods, the correct implementation of special diets (lactose- and gluten-free) and the nutritional value of foods were assessed. Thirty-six samples of lactose-free and 87 samples of gluten-free special diet food preparations were subjected to microbiological, chemical, and nutritional analyses. The data collected demonstrate the effectiveness of the HACCP plan in reducing the occurrence of microbial and chemical (lactose and gluten) cross-contamination. The data obtained from the nutritional analyses showed that the dietary intake provided by the meals under study was satisfactory.

Implementation of a biotechnological process for vat dyeing with woad.

The traditional process for vat dyeing with woad (Isatis tinctoria L.) basically relies on microbial reduction of indigo to its soluble form, leucoindigo, through a complex fermentative process. In the 19th century, cultivation of woad went into decline and use of synthetic indigo dye and chemical reduction agents was established, with a consequent negative impact on the environment due to the release of polluting wastewaters by the synthetic dyeing industry. Recently, the ever-growing demand for environmentally friendly dyeing technologies has led to renewed interest in ecological textile traditions. In this context, this study aims at developing an environmentally friendly biotechnological process for vat dyeing with woad to replace use of polluting chemical reduction agents. Two simple broth media, containing yeast extract or corn steep liquor (CSL), were comparatively evaluated for their capacity to sustain the growth and reducing activity of the strain Clostridium isatidis DSM 15098(T). Subsequently, the dyeing capacity of the CSL medium added with 140 g L⁻¹ of woad powder, providing 2.4 g L⁻¹ of indigo dye, was evaluated after fermentation in laboratory bioreactors under anaerobic or microaerophilic conditions. In all fermentations, a sufficiently negative oxidation/reduction potential for reduction of indigo was reached as early as 24 h and maintained up to the end of the monitoring period. However, clearly faster indigo dye reduction was seen in the broth cultures fermented under strict anaerobiosis, thus suggesting the suitability of the N2 flushing strategy for enhancement of bacterial-driven indigo reduction.

Recent investigations and updated criteria for the assessment of antibiotic resistance in food lactic acid bacteria.

The worldwide use, and misuse, of antibiotics for about sixty years in the so-called antibiotic era, has been estimated in some one to ten million tons, a relevant part of which destined for non-therapeutic purposes such as growth promoting treatments for livestock or crop protection. As highly adaptable organisms, bacteria have reacted to this dramatic change in their environment by developing several well-known mechanisms of antibiotic resistance and are becoming increasingly resistant to conventional antibiotics. In recent years, commensal bacteria have become a cause of concern since they may act as reservoirs for the antibiotic resistance genes found in human pathogens. In particular, the food chain has been considered the main route for the introduction of animal and environment associated antibiotic resistant bacteria into the human gastrointestinal tract (GIT) where these genes may be transferred to pathogenic and opportunistic bacteria. As fundamental microbial communities in a large variety of fermented foods and feed, the anaerobe facultative, aerotolerant lactic acid bacteria (LAB) are likely to play a pivotal role in the resistance gene exchange occurring in the environment, food, feed and animal and human GIT. Therefore their antibiotic resistance features and their genetic basis have recently received increasing attention. The present article summarises the results of the latest studies on the most typical genera belonging to the low G + C branch of LAB. The evolution of the criteria established by European regulatory bodies to ensure a safe use of microorganisms in food and feed, including the assessment of their antibiotic resistance is also reviewed.

An eight-year report on the implementation of HACCP in a university canteen: impact on the microbiological quality of meals.

An investigation aimed at assessing the microbiological quality of meals consumed at a university canteen after implementation of the HACCP system and personnel training was carried out. Cooked and warm-served products (74 samples), cooked and cold-served products (92 samples) and cold gastronomy products (63 samples) sampled from 2000 to 2007 underwent microbiological analyses. All the samples were tested for: Samonella spp., Listeria monocytogenes, total mesophilic aerobes, coliforms, Escherichia coli, Staphylococcus aureus, Bacillus cereus, and sulphite-reducing clostridia. The microbiological contamination of work surfaces (tables, tablewares, cutters, ladles, slicing machines, wash-basins, etc.), hands and white coats of members of the canteen staff was also assessed. The microbiological results clearly demonstrated the success of the HACCP plan implementation, through a general improvement of the hygiene conditions of both meals and work surfaces.

Microbial dynamics in rearing trials of Hermetia illucens larvae fed coffee silverskin and microalgae.

In the present study, Hermetia illucens larvae were reared on a main rearing substrate composed of a coffee roasting byproduct (coffee silverskin, Cs) enriched with microalgae (Schizochytrium limacinum or Isochrysis galbana) at various substitution levels. The microbial diversity of the rearing substrates, larvae, and frass (excrement from the larvae mixed with the substrate residue) were studied by the combination of microbial culturing on various growth media and metataxonomic analysis (Illumina sequencing). High counts of total mesophilic aerobes, bacterial spores, presumptive lactic acid bacteria, coagulase-positive cocci, and eumycetes were detected. Enterobacteriaceae counts were low in the rearing diets, whereas higher counts of this microbial family were observed in the larvae and frass. The microbiota of the rearing substrates was characterized by the presence of lactic acid bacteria, including the genera Lactobacillus, Leuconostoc and Weissella. The microbiota of the H. illucens larvae fed Cs was characterized by the dominance of Paenibacillus. H. illucens fed diets containing I. galbana were characterized by the presence of Enterococcus, Lysinibacillus, Morganella, and Paenibacillus, depending on the algae inclusion level, while H. illucens fed diets containing S. limacinum were characterized by high relative abundances of Brevundimonas, Enterococcus, Paracoccus, and Paenibacillus, depending on the algae inclusion level. Brevundimonas and Alcaligenes dominated in the frass from larvae fed I. galbana; the predominance of Brevundimonas was also observed in the frass from larvae fed Schyzochitrium-enriched diets. Based on the results of the present study, an effect of algae nutrient bioactive substances (e.g. polysaccharides, high-unsaturated fatty acids, taurine, carotenoids) on the relative abundance of some of the bacterial taxa detected in larvae may be hypothesized, thus opening new intriguing perspectives for the control of the entomopathogenic species and foodborne human pathogens potentially occurring in edible insects. Further studies are needed to support this hypothesis. Finally, new information on the microbial diversity occurring in insect frass was also obtained.

Occurrence of Antibiotic Resistance Genes in Hermetia illucens Larvae Fed Coffee Silverskin Enriched with Schizochytrium limacinum or Isochrysis galbana Microalgae.

Hermetia illucens larvae are among the most promising insects for use as food or feed ingredients due to their ability to convert organic waste into biomass with high-quality proteins. In this novel food or feed source, the absence of antibiotic-resistant bacteria and their antibiotic resistance (AR) genes, which could be horizontally transferred to animal or human pathogens through the food chain, must be guaranteed. This study was conducted to enhance the extremely scarce knowledge on the occurrence of AR genes conferring resistance to the main classes of antibiotics in a rearing chain of H. illucens larvae and how they were affected by rearing substrates based on coffee silverskin supplemented with increasing percentages of Schizochytrium limacinum or Isochrysis galbana microalgae. Overall, the PCR and nested PCR assays showed a high prevalence of tetracycline resistance genes. No significant effect of rearing substrates on the distribution of the AR genes in the H. illucens larvae was observed. In contrast, the frass samples were characterized by a significant accumulation of AR genes, and this phenomenon was particularly evident for the samples collected after rearing H. illucens larvae on substrates supplemented with high percentages (>20%) of I. galbana. The latter finding indicates potential safety concerns in reusing frass in agriculture.

Selection of cereal-sourced lactic acid bacteria as candidate starters for the baking industry.

The quality of sourdough bread mainly depends on metabolic activities of lactic acid bacteria (LAB). The exopolysaccharides (EPS) produced by LAB affect positively the technological and nutritional properties of the bread, while phytases improve the bioavailability of the minerals by reducing its phytate content. In the present study, a pool of 152 cereal-sourced LAB were screened for production of phytases and EPS for potential use as sourdough starter cultures for the baking industry. There was large heterogeneity in the phytase activity observed among the screened isolates, with 95% showing the ability to degrade sodium phytate on plates containing Sourdough Simulation Medium (SSM). The isolates Lactobacillus brevis LD65 and Lactobacillus plantarum PB241 showed the highest enzymatic activity, while the isolates ascribed to Weissella confusa were characterized by low or no phytase activity. Only 18% of the screened LAB produced EPS, which were distinguished as ropy or mucoid phenotypes on SSM supplemented with sucrose. Almost all the EPS producers carried one or more genes (epsD/E and/or epsA) involved in the production of heteropolysaccharides (HePS), whereas the isolates ascribed to Leuconostoc citreum and W. confusa carried genes involved in the production of both HePS and homopolysaccharides (HoPS). Monosaccharide composition analysis of the EPS produced by a selected subset of isolates revealed that all the HePS included glucose, mannose, and galactose, though at different ratios. Furthermore, a few isolates ascribed to L. citreum and W. confusa and carrying the gtf gene produced ß-glucans after fermentation in an ad hoc formulated barley flour medium. Based on the overall results collected, a subset of candidate sourdough starter cultures for the baking industry was selected, including Lb. brevis LD66 and L. citreum PB220, which showed high phytase activity and positive EPS production.

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.