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 (Kielbasa polska wedzona) 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 (Kielbasa polska wedzona) 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.

Chemical, microbiological, textural, and sensory characteristics of pilot-scale Caciofiore cheese curdled with commercial Cynara cardunculus rennet and crude extracts from spontaneous and cultivated Onopordum tauricum.

The aim of this study was the chemical, microbiological, textural, and sensory characterization of pilot-scale prototypes of an Italian ewe's raw milk cheese (Caciofiore) curdled with commercial Cynara cardunculus rennet, used as a control, and crude extracts obtained from flowers of either spontaneous or cultivated Onopordum tauricum. Hence, the control and experimental cheese prototypes produced in two rounds of cheesemaking trials were assayed, at the end of their 60-day maturation, for the following features: pH, titratable acidity, dry matter, fat, total and soluble nitrogen (TN and SN, respectively), ash, salt, protein, lactose, viable plate counts and composition of the bacterial and fungal populations, color, texture, volatile organic compounds (VOCs), and olfactory attributes by sensory analysis (the latter for the sole prototypes curdled with the commercial rennet and the extract obtained from cultivated O. tauricum). The data overall collected showed a very low impact of the type of thistle rennet on the analyzed cheese traits, with significant differences being exclusively found for SN/TN%, titratable acidity, color, and adhesiveness. By contrast, a higher impact of the cheesemaking round was seen, with significant differences being observed for salt content, load of presumptive lactobacilli, thermophilic cocci, and Escherichia coli, and levels of the following VOCs: 2,3-butanedione, 2-pentanone, 1-butanol, 2-heptanone, 3-methyl-1-butanol, 2-heptanol, 2-nonanone, dimethyl trisulfide, 2-methyl propanoic acid, butanoic acid, and 3-methyl butanoic acid. Sensory analysis revealed a strong ewe's cheese odor, accompanied by other olfactory notes, such as pungent, sour curd, sweet, and Parmesan cheese-like notes, in all the analysed cheese prototypes. Moreover, key odor active compounds, including butanoic acid, ethyl butanoate, 2,3-butanedione, 1-octen-3-one, and dimethyl trisulfide, were identified by GC-olfactometry analysis. Regarding the odor attributes as determined by sensory analysis, again the type of rennet had an almost negligible impact, with significant differences being only perceived for 1 or 2 out of 20 odor attributes, depending on the analytical conditions applied. Although some aspects deserve further investigation, the results herein collected confirm that O. tauricum can be regarded as an alternative source of thistle rennet for the manufacture of Caciofiore cheese, and more in general, Mediterranean ewe's milk cheeses.

Croatian white grape variety Marastina: First taste of its indigenous mycobiota.

The indigenous vineyard mycobiota contribute both to wine quality and vineyard sanitary status. Wines made from same grape variety but from different geographical locations are appreciated for their diversity. Because no information on indigenous mycobiota of Croatian grapevines is available, the aim of the present study was to start filling this knowledge gap by characterizing the indigenous mycobiota of Marastina variety. The use of metataxonomic approach has enabled the identification of 25 different fungal genera present on Marastina grape berries collected from 11 vineyards located within the Croatian coastal winegrowing region of Dalmatia (Northern Dalmatia, Dalmatian hinterland, Central and Southern Dalmatia). The substantial regional and local scale differences in their distribution were observed. Overall, Aureobasidium was the dominant genus followed by Cladosporium and Metschnikowia. Botrytis and Plenodomus were associated with the vineyards located in Central and Southern Dalmatia, whereas Pichia was associated with Northern Dalmatia vineyards. The largest abundance of Buckleyzyma, Cladosporium, Eremothecium, Fusarium, Papiliotrema, and Rhodotorula was observed in Dalmatian hinterland. Moreover, data suggested that climate conditions and soil type partially influenced the distribution of fungal communities. The local-scale differences emerged also for the physicochemical characteristics of fresh musts. The high malic acid content supported the development of Metschnikowia, and inhibited Fusarium growth, whereas a positive correlation between Erysiphe and pH values was observed. Sporobolomyces and Cystobasidium were negatively associated with high glucose concentration. The revealing of Marastina indigenous mycobiota provided information on the members of fungal community negatively influencing the grapevine sanitary status as well as those which could be employed in disease biocontrol.

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.

Autochthonous starter culture selection for Salame Piemonte PGI production.

Salame Piemonte is a dry-fermented meat product typical of the Piedmont region in Italy, manufactured using commercial starter cultures. This study aimed to select autochthonous starter cultures (ASCs) that could be used for sausage fermentation in order to strengthen the link with the geographical area of production and improve the sensory properties of the final product. A culture-dependent approach was adopted during three different spontaneous sausage fermentation processes to isolate and characterise the main bacterial resources involved. Dominant lactic acid bacteria (LAB) in each batch were Pediococcus pentosaceus, Latilactobacillus sakei, and Latilactobacillus curvatus; Staphylococcus xylosus was the most dominant coagulase-negative staphylococci (CNS) in all the studied batches. LAB and presumptive CNS isolates were further evaluated for their physiological properties and biotechnological potential. Thereafter, 11 strains were selected and evaluated for safety. Five selected strains (two P. pentosaceus, two L. sakei, and one S. xylosus strain) were used for pilot-scale Salame Piemonte production with seven different strain combinations. Based on the liking test, three ASC combinations led to the highest liking score compared to industrial products. These three ASCs were then used for the second pilot-scale sausage production confirming the high liking score. In summary, the use of P. pentosaceus and S. xylosus ASC significantly improved product sensory properties compared with that obtained using commercial starter cultures.

Effects of Dietary Supplementation of Lactobacillus acidophilus on Blood Parameters and Gut Health of Rabbits.

This study aimed to evaluate the effects of Lactobacillus acidophilus D2/CSL (L-1 × 109 cfu/kg feed/day) on biochemical parameters, faecal score (FS), cecal pH, gut morphometry, microbiota and cecal short-chain fatty acid (SCFAs) in rabbits. Three zootechnical trials were performed and in each trial 30 rabbits were allotted to two groups; a probiotic group (L) and a control group (C). At slaughter (day 45), samples of blood, duodenum, jejunum, ileum, liver and spleen were collected and submitted to histomorphometric analyses. Blood biochemical analyses, cecal microbiota and SCFAs determination were also performed. In trial 1 and 3, L. acidophilus D2/CSL did not affect productive parameters (p > 0.05). However, L group of trial 1 showed a lower morbidity and mortality compared to the control. In trial 2, C group showed a higher daily feed intake (p = 0.018) and a positive statistical tendency for live weight and average daily gain (p = 0.068). On the contrary, albumin was higher and ALFA-1 globulin was lower in the C group compared to L (p < 0.05). In all the trials, FS, cecal pH, histomorphometry, microbiota and SCFAs were unaffected. In conclusion, L. acidophilus D2/CSL did not impair growth performances, gut and rabbit's health, reducing morbidity and mortality.

Mycobiota composition and changes across pregnancy in patients with gestational diabetes mellitus (GDM).

The gut mycobiota has never been studied either during pregnancy or in patients with gestational diabetes (GDM). This study aimed to analyze the fecal mycobiota of GDM patients during the second (T2) and third (T3) trimester of pregnancy and to compare it with the mycobiota of pregnant normoglycemic women (controls). Forty-one GDM patients and 121 normoglycemic women were studied. GDM mycobiota was composed almost exclusively by the Ascomycota phylum; Basidiomicota accounted for 43% of the relative frequency of the controls. Kluyveromyces (p < 0.001), Metschnikowia (p < 0.001), and Pichia (p < 0.001) showed a significantly higher frequency in GDM patients, while Saccharomyces (p = 0.019), were more prevalent in controls. From T2 to T3, a reduction in fungal alpha diversity was found in GDM patients, with an increase of the relative frequency of Candida, and the reduction of some pro-inflammatory taxa. Many associations between fungi and foods and nutrients were detected. Finally, several fungi and bacteria showed competition or co-occurrence. Patients with GDM showed a predominance of fungal taxa with potential inflammatory effects when compared to normoglycemic pregnant women, with a marked shift in their mycobiota during pregnancy, and complex bacteria-fungi interactions.

Metataxonomic signature of beef burger perishability depends on the meat origin prior grinding.

Spoilage dynamics of two beef burger batches from different beef origins were followed from their shared processing run until the use-by date and beyond. Amplicon based sequencing of bacterial and fungal communities were compared with microbial counts and volatilome profile in order to determine whether and at which extent their perishability was related to the batch origin. Microbiological counts did not differ between batch A and B, whereas Volatile Organic Compounds (VOCs) profiles were only distinguishable after the use-by date. Metataxonomic analysis showed that both batches shared the same initial fungal and bacterial community, which however represented a transient signature of the processing run. Indeed, it was rapidly replaced by batch-autochthonous species of fungi and bacteria. Different temporal succession patterns of psychotropic lactic acid bacteria (LAB) were observed between the batches from the fourth day of vacuum storage. In particular, the sequential dominance of Carnobacterium divergens and Leuconostoc piscium in batch B was correlated with a more heterogeneous volatilome and greater production of VOCs linked to off-odours, such as the acetoin. The metataxonomic survey was able to discriminate between the two batches of hamburgers in relation to their origin and regardless of the initially shared processing-derived contamination.

Fate of Escherichia coli artificially inoculated in Tenebrio molitor L. larvae rearing chain for human consumption.

The edible insect food chain represents a relatively novel food-producing system; hence, associated biological risks still need to be exhaustively evaluated. In the present study, the dynamics of Escherichia coli during the whole living period of Tenebrio molitor larvae (from eggs to pupae) were studied. To this end, a rearing substrate consisting of organic wheat middlings was spiked with E. coli cells at two initial contamination levels: 1 log cfu g-1 (low) and 6 log cfu g-1 (high). Microbial viability counting coupled with metataxonomic analyses was used to assess i) the persistence and growth of E. coli in the rearing substrate (wheat middlings); ii) the colonization and growth of E. coli in the insect larvae; and iii) the occurrence and load of E. coli in the frass (excrement from larvae mixed with substrate residues). The results highlighted a very limited persistence of the pathogen in all analyzed samples. In more detail, the results suggested that when E. coli was present at very low levels in the eggs of the insect, the pathogen was not able to reach concerning levels in the larvae. Moreover, when E. coli was present in the wheat middlings used for rearing, the environmental conditions of the substrate (low aw values) were not favorable for its survival and multiplication, irrespective of the presence of the larvae and their frass. Surprisingly, under the conditions applied in the present study, the larvae fed wheat middlings contaminated with E. coli seemed to be inhospitable or even hostile environments for microbial survival or multiplication. To explain the low levels of E. coli cells in the larvae reared in the present study, many factors can be considered, including the immune response of the host, microbial composition and interactions established in the gut of larvae, and insect species. Of note, part of the major fraction of the microbiota of larvae at the end of rearing was represented by Lactococcus, thus suggesting a possible effect of this lactic acid bacterium on E. coli decay. Further research is needed to better clarify the interactions between E. coli and the insect gut, as well as the interactions established among the target microorganism and those naturally harbored by the insect gut.

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.

Queijo Serra da Estrela PDO Cheese: Investigation into Its Morpho-Textural Traits, Microbiota, and Volatilome.

Queijo Serra da Estrela is a PDO Portuguese cheese produced through coagulation of raw ewe's milk using cardoon (Cynara cardunculus L.) flowers. The present research was aimed at depicting an up-to-date and comprehensive overview of the microbiota of Queijo Serra da Estrela cheese. To this end, viable counting and metataxonomic analysis were carried out on cheeses sampled from four Portuguese artisan producers. Physico-chemical and morpho-textural analyses were also performed, together with the analysis of volatile organic compounds (VOCs). Finally, non-starter lactic acid bacteria (NSLAB) isolated from the cheeses were characterized for their enzymatic activities using a semi-quantitative method. According to the metataxonomic analysis, Lactococcus lactis and Lactococcus piscium were the species occurring at the highest relative abundance. The isolates collected from the cheeses were assigned to Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus lactis, Levilactobacillus brevis, Latilactobacillus graminis, Leuconostoc mesenteroides, and the Lacticaseibacillus casei group. The enzymatic characterization of these cultures highlighted esterase, aminopeptidase, acid phosphatase, beta-galactosidase, alpha-glucosidase, and beta-glucosidase among the major enzymatic activities. Fungal populations were dominated by Debaryomyces hansenii and Kurtzmaniella zeylanoides; however, species rarely found in cheese (e.g., Candida boidinii, Vishniacozyma victoriae, and Starmerella) were also detected. The volatile compounds characterizing the analyzed cheeses were carboxylic acids and esters, followed by carbonyl compounds and alcohols.

Technological, functional and safety properties of lactobacilli isolates from soft wheat sourdough and their potential use as antimould cultures.

Bakery products are a common medium for fungal growth due to their high-water activity and nutrients availability. The application of lactic acid bacteria (LAB) isolated from wheat bran or other cereals has shown great potential in controlling the growth of spoilage fungi, guarantee quality and prolong the shelf life of bakery products. This study outlines the antifungal, technological, functional and safety properties of autochthonous LAB microbiota isolated from type 0 soft wheat sourdough fermentation. Antifungal activity of 77 LAB belonging to Lactiplantibacillus plantarum and Lacticaseibacillus casei species isolated from spontaneous sourdough fermentation was tested in vitro against 16 spoilage fungi. Our findings demonstrated that the antifungal activity, enzymatic and safety properties of LAB isolates vary strain-dependently. Four LAB isolates (Lp. plantarum A16, A25, B11, and B15) showed the best traits, in particular strong antifungal activity and good capabilities to produce exopolysaccharides from different carbon sources in vitro. Care should be taken when using Lp. plantarum A310 and B18 and Lc. casei A23, as starter cultures, since these isolates exhibited a multiple antibiotic-resistance. Here we showed the promising potential of different LAB isolates as bio-preservative agents and to provide new insights regarding their prospective use as starter cultures to guarantee safety and palatability.

Specific metagenomic asset drives the spontaneous fermentation of Italian sausages.

Metagenomics is a powerful tool to study and understand the microbial dynamics that occur during food fermentation and allows to close the link between microbial diversity and final sensory characteristics. Each food matrix can be colonized by different microbes, but also by different strains of the same species. In this study, using an innovative integrated approach combining culture-dependent method with a shotgun sequencing, we were able to show how strain-level biodiversity could influence the quality characteristics of the final product. The attention was placed on a model food fermentation process: Salame Piemonte, a Protected Geographical Indication (PGI) Italian fermented sausage. Three independent batches produced in February, March and May 2018 were analysed. The sausages were manufactured, following the production specification, in a local meat factory in the area of Turin (Italy) without the use of starter cultures. A pangenomic approach was applied in order to identify and evaluate the lactic acid bacteria (LAB) population driving the fermentation process. It was observed that all batches were characterized by the presence of few LAB species, namely Pediococcus pentosaceus, Latilactobacillus curvatus and Latilactobacillus sakei. Sausages from the different batches were different when the volatilome was taken into consideration, and a strong association between quality attributes and strains present was determined. In particular, different strains of L. sakei, showing heterogeneity at genomic level, colonized the meat at the beginning of each production and deeply influenced the fermentation process by distinctive metabolic pathways that affected the fermentation process and the final sensory aspects.

A new practical approach for the biological treatment of a mixture of cheese whey and white wastewaters using Kefir grains.

Kefir grains are a microbial consortium of different genera of bacteria and yeasts. In this study, the performance of Tunisian Kefir grains during the biological treatment of a mixture of Gouda cheese whey and white wastewaters (GCW) in ratio 1:1 with very high organic matter concentration is investigated. The biological process was evaluated and optimized through the response surface methodology. Under the optimum conditions, Kefir grains concentration of 1.02%, temperature at 36.68 °C, and incubation time of 5.14 days, the removal efficiencies of COD, PO43-, and NO3- were 87, 37.48, and 39.5%, respectively. Interestingly, the reusability tests of the grains proved not only their high resistance to harsh environmental conditions but also their great potential for more practical applications. Particularly, different strains were isolated from the grains and identified as Kluyveromyces marxianus, Lactoccocus lactis, Lactobacillus kefiri, and Bacillus spp. using 16S rDNA sequence analysis and rep-PCR fingerprinting. At the biological level, the raw GCW (RGCW) has a negative impact on the Hordeum vulgare both on seed germination, and on the growth parameters of seedlings. Interestingly, after Kefir grains treatment, the treated GCW (TGCW) allow a seedlings growth and germination rate similar to those soaked in water.

Mixed culture of Lactococcus lactis and Kluyveromyces marxianus isolated from kefir grains for pollutants load removal from Jebel Chakir leachate.

The wastewater from the dumping site usually contains high pollutant levels. Biological process and physico-chemical treatments are among several technologies for wastewater treatment. Using microorganisms in the treatment of landfill leachate is an emerging research issue. Furthermore, bioremediation is a feasible approach for pollutants removal from landfill leachate which would provide an efficient way to resolve the issue of landfill leachate. In this study, the performance of yeast and bacteria isolated from kefir grains was assessed for landfill leachate treatment. Kefir grains microbial composition was evaluated by molecular approaches (Rep-PCR and 16S rRNA gene sequencing). The obtained outcomes denoted that high concentrations of lactic acid bacteria and yeast populations (over 107  CFU/ml) were found in the kefir grains and were essentially composed of Lactococcus lactis, Lactobaccillus kefirien, bacillus sp., L. lactis, and Kluyveromyces marxianus. The co-culture with 1% of inoculum size was demonstrated as the most efficient in the degradation of different contaminants. The overall abatement rate of chemical oxygen demand (COD), ammonium nitrogen ( NH 4 + - N ), and salinity were 75.8%, 85.9%, and 75.13%, respectively. The bioremediation process resulted in up of 75% removal efficiency of Ni and Cd, and a 73.45%, 68.53%, and a 58.17% removal rates of Cu, Pb, and Fe, respectively. The research findings indicate the performance of L. lactis and K. marxianus co-culture isolated from kefir grains for the bioremediation of LFL. PRACTITIONER POINTS: Isolation and identification of microorganisms from kefir grains was carried out. Biological treatment of LFL using monoculture of (Lactoccocus lactis; Kluyveromyces marxianus) and co-culture (5% of L. lactis and 5% K. marxianus) has been performed. Biological treatment using co-culture strain is an effective approach to remove organic matter, NH 4 + - N and heavy metals.

Sausage fermentation and starter cultures in the era of molecular biology methods.

Fermented sausages have a long tradition originating from Europe and they constitute a significant part of the Mediterranean diet. This kind of products has a specific microbiota that is typical of the region or area where they are produced. Therefore, in order to protect the traditional aspect of these products, it is essential to understand the microbial ecology during fermentation by studying the dynamic changes that occur and to select autochthonous starter cultures that can be used in the production. In this paper we summarize the state of the art concerning the selection and use of starter cultures and ecology aspects of naturally fermented sausages. We pay particular attention to the application of bacteriocinogenic strains as they could provide an additional tool in the prevention of foodborne pathogens as well as enhancing the competitiveness of the starter organisms. Microbial ecology of fermented sausages has been determined by traditional microbiological methods, but the introduction in food microbiology of new molecular techniques complements the studies carried out so far and allows scientists to overcome the limitations of traditional methods. Next Generation Sequencing (NGS) techniques represent a change in the way microbiologists address ecology and diversity in foods. Indeed the application of metataxonomics and metagenomics will permit a detailed understanding of microbial ecology. A thorough knowledge of the mechanisms behind the biological processes will enhance meat fermentation control and modulation to obtain products with desired organoleptic properties.