Reduction of PDO Pecorino Siciliano cheese making duration: Microbial dynamics and quality attributes deriving from replacing whey permeate with hot water during cooking.

This work was carried out with the aim to reduce the transformation duration of Protected Designation of Origin (PDO) Pecorino Siciliano cheese. To this purpose, the cooking in hot water (experimental production, EXP) was compared to the traditional cheese cooking under whey permeate (control production, CTR). The microbiological composition of under rind (UR) and core (Co) section of CTR and EXP cheeses was determined by a combined culture-dependent and -independent approach. Total mesophilic microorganisms and lactic acid bacteria (LAB) present in raw ewes' milk (5.0 log CFU/mL) increased during cheese making and reached values of about 8.0 log CFU/g in both sections (UR and Co) of 5-month ripened cheeses of both productions (CTR and EXP) monitored. The identification of the viable LAB populations in ripened cheeses showed that Enterococcus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Limosilactobacillus and Streptococcus dominated UR and Co sections of all cheeses. MiSeq Illumina analysis demonstrated that LAB populations (lactobacilli, lactococci and streptococci) dominated the bacterial community of cheeses at 95.63-98.41 % of relative abundance. The two different cooking operations did not influence the physicochemical characteristics of PDO Pecorino Siciliano cheeses. Sensory evaluation performed by artificial senses analysis and trained panelists confirmed that the modification of PDO Pecorino Siciliano cheese production protocol did not significantly affect product characteristics and overall acceptance. Thus, data of this work confirmed that cooking under hot water allowed to reduce transformation duration and safeguard typicality of PDO Pecorino Siciliano cheese.

Metagenomic, microbiological, chemical and sensory profiling of Caciocavallo Podolico Lucano cheese.

In this study, Caciocavallo Podolico Lucano (CPL) cheese was deeply characterized for its bacterial community, chemical composition and sensory aspects. The entire cheese making process (from milk collection to ripened cheese) was performed by strictly applying the traditional protocol for CPL production in four dairy factories (A-D) representative of the production area. The vat made of wood represents the main transformation tool for CPL cheese production and the biofilms hosted onto the internal surfaces of all vats analyzed in this study were dominated by lactic acid bacteria. Total mesophilic microorganisms present in bulk milk (4.7-5.0 log CFU/ml) increased consistently after contact with the wooden vat surfaces (5.4-6.4 log CFU/ml). The application of Illumina sequencing technology identified barely 18 taxonomic groups among processed samples; streptococci and lactobacilli constituted the major groups of the wooden vat biofilms [94.74-99.70 % of relative abundance (RA)], while lactobacilli dominated almost entirely (94.19-100 % of total RA) the bacterial community of ripened cheeses. Except coagulase positive staphylococci, undesirable bacteria were undetectable. Among chemical parameters, significant variations were registered for unsaturated, monounsaturated, polyunsaturated fatty acids and antioxidant properties (significantly lower for CPL cheeses produced in factory B). The cheeses from factories A, C and D were characterized by a higher lactic acid and persistence smell attributes than factory B. This work indicated that the strict application of CPL cheese making protocol harmonized the main microbiological, physicochemical and sensory parameters of the final cheeses produced in the four factories investigated.

Reuse of almond by-products: Functionalization of traditional semolina sourdough bread with almond skin.

Almond production generates large amounts of by-products rich in polyphenols. In this study, almond skin was explored as a valuable food ingredient in bread making. To this purpose, almond skin was used to produce functional products modifying a traditional sourdough bread recipe. The doughs were prepared replacing semolina with powdered almond skin (PAS) at 5 and 10 % (w/w). Sourdough inoculum was started with a mix of lactic acid bacteria (LAB) and propagated in semolina until reaching pH 3.7. The pH of PAS added breads was higher than that of control (CTR) breads before and after fermentation. Plate counts showed a similar evolution of LAB and total mesophilic microorganisms, but members of Enterobacteriaceae and coliform were detectable in PAS doughs. Illumina data clearly showed a dominance of lactobacilli in all trials, but PAS doughs displayed the presence of Bacillus. The final bread characteristics were influenced by PAS and its addition percentage; in particular, crust and crumb colour resulted darker, the alveolation decreased and, regarding sensory attributes, odour intensity increased, while bread odour diminished. In presence of PAS, bread emissions were characterized by lower percentages of alcohols and aromatic hydrocarbons and higher percentages of the other volatile compound classes, especially terpenoids like ß-pinene, ß-myrcene and limonene than CTR trial. After in vitro simulated digestion, the final release of phytochemicals from 10 % PAS bread was almost 100 %. Thus, PAS determined an increase of the antioxidant capacity of the breads. Phytochemicals released from digested PAS-fortified bread can provide antioxidant protection in a complex biological environment such as human intestinal-like cells. Besides the positive functional properties of PAS, this work also evidenced the hygienic issues of almond skin and, in order to avoid potential risks for the human health, highlighted the need to preserve its microbiological characteristics during storage for their reuse in bread production.

Effect of commission implementing regulation (EU) 2020/1319 on the bacterial composition of PDO Provola dei Nebrodi cheese.

In this study, PDO Provola dei Nebrodi cheese was deeply characterized for its bacterial community and chemical composition. Four dairy factories (A-D) were monitored from milk to ripened cheese. Wooden vat biofilms were dominated by thermophilic rod LAB (4.6-6.5 log CFU/cm2). Bulk milk showed consistent levels of total mesophilic microorganisms (TMM) (5.0-6.0 log CFU/mL) and, after curdling, a general increase was recorded. The identification of the dominant LAB in wooden vat biofilms and ripened cheeses showed that the majority of wooden vat LAB were lactococci and Streptococcus thermophilus, while cheese LAB mainly belonged to Lacticaseibacillus paracasei and Enterococcus. Illumina sequencing identified 22 taxonomic groups; streptococci, lactococci, lactobacilli and other LAB constituted the majority of the total relative abundance % of the wooden vat (69.01-97.58 %) and cheese (81.57-99.87 %) bacterial communities. Regarding chemical composition, the effect of dairy factories was significant only for protein content. Inside cheese color was lighter and yellower than surface. Differences in fatty acids regarded only myristic acid and total amount of monounsaturated fatty acids. The sensory evaluation indicated some differences among cheeses produced in the four dairies regarding color, homogeneity of structure, overall intensity, salty, spicy, and hardness. The integrated approach applied in this study showed that PDO Provola dei Nebrodi cheese characteristics are quite stable among the dairy factories analyzed and this has to be unavoidably imputed to the application of the same cheese making protocol among different dairies.

Milk metagenomics and cheese-making properties as affected by indoor farming and summer highland grazing.

The study of the complex relationships between milk metagenomics and milk composition and cheese-making efficiency as affected by indoor farming and summer highland grazing was the aim of the present work. The experimental design considered monthly sampling (over 5 mo) of the milk produced by 12 Brown Swiss cows divided into 2 groups: the first remained on a lowland indoor farm from June to October, and the second was moved to highland pastures in July and then returned to the lowland farm in September. The resulting 60 milk samples (2 kg each) were used to analyze milk composition, milk coagulation, curd firming, and syneresis processes, and to make individual model cheeses to measure cheese yields and nutrient recoveries in the cheese. After DNA extraction and Illumina Miseq sequencing, milk microbiota amplicons were also processed by means of an open-source pipeline called Quantitative Insights Into Microbial Ecology (Qiime2, version 2018.2; https://qiime2.org). Out of a total of 44 taxa analyzed, 13 bacterial taxa were considered important for the dairy industry (lactic acid bacteria, LAB, 5 taxa; and spoilage bacteria, 4) and for human (other probiotics, 2) and animal health (pathogenic bacteria, 2). The results revealed the transhumant group of cows transferred to summer highland pastures showed an increase in almost all the LAB taxa, bifidobacteria, and propionibacteria, and a reduction in spoilage taxa. All the metagenomic changes disappeared when the transhumant cows were moved back to the permanent indoor farm. The relationships between 17 microbial traits and 30 compositional and technological milk traits were investigated through analysis of correlation and latent explanatory factor analysis. Eight latent factors were identified, explaining 75.3% of the total variance, 2 of which were mainly based on microbial traits: pro-dairy bacteria (14% of total variance, improving during summer pasturing) and pathogenic bacteria (6.0% of total variance). Some bacterial traits contributed to other compositional-technological latent factors (gelation, udder health, and caseins).

Spatial and Seasonal Structure of Bacterial Communities Within Alpine Vineyards: Trentino as a Case Study.

Bacteria have a fundamental role in determining the fitness of grapevine, the composition of grapes and the features of wines but at present, little information is available. In this work, the bacteria colonizing the different portions of grapevine (bark, leaves and grapes) were explored in the vineyards of the Alpine region of Trentino, considering the impact of different environmental and agronomical variables. The vineyards included in the work were selected based on their different geographical positions (altitude) and grapevine training systems in order to explore the whole variability of the grapevine ecosystem. Moreover, the surface amount of copper was measured on grapes and leaves during the vegetative growth. Bacterial analysis, performed using plate counts and Illumina MiSeq, revealed an increase in the concentration of grape bacteria proportional to the progress of the ripening stage. Conversely, the peak of bacterial concentration onto leaf and bark samples occurred in August, probably due to the more favourable environmental conditions. In bark samples, the bacterial microbiota reached the 7 log CFU/cm2, while 6 log UFC/g were measured in grape samples. A remarkable biodiversity was observed, with 13 phyla, 35 classes, 55 orders, 78 families and 95 genera of bacteria present. The presence of some taxa (Alphaproteobacteria, Desulfovibrionaceae, Clostriadiales, Oscillospira, Lachnospiraceae and Bacteroidales) was ubiquitous in all vineyards, but differences in terms of relative abundance were observed according to the vegetative stage, altitude of the vineyard and training system. Bacteria having oenological implication (Lactobacillus, Pediococcus and Oenococcus) were detected in grape samples collected in August, in low abundance. The data revealed a complex bacterial ecosystem inside the vineyard that, while maintaining common traits, evolves according to environmental and agronomical inputs. This study contributes to define the role of bacteria in the complex balance established in each vineyard between human actions and agricultural environment, known as terroir.

Microbial and metabolic characterization of organic artisanal sauerkraut fermentation and study of gut health-promoting properties of sauerkraut brine.

Sauerkraut is a traditionally fermented cabbage, and recent evidence suggests that it has beneficial properties for human health. In this work, a multi-disciplinary approach was employed to characterize the fermentation process and gut health-promoting properties of locally produced, organic sauerkraut from two distinct producers, SK1 and SK2. 16S rRNA metataxonomics showed that bacterial diversity gradually decreased as fermentation progressed. Differences in sauerkraut microbiota composition were observed between the two producers, especially at the start of fermentation. Lactic acid bacteria (LAB) dominated the microbiota after 35 days, with Lactiplantibacillus being the dominant genus in both sauerkraut products, together with Leuconostoc and Paucilactobacillus in SK1, and with Pediococcus, Levilactibacillus, and Leuconostoc in SK2. LAB reached between 7 and 8 Log CFU/mL brine at the end of fermentation (35 days), while pH lowering happened within the first week of fermentation. A total of 220 LAB strains, corresponding to 133 RAPD-PCR biotypes, were successfully isolated. Lactiplantibacillus plantarum and Lactiplantibacillus pentosus accounted for 67% of all SK1 isolates, and Lactiplantibacillus plantarum/paraplantarum and Leuconostoc mesenteroides represented 72% of all the isolates from SK2. 1H-NMR analysis revealed significant changes in microbial metabolite profiles during the fermentation process, with lactic and acetic acids, as well as amino acids, amines, and uracil, being the dominant metabolites quantified. Sauerkraut brine did not affect trans-epithelial electrical resistance through a Caco-2 cell monolayer as a measure of gut barrier function. However, significant modulation of inflammatory response after LPS stimulation was observed in PBMCs-Caco-2 co-culture. Sauerkraut brine supported a robust inflammatory response to endotoxin, by increasing TNF-α and IL-6 production while also stimulating the anti-inflammatory IL-10, therefore suggesting positive resolution of inflammation after 24 h and supporting the potential of sauerkraut brine to regulate intestinal immune function.

Evaluation of the variations in chemical and microbiological properties of the sourdoughs produced with selected lactic acid bacteria strains during fermentation.

This research aimed to analyze variations in chemical properties, microbiological characteristics and generated volatile organic compounds (VOCs) profile during sourdough fermentation. Sourdoughs were collected from different cities in Turkey at two different times and lactic acid bacteria (LAB) in the samples were identified with culture-independent and culture-dependent molecular methods. According to culture-dependent methodology, thirteen LAB species were identified. Lactobacillus spp. were identified as the major group according to MiSeq Illumina analysis. Technological potential of commonly isolated LAB species was evaluated. Due to high frequency of isolation, Fructilactobacillus sanfranciscensis and Lactiplantibacillus plantarum strains were better investigated for their technological traits useful in sourdough production. Experimental sourdoughs were produced with mono- and dual-culture of the selected strains and chemical properties and microbiological characteristics, as well as VOCs profile of the sourdoughs, were subjected to multivariate analysis which showed the relevance of added starter, in terms of acidification and VOCs profile.

Preliminary Investigation of Biogenic Amines in Type I Sourdoughs Produced at Home and Bakery Level.

During a survey for isolating sourdough lactic acid bacteria (LAB), 20 dough samples produced at the bakery level (BL) or home-made (HM) were collected. An enzyme-based colorimetric method revealed a total biogenic amines (BAs) concentration in the range 41.4-251.8 ppm for six (three BL and three HM) sourdoughs characterised by unpleasant odours. Eight BAs generally investigated in foods were identified and quantified from these six samples by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Only one HM sample contained almost all analysed BAs. Tryptamine was exclusively detected in HM sourdoughs (0.71-24.1 ppm). Putrescine, tryptamine, spermidine, and spermine were the only BAs detected in BL sourdoughs. MiSeq Illumina analysis was applied to study the total bacterial community of sourdoughs. LAB accounted from 67.89 to 92.17% of total bacterial diversity, and Levilactobacillus brevis was identified in all six sourdoughs. Leuconostoc, Pediococcus, and Weissella were also dominant. Plate counts detected neither the presence of Pseudomonas nor members of the Enterobacteriaceae family, and LAB levels were, on average, barely 5.89 Log CFU/g for BL, and 7.33 Log CFU/g for HM sourdoughs. Data suggested that the microorganisms mainly imputable of BAs formation in sourdough are members of the LAB community.

Improvement of Caciotta-like cheese nutritional value by means of enrichment with blackcurrant (Ribes nigrum) and Cornelian cherry (Cornus mas).

Introduction: In this study, we supplemented models of Caciotta-like cheese with blackcurrant (Ribes nigrum) and Cornelian cherry (Cornus mas), as they have a high content of polyphenols, known as phytochemicals associated with health benefits. We evaluated the microbial composition, organoleptic aspects, total phenolic content, and chemical composition of model cheeses enriched with blackcurrant and Cornelian cherry. Methods: Two different suppliers have been tested: a conventional and an organic one. Two different conditions of preparation (freeze-dried and not freeze-dried) were tested in two different amounts (0.3 and 0.6% dry weight w/v milk volume). Polyphenols were determined using Folin-Ciocalteu reaction and spectrometry; microbial community was determined with selective 24 media and plate counts; composition was determined using nuclear magnetic resonance spectrometry. Organoleptic tests with an untrained panel have been performed. Results: The enrichments with blackcurrant and Cornelian cherry increased the total polyphenol content in model cheeses, in particular, when blackcurrant and Cornelian cherry were from conventional farming. Blackcurrant-enriched cheeses showed higher counts of lactic acid bacteria, higher levels of organic acids, amino acids, gamma-aminobutyric acid, histamine, and lower amount of monosaccharides deriving from bacterial lactose fermentation in cheese, suggesting a positive effect of blackcurrant compounds on the growth and activity of lactic acid bacteria. The enrichments did not affect the acceptance of the cheese, neither by blackcurrant nor by Cornelian cherry incorporation, with the exception of the appearance. Discussion: Overall, we showed that cheeses enriched with blackcurrant or Cornelian cherry from conventional farming increased the bioactive potential of the dairy product without having an adverse effect on the microbial community, physiochemical properties, or organoleptic properties.

In-Depth Investigation of the Safety of Wooden Shelves Used for Traditional Cheese Ripening.

The main goal of this research was to characterize the bacterial diversity of the wooden boards used for aging traditional Sicilian cheeses and to evaluate whether pathogenic bacteria are associated with these surfaces. Eighteen cheese dairy factories producing three traditional cheese typologies (PDO Pecorino Siciliano, PDO Piacentinu Ennese, and Caciocavallo Palermitano) were selected within the region of Sicily. The wooden shelf surfaces were sampled by a destructive method to detach wood splinters as well as by a nondestructive brushing to collect microbial cells. Scanning electron microscopy showed the presence of almost continuous bacterial formations on the majority of the shelves analyzed. Yeasts and fungal hyphae were also visualized, indicating the complexity of the plank communities. The amplicon library of the 16S rRNA gene V3-V4 region was paired-end sequenced using the Illumina MiSeq system, allowing the identification of 14 phyla, 32 classes, 52 orders, 93 families, and 137 genera. Staphylococcus equorum was identified from all wooden surfaces, with a maximum abundance of 64.75%. Among cheese-surface-ripening bacteria, Brevibacterium and Corynebacterium were detected in almost all samples. Several halophilic (Halomonas, Tetragenococcus halophilus, Chromohalobacter, Salimicrobium, Marinococcus, Salegentibacter, Haererehalobacter, Marinobacter, and Idiomarinaceae) and moderately halophilic (Salinicoccus, Psychrobacter, and Salinisphaera) bacteria were frequently identified. Lactic acid bacteria (LAB) were present at low percentages in the genera Leuconostoc, Lactococcus, Lactobacillus, Pediococcus, and Streptococcus. The levels of viable microorganisms on the wooden shelves ranged between 2.4 and 7.8 log CFU/cm2. In some cases, LAB were counted at very high levels (8.2 log CFU/cm2). Members of the Enterobacteriaceae family were detected in a viable state for only six samples. Coagulase-positive staphylococci, Salmonella spp., and Listeria monocytogenes were not detected. Seventy-five strains belonged to the genera Leuconostoc, Lactococcus, Pediococcus, Enterococcus, Lactobacillus, and Weissella. IMPORTANCE This study provides evidence for the lack of pathogenic bacteria on the wooden shelves used to ripen internal bacterially ripened semihard and hard cheeses produced in Sicily. These three cheeses are not inoculated on their surfaces, and surface ripening is not considered to occur or, at least, does not occur at the same extent as surface-inoculated smear cheeses. Several bacterial groups identified from the wooden shelves are typically associated with smear cheeses, strongly suggesting that PDO Pecorino Siciliano, PDO Piacentinu Ennese, and Caciocavallo Palermitano cheese rind contributes to their final organoleptic profiles.

Massive Survey on Bacterial-Bacteriophages Biodiversity and Quality of Natural Whey Starter Cultures in Trentingrana Cheese Production.

This study focused on the microbial and bacteriophages identification and characterization in cheese-production facilities that use natural whey starter (NWS) cultures for Trentingrana production. Bacterial and phage screening was carried out on cooked not acidified whey and NWS samples isolated from six dairy factories, for 4 consecutive days in four different months. By means of a combined approach, using plate counts, bacterial isolation, and metataxonomic analysis Lactobacillus helveticus was found occurring as the dominant species in NWS cultures and Levilactobacillus brevis as codominant in the cheese factories where the temperature of NWS production was mainly lower than 40°C, suggesting that the variability in the parameters of the NWS culture preparation could differently modulate the bacterial species in NWS cultures. Using turbidity test approach on 303 bacterial isolates from the NWS cultures, 120 distinct phages were identified. L. helveticus phage contamination of NWS cultures was revealed in most of the analyzed samples, but despite the great recovery of bacteriophage contamination cases, the microbial quality of NWS cultures was high. Our results support the presence of natural bacteriophage resistance mechanisms in L. helveticus. The use of NWS cultures probably creates an ideal environment for the proliferation of different L. helveticus strains balanced with their phages without a clear dominance. It is evident, from this study, that the presence of a high biodiversity of NWS bacterial strains is relevant to avoid phages dominance in NWS cultures and consequently to keep a good acidification ability.

Selenium bio-enrichment of Mediterranean fruit juices through lactic acid fermentation.

This work was carried out to elaborate selenium (Se) bio-enriched fermented Mediterranean fruit juices. To this purpose, pomegranate and table red grape juices were added with sodium selenite (Na2SeO3) and fermented by Levilactobacillus brevis CRL 2051 and Fructobacillus tropaeoli CRL 2034 individually or combined. To better evaluate the effect of selenite addition and starter strain inoculums on the total bacterial community of the fruit juices, fermentation trials were performed with raw and pasteurized fruit juices. No statistical significant differences were observed for total mesophilic microorganisms (TMM) and rod-shaped lactic acid bacteria (LAB) levels among raw and pasteurized juices inoculated with the starter strains, while significant differences between those juices with and without selenite were registered. LAB cocci, Pseudomonadaceae and yeasts were detected only for the raw juice preparations. The dominance of L. brevis CRL 2051 and F. tropaeoli CRL 2034 was confirmed by randomly amplified polymorphic DNA (RAPD)-PCR analysis. After fermentation, pH dropped for all inoculated trials and control raw juices. The soluble solid content (SSC) levels of the raw juices were higher than the corresponding pasteurized trials. The thermal treatment affected consistently yellowness of grape juice trials and redness of pomegranate juices. No microbial Se accumulation was registered for pomegranate juices, while F. tropaeoli CRL 2034 accumulated the highest amount of Se (65.5 µg/L) in the grape juice. For this reason, only trials carried out with raw grape juices were investigated by metagenomics analysis by Illumina MiSeq technology. Non-inoculated grape juices were massively fermented by acetic acid bacteria while Fructobacillus and Lactobacillus (previous genus name of Levilactobacillus) represented the highest operational taxonomy units (OTUs) relative abundance % of the trials inoculated with the starter strains as confirmed by this technique.

Bacterial Complexity of Traditional Mountain Butter Is Affected by the Malga-Farm of Production.

Bacteria can play different roles affecting flavors and food characteristics. Few studies have described the bacterial microbiota of butter. In the present paper, next-generation sequencing was used to determine bacterial diversity, together with aromatic characteristics, in raw cow milk butter processed by traditional fermentation, in fourteen small farms called "Malga", located in the Trentino province (Alpine region, North-East of Italy). The physicochemical and aromatic characterization of traditional mountain butter (TMB) showed a low moisture level depending on the Malga producing the butter. Counts of lactic acid bacteria, Staphylococci, and coliforms, as well as diacetyl/acetoin concentrations exhibited changes according to the geographical origin of Malga and the residual humidity of butter. MiSeq Illumina data analysis revealed that the relative abundance of Lactococcus was higher in TMB samples with the highest values of acetoin (acetoin higher than 10 mg/kg). The traditional mountain butter bacterial community was characterized by a "core dominance" of psychrotrophic genera, mainly Acinetobacter and Pseudomonas, but according to ANCOM analysis, a complex bacterial population emerged and specific bacterial genera were able to characterize the TMB bacteria community, with their high abundance, based on the Malga producing the butter.

Microbial community dynamics in phyto-thermotherapy baths viewed through next generation sequencing and metabolomics approach.

Phyto-thermotherapy is a treatment consisting in immersing oneself in baths of self-heating alpine grass, to benefit of the heat and rich aromatic components released by the process. The aim of this study was to characterize the bacterial and fungal diversity of three phyto-thermal baths (PTB) performed in three different months, and to compare the data with the profile of the volatile organic compounds (VOCs) of the process. All the data collected showed that PTBs were structured in two stages: the first three days were characterised by an exponential rise of the temperature, a fast bacterial development, higher microbial diversity and higher concentrations of plant aliphatic hydrocarbons. The second stage was characterised by a stable high temperature, shrinkage of the microbial diversity with a predominance of few bacterial and fungi species and higher concentrations of volatiles of microbial origin. Erwinia was the dominant microbial species during the first stage and probably responsible of the self-heating process. In conclusion, PTBs has shown both similarities with common self-heating processes and important peculiarities such as the absence of pathogenic bacteria and the dominance of plant terpenoids with health characteristics among the VOCs confirming the evidence of beneficial effects in particular in the first three days.

Addition of selected starter/non-starter lactic acid bacterial inoculums to stabilise PDO Pecorino Siciliano cheese production.

The present study was carried out to produce Protected Denomination of Origin (PDO) Pecorino Siciliano cheese with a multi-species lactic acid bacteria (LAB) culture, composed of starter and non-starter strains in order to reduce the microbiological variability of the products derived without LAB inoculums. To this end, cheese samples produced in six factories located in five provinces (Agrigento, Catania, Enna, Palermo and Trapani) of Sicily, and previously characterised for physicochemical, microbiological and sensory aspects, have been investigated in this work for bacterial microbiome, fatty acid (FA) composition as well as volatile organic compound (VOC) profiles. Analysis of the cheese microbiomes indicated that streptococci (30.62-77.18% relative abundance) and lactobacilli (on average 25.90% relative abundance) dominated the bacterial communities of control cheeses, produced without exogenous inoculums, whereas the cheeses produced with the selected multi-strain culture saw the dominance of lactococci (in the range 6.49-14.92% relative abundance), streptococci and lactobacilli. After the addition of the selected mixed culture, Shannon index increased in all cheeses, but only the cheeses produced with the selected LAB mixed culture in the factory 2 showed Gini-Simpson diversity index (0.79) closer to the reference value (0.94) for a perfect even community. FA composition, mainly represented by saturated FA (on average 69.60% and 69.39% in control cheeses and experimental cheeses, respectively), was not affected by adding LAB culture. The presence of polyunsaturated FA ranged between 7.93 and 8.03% of FA. VOC profiles were different only for the content of butanoic acid, registered for the experimental cheeses at higher concentrations (on average 662.54 mg/kg) than control cheeses (barely 11.96 mg/kg). This study validated addition of the ad hoc starter/non-starter culture for PDO Pecorino cheese production.

Shift in the cow milk microbiota during alpine pasture as analyzed by culture dependent and high-throughput sequencing techniques.

In the present study, two groups of cows from a permanent lowland farm (PF) were divided during summer and reared in the PF or in a temporary alpine farm (ALP), respectively. Microbiological analyses were performed with the objective to investigate the microbial evolution of milk before, during, and after summer transhumance comparing, in particular, the two groups of cows to determine whether the alpine pasture could directly influence the milk microbiota. A significant increase of all microbial groups was registered in milk samples collected in the ALP. Interestingly, many strains belonging to species with well reported technological and probiotic activities were isolated from Alpine milk (20% Lactococcus lactis subsp. lactis/cremoris, 18% Lactobacillus paracasei, 14% Bifidobacterium crudilactis and 18% Propionibacterium sp.), whereas only 16% of strains isolated from the permanent farm milk belonged to the species Lactococcus lactis subsp. lactis/cremoris, 6% to Lactobacillus paracasei, 2% to Bifidobacterium crudilactis and 5% to Propionibacterium sp. The MiSeq Illumina data showed that Alpine milk presented a significant reduction of Pseudomonas and an increase of Lactococcus, Bifidobacterium and Lactobacillus genera. These data confirmed the practice of Alpine pasture as one of the main drivers affecting the milk microbiota. All the microbial changes disappeared when cows were delivered back from Alpine pasture to the indoor farm.

Molecular analysis of the dominant lactic acid bacteria of chickpea liquid starters and doughs and propagation of chickpea sourdoughs with selected Weissella confusa.

Fermented chickpea liquid is used as a leavening agent in chickpea bread production. In the present study, traditional chickpea liquid starter and dough samples were collected from bakeries in Turkey and microbiologically investigated. Culture-independent analysis for microbiota diversity, performed by MiSeq Illumina, identified Clostridium perfringens as major group in all samples, while Weissella spp. Dominated LAB community. A culture-dependent methodology was applied and 141 isolates were confirmed to be members of the LAB group based on 16s rRNA gene sequence analysis. In particular, 11 different LAB species were identified confirming the high frequency of isolation of weissellas, since Weissella confusa and Weissella cibaria constituted 47.8 and 12.4%, respectively, of total LAB isolated. The other species were Enterococcus faecium, Enterococcus lactis, Lactobacillus brevis, Lactobacillus plantarum, Leuconostoc mesenteroides, Leuconostoc mesenteroides subsp. Dextranium, Pediococcus acidilactici, Pediococcus pentosaceus and Streptococcus lutetiensis. Due to high frequency of isolation, W. confusa strains were investigated at technological level and W. confusa RL1139 was used as mono-culture starter in the experimental chickpea sourdough production. Chemical and microbiological properties, as well as volatile organic compounds (VOCs) of the chickpea liquid starters and doughs were subjected to a multivariate analysis. Control and W. confusa inoculated chickpea liquid starter and dough samples were close to each other in terms of some characteristics related to chemical, microbiological and VOCs profile, but the inoculated sourdough showed a higher generation of certain VOCs, like butanoic acid (81.52%) and ethyl acetate (8.15%) than control sourdough. This is important in order to maintain typical characteristics of the traditional chickpea dough, but at the same time improving the aroma profile. This work demonstrated that W. confusa RL1139 can be applied at large scale production level without compromising the typical characteristics of the final product.

Microbial dynamics in durum wheat kernels during aging.

In the present work the microbial dynamics in wheat kernels were evaluated over time. The main aim of this research was to study the resistance of lactic acid bacteria (LAB) and yeasts associated to unprocessed cereals used for bread making during long term conservation. To this purpose four Triticum durum Desf. genotypes including two modern varieties (Claudio and Simeto) and two Sicilian wheat landraces (Russello and Timilia) were analysed by a combined culture-independent and -dependent microbiological approach after one, two or three years from cultivation and threshing. DNA based MiSeq Illumina technology was applied to reveal the entire bacterial composition of all semolina samples. The samples showed a different distribution of bacterial taxa per variety and time of storage. The groups mostly represented were Stenotrophomonas, Pseudomonas, Erwinia, Delftia and Sphingomonas genera, Enterobacteriaceae and Oxalobacteriaceae families, and Actinobacteria phylum. Among LAB, only Enterococcus genus was detected barely in a single sample (Simeto stored for one year) by the next generation sequencing, indicating that LAB remained unassigned or their abundances were below 0.1% or their DNAs were rendered inaccessible. Plate counts showed consistent differences in relation to genotypes and duration of storage, with the highest levels found for total mesophilic microorganisms detected up 6.8 Log CFU/g. Colonies of presumptive sourdough LAB were detected only in a few samples. Cocci constituted the major group of LAB in almost all samples. Following the enrichment procedure, almost all samples were characterised by the presence of acidifying microorganisms. All isolates collected before and after enrichment represented 28 different strains belonging to 10 species of Enterococcus, Lactobacillus and Pediococcus genera. The most resistant species during aging were Enterococcus faecium, Enterococcus durans, Lactobacillus brevis, Lactobacillus pentosus and Lactobacillus paracasei demonstrating that lactobacilli and enterococci are able to overcome the stressing conditions represented by cereal storage better than other LAB genera commonly found associated to cereals after harvest. Yeast community included mostly species with no interest in bread making.

Evolution of indigenous starter microorganisms and physicochemical parameters in spontaneously fermented beef, horse, wild boar and pork salamis produced under controlled conditions.

The present work was carried out to evaluate the microbiological and physicochemical composition of salamis produced with the meat of beef, horse, wild boar and pork. Salami productions occurred under controlled laboratory conditions to exclude butchery environmental contaminations, without the addition of nitrate and nitrite. All trials were monitored during the ripening (13 °C and 90% relative humidity) extended until 45 d. The evolution of physicochemical parameters showed that beef and pork salamis were characterized by a higher content of branched chain fatty acids (FA) and rumenic acid than horse and wild boar salamis, whereas the last two productions showed higher values of secondary lipid oxidation. Plate counts showed that lactic acid bacteria (LAB), yeasts and coagulase-negative staphylococci (CNS) populations dominated the microbial community of all productions with Lactobacillus and Staphylococcus as most frequently isolated bacteria. The microbial diversity evaluated by MiSeq Illumina showed the presence of members of Gammaproteobacteria phylum, Moraxellaceae family, Acinetobacter, Pseudomonas, Carnobacterium and Enterococcus in all salamis. This study showed the natural evolution of indigenous fermented meat starter cultures and confirmed a higher suitability of horse and beef meat for nitrate/nitrite free salami production due to their hygienic quality at 30 d.