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

Alpine grazing management, breed and diet effects on coagulation properties, composition, and microbiota of dairy cow milk by commercial mountain based herds

Cow milk microbiota has received increased attention in recent years, not only because of its importance for human health but also because of its effect on the quality and technological properties of milk. Several studies, therefore, have investigated the effect of various production factors on the microbial composition of milk. However, most of the previous studies considered a limited number of animals from experimental or single farm, which could have biased the results. Therefore, this study aimed to understand the effect of different alpine production systems on the compositional and microbiological quality of milk, considering commercial herds with different feeding intensities and cattle breeds. The results obtained in this work indicated that the month/season of sampling (July for summer or February for winter) more than farm, breed and cow diet exerted significant effects on cow milk parameters and microbiota. In particular, significant differences were observed for urea content in milk between sampling seasons. Differences in milk fat were mainly related to breed specific effects. From a microbiological point of view, statistically significant differences were found in presumptive lactic acid bacteria counts. Based on a culture-independent method, milk obtained in February harbored the highest number of Firmicutes (e.g., Lactobacillus) and the lowest number of Actinobacteria (e.g., Corynebacterium). Moreover, bacterial richness and diversity were higher in July/summer during alpine pasture season indicating a significant effect of pasture feed on the growth of bacterial communities. The results of this study highlighted the effect of month/season mainly related to differences in feeding management (e.g., access to pasture during vegetation period, concentrates supplementation) on composition and microbiota in milk

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 propertie

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

7openInternationalBothThis 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.openBoyaci Gunduz, Cennet Pelin; Agirman, Bilal; Gaglio, Raimondo; Franciosi, Elena; Francesca, Nicola; Settanni, Luca; Erten, HuseyinBoyaci Gunduz, C.P.; Agirman, B.; Gaglio, R.; Franciosi, E.; Francesca, N.; Settanni, L.; Erten, H

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)

In-depth investigation of the safety of wooden shelves used for traditional cheese ripening

9openInternationalItalian coauthor/editorThe 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.openSettanni, Luca; Busetta, Gabriele; Puccio, Valeria; Licitra, Giuseppe; Franciosi, Elena; Botta, Luigi; Di Gerlando, Rosalia; Todaro, Massimo; Gaglio, RaimondoSettanni, L.; Busetta, G.; Puccio, V.; Licitra, G.; Franciosi, E.; Botta, L.; Di Gerlando, R.; Todaro, M.; Gaglio, R

Massive survey on bacterial–bacteriophages biodiversity and quality of natural whey starter cultures in Trentingrana cheese production

9openInternationalItalian coauthor/editorThis 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.openMancini, A.; Cid Rodriguez, M.; Zago, M.; Cologna, M.; Goss, A.; Carafa, I.; Tuohy, K.; Merz, A.; Franciosi, E.Mancini, A.; Cid Rodriguez, M.; Zago, M.; Cologna, M.; Goss, A.; Carafa, I.; Tuohy, K.; Merz, A.; Franciosi, E

Preliminary investigation of biogenic amines in type I sourdoughs produced at home and bakery level

10openInternationalItalian coauthor/editorDuring 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 communityopenMannino, Giuseppe; Cirlincione, Fortunato; Gaglio, Raimondo; Franciosi, Elena; Francesca, Nicola; Moschetti, Giancarlo; Asteggiano, Alberto; Medana, Claudio; Gentile, Carla; Settanni, LucaMannino, G.; Cirlincione, F.; Gaglio, R.; Franciosi, E.; Francesca, N.; Moschetti, G.; Asteggiano, A.; Medana, C.; Gentile, C.; Settanni, L

The wooden shelf surface and cheese rind mutually exchange microbiota during the traditional ripening process

The rind acts as a protective barrier for internally-bacterial ripened cheeses. Unlike surface-inoculated smear cheeses, centripetal maturation is not assumed to occur in these cheeses. This research was aimed to evaluate the microbial diversity of the wooden shelves used for the ripening of Protected Denomination of Origin (PDO) Pecorino di Filiano and Protected Geographical Indication (PGI) Canestrato di Moliterno cheeses. The microorganisms associated with the rind of these cheeses were also investigated. Both wooden shelf surfaces and cheese rinds were sampled by brushing method to collect their biofilms. Wooden shelves showed levels of total mesophilic microorganisms (TMM) between 5.6 and 7.2 log CFU/cm2, while cheese rinds between 6.1 and 7.8 log CFU/cm2. The major dairy pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus) were never detected, while mesophilic and thermophilic bacteria dominated the surfaces of all wooden shelves and cheese rinds. LAB community was represented by Enterococcus spp., Leuconostoc spp., and Marinilactibacillus spp. Among yeasts, Debaryomyces spp., Candida spp., were identified, while Aspergillus spp., and Penicillium spp., dominated the community of filamentous fungi. MiSeq Illumina analysis identified 15 phyla, 13 classes, 28 orders, 54 families, and 56 genera among bacteria. Staphylococcus spp. was identified from all wooden surfaces, with a maximum abundance of 71 %. Brevibacterium, Corynebacterium and halophilic bacteria were detected in almost all samples. Regarding fungi, wooden shelves mainly hosted Aspergillus, Penicillium and Debaryomyces hansenii, while cheese rinds especially Penicillium and D. hansenii. Alpha diversity confirmed a strict correlation between the microbiota of wooden shelves and that of cheese rinds for the majority of factories. This study confirmed that the wooden shelves used for cheese ripening are microbiologically active and represent safe systems. Furthermore, the results of this work clarified the transfer flow between wooden shelves and PDO Pecorino di Filiano and PGI Canestrato di Moliterno cheese surfaces: smear-active microorganisms are mainly transferred from wooden shelves to cheese rind, which potentially contribute to the development of the final organoleptic characteristics; meanwhile, cheeses transfer LAB that are potentially involved in defining the safety aspects of the shelve