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The food industry constantly seeks new starter cultures with superior characteristics to enhance the sensory and overall quality of final products. Starting from a collection of Algerian dairy (goat and camel) lactic acid bacteria, this work focused on the exploration of the technological and probiotic potential of Weissella cibaria (VR81 and LVT1) and Lactiplantibacillus plantarum R12 strains isolated from raw camel milk and fermented milk, respectively. These bioactive strains were selected for their high performance among ten other LAB strains and were used as starter cultures to develop a novel and nutritionally enhanced dairy-like plant-based yogurt using quinoa (Chenopodium quinoa Willd) as a raw matrix. The strains were evaluated for their antagonistic effects against Listeria innocua, Listeria ivanovii, Staphylococcus aureus, Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, resilience to acidic and osmotic challenges, and tolerance to gastrointestinal mimicking conditions (i.e., pepsin and bile salt). Their aggregation and adhesion profiles were also analyzed. Furthermore, L. plantarum and W. cibaria were tested in single and co-culture for the fermentation and biocontrol of quinoa. The strains exhibited probiotic properties, including a high potential for biocontrol applications, specifically against L. innocua and P. aeruginosa (20 mm diameter zone with the neutralized cell-free supernatant), which disappeared after protease treatment, suggesting that bioactive peptides might be responsible for the observed antimicrobial effect. Additionally, they demonstrated resilience to acidic (pH 2) and osmotic challenges (1M sucrose), tolerance to gastro-intestinal conditions, as well as good aggregation and adhesion profile. Furthermore, the strains were able to produce metabolites of interest, such as exopolysaccharide (yielding up to 4.7 mg/mL) and riboflavin, reaching considerable production levels of 2.5 mg/L upon roseoflavin selection. The application of W. cibaria and L. plantarum as primary starters (both in single and co-culture) for fermenting quinoa resulted in effective acidification of the matrix (ΔpH of 2.03 units) and high-quality beverage production. in vivo challenge tests against L. innocua showed the complete inhibition of this pathogen when L. plantarum was included in the starter, either alone or in combination with W. cibaria. Both species also inhibited Staphylococcus and filamentous fungi. Moreover, the co-culture of mutant strains of L. plantarum R12d and W. cibaria VR81d produced riboflavin levels of 175.41 µg/100 g in fermented quinoa, underscoring their potential as starters for the fermentation, biopreservation, and biofortification of quinoa while also displaying promising probiotic characteristics.

The ongoing occurrence of foodborne diseases and the imperative need for efficient spoilage and pathogen control in food products constitute a critical challenge for the food industry. The rising demands of consumers for safe, healthy, and clean-label food products have led to an increased interest in natural antimicrobial alternatives. Lactic acid bacteria (LAB) have proven their value in the food industry in recent years, also in reason of their antagonistic properties against undesired microbes and their significant related protechnological attributes. The natural antimicrobial compounds produced by LAB exhibit inhibitory effects on pathogens and effectively inhibit the activities of food spoilage-related organisms. Applying secondary metabolites of LAB, notably bacteriocins, organic acids, and others, has found commercial utility across multiple food sectors, effectively preventing the proliferation of undesirable microorganisms and simultaneously enhancing the sensory properties and overall quality of various food products. This review comprehensively explores the natural microbial compounds produced by LAB, specifically focusing on their antimicrobial action in supporting effective and sustainable microbial management. Additionally, it highlights their strategic application across various technological contexts within the food industry.

The use of lactic acid bacteria (LAB) for the probiotic enrichment of minimally processed fruit is a well-established practice in the literature. In addition, several LAB demonstrated a strain-specific ability to control harmful microorganisms and decay agents, improving shelf life, maintaining quality, and promoting the safety of fruits and vegetables. Edible coatings can help modulate the phenomena of gas exchange and water loss by fruits, representing protection from physical damage and spoilage phenomena linked to oxidation and the development of undesired microorganisms. At the same time, the coating can represent an innovative delivery matrix for the LAB strains of potential interest to improve safety and quality in the postharvest management of fruits. In this work, five Lactiplantibacillus plantarum strains, previously characterised for their probiotic and antifungal activity, were incorporated into a sodium alginate coating to develop edible probiotic coatings with antifungal properties for table grapes cv. Italia. The bacterial transfer and their survival were evaluated by comparing coated and uncoated table grapes during 14 days of cold storage at 4 °C. The alginate edible coating increased the number of viable cells transferred to the surface of the berries from about 5 to more than 7 Log CFU/g, with a crucial impact on the potential functional attributes of the final product. The ability of the functionalised coatings to counteract the decay development was evaluated on table grape berries artificially contaminated with Aspergillus niger CECT 2805. A significant reduction in lesion diameter was observed in the alginate coating with L. plantarum 11-A, with a reduction from 15.40 ± 1.14 mm of uncoated berries to 8.40 ± 1.14 mm of berries coated with L. plantarum 11-A. The lesion diameter reduction was also accompanied by a reduction in the symptoms of infection, such as browning around the wound. These results suggest the application of selected strains of L. plantarum as promising bio-resources to enhance the overall value of ready-to-eat fruits and vegetables, particularly in combination with edible coating as a carrier matrix. While a strain-dependent effect was not detected with respect to the improvement in the number of cells in the edible coating, a variability depending on the biotype used was detected for the properties linked to biocontrol, suggesting that the inclusion in edible packaging may represent an innovative criterion in the selection of lactobacilli to be applied postharvest.

Gluten consumption causes several immunological and non-immunological intolerances in susceptible individuals. In this study, the dextran-producing Weissella cibaria BAL3C-5 and its derivative, the riboflavin-overproducing strain BAL3C-5 C120T, together with a commercial bakery yeast, were used to ferment gluten-free (GF)-doughs obtained from corn and rice flours at two different concentrations and supplemented with either quinoa, buckwheat, or chickpea to obtain laboratory-scale GF bread. The levels of dextran, riboflavin, and total flavins were determined in the fermented and breads. Both strains grew in fermented doughs and contributed dextran, especially to those made with corn plus quinoa (~1 g/100 g). The highest riboflavin (350–150 µg/100 g) and total flavin (2.3–1.75 mg/100 g) levels were observed with BAL3C-5 C120T, though some differences were detected between the various doughs or breads, suggesting an impact of the type of flour used. The safety assessment confirmed the lack of pathogenic factors in the bacterial strains, such as hemolysin and gelatinase activity, as well as the genetic determinants for biogenic amine production. Some intrinsic resistance to antibiotics, including vancomycin and kanamycin, was found. These results indicated the microbiological safety of both W. cibaria strains and indicated their potential application in baking to produce GF bread.

Summary Manufacturing of probiotics and functional foods using lactic acid bacteria (LAB) that overproduce vitamin B2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin‐producing parental strain (UFG9) and a roseoflavin‐resistant strain (B2) that carries a mutation in the FMN‐aptamer of the potential rib operon riboswitch, were analysed for production and intra‐ and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for LAB, exceeding by ~ 25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~ 8‐fold higher in B2. Mutation B2 could decrease the stability of the aptamer’s regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2‐mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavin, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods.

The continued exploration of the diversity of lactic acid bacteria in little-studied ecological niches represents a fundamental activity to understand the diffusion and biotechnological significance of this heterogeneous class of prokaryotes. In this study, Lactiplantibacillus plantarum (Lpb. plantarum) strains were isolated from Tunisian vegetable sources, including fermented olive and fermented pepper, and from dead locust intestines, which were subsequently evaluated for their antimicrobial activity against foodborne pathogenic bacteria, including Escherichia coli O157:H7 CECT 4267 and Listeria monocytogenes CECT 4031, as well as against some fungi, including Penicillium expansum, Aspergilus niger, and Botrytis cinerea. In addition, their resistance to oro-gastro-intestinal transit, aggregation capabilities, biofilm production capacity, adhesion to human enterocyte-like cells, and cytotoxicity to colorectal adenocarcinoma cell line were determined. Further, adhesion to tomatoes and the biocontrol potential of this model food matrix were analyzed. It was found that all the strains were able to inhibit the indicator growth, mostly through organic acid production. Furthermore, these strains showed promising probiotic traits, including in vitro tolerance to oro-gastrointestinal conditions, and adhesion to abiotic surfaces and Caco-2 cells. Moreover, all tested Lpb. plantarum strains were able to adhere to tomatoes with similar rates (4.0–6.0 LogCFU/g tomato). The co-culture of LAB strains with pathogens on tomatoes showed that Lpb. plantarum could be a good candidate to control pathogen growth. Nonetheless, further studies are needed to guarantee their use as probiotic strains for biocontrol on food matrices.

Table grapes are characterized by high susceptibility to mold development during post-harvest, mostly due to Botrytis cinerea. Microwave application on ready-to-eat product can represent an alternative to antifungal treatment. With the aim of identifying the maximum energy that can be applied on grape without detrimental effects a Central Composite Design was developed testing the application of 10 combinations of treatment time (seconds) and microwave power (Watt). As a result, energies above 8000 kJ negatively affected the sensorial quality of fresh product, both in the presence and absence of B. cinerea inoculum (106 log CFU g-1). The physico-chemical parameters did not show significant differences, but two time/power combinations improved sensory quality of table grape, being selected for the subsequent packaging trial: 14 s/100 W and 80 s/100 W. Treatments were applied before or after packaging in polypropylene bags. At the end of storage period, 100 W applied for 80 seconds before packaging led to a better external appearance of the product than the other treatments, maintaining an intermediate level of mesophilic bacterial load and no significant differences in terms of nutritional quality. 80 seconds at 100 Watt combined with packaging can represent a valuable starting point for further experiments.

This work describes a method for deriving riboflavin overproducing strains of Weissella cibaria by exposing three strains (BAL3C-5, BAL3C-7, and BAL3C-22) isolated from dough to increasing concentrations of roseoflavin. By this procedure, we selected one mutant overproducing strain from each parental strain (BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2, respectively). Quantification of dextran and riboflavin produced by the parental and mutant strains in a defined medium lacking riboflavin and polysaccharides confirmed that riboflavin was only overproduced by the mutant strains, whereas dextran production was similar in both mutant and parental strains. The molecular basis of the riboflavin overproduction by the mutants was determined by nucleotide sequencing of their rib operons, which encode the enzymes of the riboflavin biosynthetic pathway. We detected a unique mutation in each of the overproducing strains. These mutations, which map in the sensor domain (aptamer) of a regulatory element (the so-called FMN riboswitch) present in the 5’ untranslated region of the rib operon mRNA, appear to be responsible for the riboflavin-overproducing phenotype of the BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2 mutant strains. Furthermore, the molecular basis of dextran production by the six W. cibaria strains has been characterized by (i) the sequencing of their dsr genes encoding dextransucrases, which synthesize dextran using sucrose as substrate, and (ii) the detection of active Dsr proteins by zymograms. Finally, the parental and mutant strains were analyzed for in situ production of riboflavin and dextran during experimental bread making. The results indicate that the mutant strains were able to produce experimental wheat breads biofortified with both riboflavin and dextran and, therefore, may be useful for the manufacture of functional commercial breads.

Since millennia, humankind has exploited microbial diversity associated to give food matrices in order to obtain fermented foods and beverages, resulting in products with improved quality and extended shelf life. This topic has received deserved and continuous interest in the scientific community, for the reason of its significance as a driver of innovation in the food and beverage sector. In this review paper, using sourdough as a model matrix, we provide some insights into the field, testifying the relevance as a transdisciplinary subject. Firstly, we encompassed the prokaryotic and eukaryotic microbial diversity associated with the sourdough ecosystems. The importance of this micro-biodiversity in the light of flour-related chemical diversity was examined. Finally, we highlighted the increasing interest in microbial-based applications oriented toward biocontrol solution in the field of sourdough-based products (i.e., bread).

This paper reports on a common experiment performed by 17 Research Units of the Italian Group of Microbiology of Vine and Wine (GMVV), which belongs to the Scientific Society SIMTREA, with the aim to validate a protocol for the characterization of wine strains of Saccharomyces cerevisiae. For this purpose, two commercial S. cerevisiae strains (EC 1118 and AWRI796) were used to carry out inter-laboratory-scale comparative fermentations using both synthetic medium and grape musts and applying the same protocol to obtain reproducible, replicable, and statistically valid results. Ethanol yield, production of acetic acid, glycerol, higher alcohols, and other volatile compounds were assessed. Moreover, the Fourier transform infrared spectroscopy was also applied to define the metabolomic fingerprint of yeast cells from each experimental trial. Data were standardized as unit of compounds or yield per gram of sugar (glucose and fructose) consumed throughout fermentation, and analyzed through parametric and non-parametric tests, and multivariate approaches (cluster analysis, two-way joining, and principal component analysis). The results of experiments carried out by using synthetic must showed that it was possible to gain comparable results from three different laboratories by using the same strains. Then, the use of the standardized protocol on different grape musts allowed pointing out the goodness and the reproducibility of the method; it showed the main traits of the two yeast strains and allowed reducing variability amongst independent batches (biological replicates) to acceptable levels. In conclusion, the findings of this collaborative study contributed to the validation of a protocol in a specific synthetic medium and in grape must and showed how data should be treated to gain reproducible and robust results, which could allow direct comparison of the experimental data obtained during the characterization of wine yeasts carried out by different research laboratories.

The topic of microbial interactions is of notable relevance in oenology, being connected with their impact on microbial biodiversity and wine quality. The interactions among different couples of microorganisms, in particular yeasts and lactic acid bacteria representative of the must/wine microbial consortium, have been tested in this study. This interaction’s screening has been implemented by means of plate assays, using culture medium, grape juice, and wine agar as substrates. Different antagonistic phenomena have been detected, belonging to the following interaction categories: yeast-yeast, yeast-bacteria, bacteria-yeast, and bacteria-bacteria. In general, the inhibitory activity has been observed in all three media agar used as substrates, resulting in more frequent on culture medium, followed by grape juice and, finally, wine. Specifically, the work is one of the first reports demonstrating the reciprocal interactions between non-Saccharomyces yeasts (NSY) and malolactic bacteria. The findings shed new light on the co-inoculation of the yeast starter culture with malolactic bacteria, as well as the biocontrol potential of Lactic Acid Bacteria (LAB) strains. Highlighted microbial interactions are relevant for the management of alcoholic fermentation, malolactic fermentation, and the development of distinctive aroma profiles, control of spoilage yeasts, and the selection of tailored mixed starter cultures. In addition, the plate assay method could be a fast, cheap, and suitable method to exclude negative interactions among Saccharomyces spp., NSY, and malolactic bacteria during trials from regional spontaneous fermentations with the aim to select tailored mixed starter cultures.

Consumers’ increasing interest in sparkling wine has enhanced the global market’s demand. The pro-technological yeasts strains selected for the formulation of microbial starter cultures are a fundamental parameter for exalting the quality and safety of the final product. Nowadays, the management of the employed microbial resource is highly requested by stakeholders, because of the increasing economic importance of this oenological sector. Here, we report an overview of the production processes of sparkling wine and the main characterisation criteria to select Saccharomyces and non-Saccharomyces strains appropriate for the preparation of commercial starter cultures dedicated to the primary and, in particular, the secondary fermentation of sparkling wines. We also focused on the possible uses of selected indigenous strains to improve the unique traits of sparkling wines from particular productive areas. In summary, the sparkling wine industry will get an important advantage from the management of autochthonous microbial resources associated with vineyard/wine microbial diversity.

Usually, the search for new candidate probiotics starts from strain isolation, followed by genotypic and phenotypic characterisations. For the best candidates, the final selection criteria, i.e., an efficient biomass production and the survival to stressful conservation processes, may often represent a bottleneck. The aim of this study is to reverse this classic bottom-up approach, thereby evaluating the in vitro probiotic properties of microbes that are already commercialized and employed in the dairy sector. The major advantage of reversing the traditional scheme is to deal with strains that are already suitable for the scale-up at the industrial level. In this work, four lactobacilli strains were analysed, belonging to the species of Lactiplantibacillus plantarum (strains PLA and PLA2) and Lacticaseibacillus rhamnosus (strains PAR4 and RHM). Both L. plantarum strains showed the best survival under simulated oro-gastrointestinal stress; PLA and PAR4 had the strongest inhibitory activity against all the tested harmful bacteria, with the latter strain showing also the highest percentage of Caco-2 adhesion; RHM was the best biofilm producer on abiotic surface. Finally, cell-free surnatants from all the strain cultures exhibited anti-inflammatory action on THP-1 macrophages. For all the studied strains, it is possible to claim beneficial functional properties other than the technological ones for which they are already marketed. The possible use of the four strains in a mixture could represent a strategy to diversify and maximize their beneficial potential. Nonetheless, future studies are necessary to validate in vivo the observed beneficial properties and to evaluate any effect of the vehicle product on the probiotic aptitude.

Listeria monocytogenes poses a major risk for the safety of food products due to the ability to persist in food products and process line surfaces as biofilm. In this work, we investigated the L. monocytogenes biofilms in relation to development factors and possible control under different conditions. In particular, the ability of six strains of L. monocytogenes from vegetable and animal sources to form biofilms was evaluated on glass or polystyrene substrates under different temperatures (15, 30 and 37°C) and availability of nutrients, by using rich (BHI) or poor (HTM) growth media. Moreover, the effectiveness of three commonly used sanitizers (benzalkonium chloride, sodium hypochlorite and hydrogen peroxide) was compared to eradicate established biofilms. Our results showed that starved conditions, hydrophilic surfaces, and high temperatures increased the L. monocytogenes ability to produce biofilms. In general, benzalkonium chloride was the most effective chemical to remove established biofilms.

Fermented dairy products are the good source of different species of live lactic acid bacteria (LAB), which are beneficial microbes well characterized for their health-promoting potential. Traditionally, dietary intake of fermented dairy foods has been related to different health-promoting benefits including antimicrobial activity and modulation of the immune system, among others. In recent years, emerging evidence suggests a contribution of dairy LAB in the prophylaxis and therapy of non-communicable diseases. Live bacterial cells or their metabolites can directly impact physiological responses and/or act as signalling molecules mediating more complex communications. This review provides up-to-date knowledge on the interactions between LAB isolated from dairy products (dairy LAB) and human health by discussing the concept of the food–gut-health axis. In particular, some bioactivities and probiotic potentials of dairy LAB have been provided on their involvement in the gut–brain axis and non-communicable diseases mainly focusing on their potential in the treatment of obesity, cardiovascular diseases, diabetes mellitus, inflammatory bowel diseases, and cancer.

Selected biological resources used as raw materials in beer production are important drivers of innovation and segmentation in the dynamic market of craft beers. Among these resources, local/regional ingredients have several benefits, such as strengthening the connection with territories, enhancing the added value of the final products, and reducing supply costs and environmental impacts. It is assumed that specific ingredients provide differences in flavours, aromas, and, more generally, sensory attributes of the final products. In particular, of interest are ingredients with features attributable and/or linked to a specific geographical origin. This review encompasses the potential contribution and exploitation of biodiversity in the main classes of beer inputs, such as cereals, hops, microbes, and adjuncts, with a specific emphasis on autochthonous biological resources, detailing the innovative paths already explored and documented in the scientific literature. This dissertation proposes an overview of the impact on beer quality for each raw material category, highlighting the benefits and limitations that influence its concrete applications and scale-up, from the field to the stain. The topics explored promote, in the sector of craft beers, trends already capitalised in the production of other alcoholic beverages, such as the preservation and revalorisation of minor and autochthonous varieties, the exploitation of yeast and bacteria strains isolated from specific sites/plant varieties, and the valorisation of the effects of peculiar terroirs on the quality of agricultural products. Finally, the examined tendencies contribute toward reducing the environmental impacts of craft beer manufacturing, and are in line with sustainable development of food systems, increasing the economic driver of biodiversity preservation.

Some strains of lactic acid bacteria (LAB) produce riboflavin, a water-soluble vitamin of the B complex, essential for human beings. Here, we have evaluated riboflavin (B2 vitamin) production by five Lactobacillus plantarum strains isolated from chicha, a traditional maize-based fermented alcoholic beverage from north-western Argentina and their isogenic riboflavin-overproducing derivatives previously selected using roseoflavin. A direct fluorescence spectroscopic detection method to quantify riboflavin production in bacterial culture supernatants has been tested. Comparison of the efficiency for riboflavin fluorescence quantification with and without prior HPLC fractionation showed that the developed method is a rapid and easy test for selection of B2 vitamin-producing strains. In addition, it can be used for quantitative detection of the vitamin production in real time during bacterial growth. On the basis of this and previous analyses, the L. plantarum M5MA1-B2 riboflavin overproducer was selected for in vitro and in vivo studies after being fluorescently labeled by transfer of the pRCR12 plasmid, which encodes the mCherry protein. The labeling did not affect negatively the growth, the riboflavin production nor the adhesion of the strain to Caco-2 cells. Thus, L. plantarum M5MA1-B2[pRCR12] was evaluated for its survival under digestive tract stresses in the presence of microbiota in the dynamic multistage BFBL gut model and in a murine model. After exposure to both models, M5MA1-B2[pRCR12] could be recovered and detected by the pink color of the colonies. The results indicated a satisfactory resistance of the strain to gastric and intestinal stress conditions but a low colonization capability observed both in vitro and in vivo. Overall, L. plantarum M5MA1-B2 could be proposed as a probiotic strain for the development of functional foods.

Small heat shock proteins (sHSPs) are ubiquitous, low molecular weight (MW) proteins that share a conserved alpha-crystallin domain. sHSPs oligomers exhibit chaperon-like activities by interacting with unfolded substrates, thereby preventing their aggregation and precipitation. Unlike most lactobacilli, which have single shsp genes, three different sHSP-encoding genes, i.e., hsp1, hsp2, and hsp3, were previously identified in the probiotic Lactobacillus plantarum WCFS1. Early studies, including the characterization of the knock out (KO) mutant for hsp2, indicated a different organization and transcriptional regulation of these genes and suggested that the three L. plantarum sHSPs might accomplish different tasks in stress response. To unravel the role of sHSPs, KO mutants of hsp1 and hsp3 were generated using a Cre-lox based system. Mutation of either genes resulted in impaired growth capacity under normal conditions, heat-stress and stresses typically found during host interactions and food technological process. However, survival to heat shock and the level of thermal stabilization of cytoplasmic proteins were similar between mutants and parental strain. Transcriptional analysis revealed that in the mutant genetic backgrounds there is an upregulated basal expression of the un-mutated mate hsps and other stress-related genes, which may compensate for the loss of HSP function, hence possibly accounting for the lack of a remarkable susceptibility to heat challenge. HSP3 seemed relevant for the induction of thermotolerance, while HSP1 was required for improved cryotolerance. Cell surface properties and plasma membrane fluidity were investigated to ascertain the possible membrane association of sHSP. Intriguingly, the loss of hsp1 was associated to a lower level of maximal membrane fluidity upon heat stress. A role for HSP1 in controlling and improving membrane fluidity is suggested which may pertains its cryoprotective function.

Botrytis cinerea, responsible for grey mold, represents the first biological cause of fruit and vegetable spoilage phenomena in post-harvest. Kiwifruit is a climacteric fruit particularly prone to this mold infestation during storage. Lactic acid bacteria (LAB) are food-grade bacteria that can synthesize several metabolites with antimicrobial activity and are, therefore, suggested as promising and eco-friendly resources for the bio-control of molds on fruits and vegetables. In this work, we propose the screening of a collection of 300 LAB previously isolated from traditional sourdoughs for their ability to counteract in vitro the growth of Botrytis cinerea CECT 20973. Only 2% of tested LAB strains belonging to Lactiplantibacillus plantarum species, exerted a strong antagonism against B. cinerea. The cell-free supernatants were partially characterized and results clearly indicated that high levels of lactic acid contributed to the antagonistic activity. PAN01 and UFG 121 cell-free supernatants were investigated as potential bio-control agents in a preliminary in vivo assay using freshly cut kiwifruits as a food model. The application of cell-free supernatants allowed to delay the growth of B. cinerea on artificially contaminated kiwifruits until two weeks. The antagonistic activity was greatly affected by the storage temperature (25 °C and 4 °C) selected for the processed fruits, suggesting the importance to include microbial-based solution in a broader framework of hurdle technologies.

The influence of oat and barley β-glucans on some features of diverse probiotic strains was investigated. Neither barley nor oat β-glucans supported the growth of any of the investigated strains when used as the sole carbon source in fermentation media. No protective effect by β-glucans was observed on bacterial survival to in vitro simulation of the human digestive tract. Moreover, the presence of β-glucans did not enhance the ability of probiotic bacteria to adhere on human intestinal cells. The immunomodulatory activities of probiotics, β-glucans, and their combinations were studied and compared in vitro by transcriptional analysis of immune-related genes on LPS-stimulated THP-1 macrophages. Immune modulating properties were evidenced. In particular, mixtures of probiotic microorganisms and barley β-glucans exhibited synergistic effects in modulating the transcriptional level of several immune-related genes, leading to an overall enhanced anti-inflammatory effect. Together, these findings suggest a promising application of probiotic bacteria and β-glucans in the preparation of dietary mixtures presenting health-promoting features such as immunomodulatory properties.

Consumers highly appreciate table grapes for their pleasant sensory attributes and as good sources of nutritional and functional compounds. This explains the rising market and global interest in this product. Along with other fruits and vegetables, table grapes are considerably perishable post-harvest due to the growth of undesired microorganisms. Among the microbial spoilers, Botrytis cinerea represents a model organism because of its degrading potential and the huge economic losses caused by its infection. The present review provides an overview of the recent primary physical, chemical, and biological control treatments adopted against the development of B. cinerea in table grapes to extend shelf life. These treatments preserve product quality and safety. This article also focuses on the compliance of different approaches with organic and sustainable production processes. Tailored approaches include those that rely on controlled atmosphere and the application of edible coating and packaging, as well as microbial-based activities. These strategies, applied alone or in combination, are among the most promising solutions in order to prolong table grape quality during cold storage. In general, the innovative design of applications dealing with hurdle technologies holds great promise for future improvements.

The management of the alcoholic fermentation (AF) in wine is crucial to shaping product quality. Numerous variables (e.g., grape varieties, yeast species/strains, technological parameters) can affect the performances of this fermentative bioprocess. The fact that these variables are often interdependent, with a high degree of interaction, leads to a huge ‘oenological space’ associated with AF that scientists and professionals have explored to obtain the desired quality standards in wine and to promote innovation. This challenge explains the high interest in approaches tested to monitor this bioprocess including those using volatile organic compounds (VOCs) as target molecules. Among direct injection mass spectrometry approaches, no study has proposed an untargeted online investigation of the diversity of volatiles associated with the wine headspace. This communication proposed the first application of proton-transfer reaction-mass spectrometry coupled to a time-of-flight mass analyzer (PTR-ToF-MS) to follow the progress of AF and evaluate the impact of the different variables of wine quality. As a case study, the assessment of VOC variability associated with different combinations of Saccharomyces/non-Saccharomyces was selected. The different combinations of microbial resources in wine are among the main factors susceptible to influencing the content of VOCs associated with the wine headspaces. In particular, this investigation explored the effect of multiple combinations of two Saccharomyces strains and two non-Saccharomyces strains (belonging to the species Metschnikowia pulcherrima and Torulaspora delbrueckii) on the content of VOCs in wine, inoculated both in commercial grape juice and fresh grape must. The results demonstrated the possible exploitation of non-invasive PTR-ToF-MS monitoring to explore, using VOCs as biomarkers, (i) the huge number of variables influencing AF in wine, and (ii) applications of single/mixed starter cultures in wine. Reported preliminary findings underlined the presence of different behaviors on grape juice and on must, respectively, and confirmed differences among the single yeast strains ‘volatomes’. It was one of the first studies to include the simultaneous inoculation on two non-Saccharomyces species together with a S. cerevisiae strain in terms of VOC contribution. Among the other outcomes, evidence suggests that the addition of M. pulcherrima to the coupled S. cerevisiae/T. delbrueckii can modify the global release of volatiles as a function of the characteristics of the fermented matrix.

The employment of multi-species starter cultures has growing importance in modern winemaking for improving the complexity and wine attributes. The assessment of compatibility for selected species/strains at the industrial-scale is crucial to assure the quality and the safety associated with fermentations. An aspect particularly relevant when the species belong to non-Saccharomyces, Saccharomyces spp. and malolactic bacteria, three categories with different biological characteristics and oenological significance. To the best of our knowledge, the present report is the first study regarding the utilization of a combined starter culture composed of three strains of non-Saccharomyces, Saccharomyces cerevisiae and Lactobacillus plantarum for production of wine at the industrial scale. More in-depth, this work investigated the oenological potential of the autochthonous characterized strains from the Apulian region (Southern Italy), Candida zemplinina (syn. Starmerella bacillaris) 35NC1, S. cerevisiae (NP103), and L. plantarum (LP44), in co-inoculation following a complete scale-up scheme. Microbial dynamics, fermentative profiles and production of volatile secondary compounds were assessed in lab-scale micro-vinification tests and then the performances of the mixed starter formulation were further evaluated by pilot-scale wine production. The above results were finally validated by performing an industrial-scale vinification on 100HL of Negroamaro cultivar grape must. The multi-starter formulation was able to rule the different stages of the fermentation processes effectively, and the different microbial combinations enhanced the organoleptic wine features to different extents. The findings indicated that the simultaneous inoculation of the three species affect the quality and quantity of several volatile compounds, confirming that the complexity of the wine can reflect the complexity of the starter cultures. Moreover, the results underlined that the same mixed culture could differently influence wine quality when tested at the lab-, pilot- and industrial-scale. Finally, we highlighted the significance of employment non-Saccharomyces and L. plantarum, together with S. cerevisiae, autochthonous strains in the design of custom-made starter culture formulation for typical regional wine production with pronounced unique quality.

Donkey milk is receiving increasing interest due to its attractive nutrient and functional properties (but also cosmetic), which make it a suitable food for sensitive consumers, such as infants with allergies, the immunocompromised, and elderly people. Our study aims to provide further information on the microbial variability of donkey milk under cold storage conditions. Therefore, we analysed by high-throughput sequencing the bacterial communities in unpasteurized donkey milk just milked, and after three days of conservation at 4 °C, respectively. Results showed that fresh donkey milk was characterized by a high incidence of spoilage Gram-negative bacteria mainly belonging to Pseudomonas spp. A composition lower than 5% of lactic acid bacteria was found in fresh milk samples, with Lactococcus spp. being the most abundant. The occurrence of microbial species belonging to risk group 2 was found in fresh milk. After three days of cold storage, the bacterial biodiversity of donkey milk was strongly reduced, since about 93% of the bacterial communities were identified as different species of psychrotrophic Pseudomonas. In conclusion, we report a preliminary description of the microbial diversity of donkey milk by using a metagenomic approach and encouraging a correct exploitation of this high-value niche product.

Pediococcus parvulus 2.6 secretes a 2-substituted (1,3)-β-D-glucan with prebiotic and immunomodulatory properties. It is synthesized by the GTF glycosyltransferase using UDP-glucose as substrate. Analysis of the P. parvulus 2.6 draft genome revealed the existence of a sorbitol utilization cluster of six genes (gutFRMCBA), whose products should be involved in sorbitol utilization and could generate substrates for UDP-glucose synthesis. Southern blot hybridization analysis showed that the cluster is located in a plasmid. Analysis of metabolic fluxes and production of the exopolysaccharide revealed that: (i) P. parvulus 2.6 is able to metabolize sorbitol, (ii) sorbitol utilization is repressed in the presence of glucose and (iii) sorbitol supports the synthesis of 2-substituted (1,3)-β-D-glucan. The sorbitol cluster encodes two putative regulators, GutR and GutM, in addition to a phosphoenolpyruvate-dependent phosphotransferase transport system and sorbitol-6-phosphate dehydrogenase. Therefore, we investigated the involvement of GutR and GutM in the expression of gutFRMCBA. The promoter-probe vector pRCR based on the mrfp gene, which encodes the fluorescence protein mCherry, was used to test the potential promoter of the cluster (Pgut) and the genes encoding the regulators. This was performed by transferring by electrotransformation the recombinant plasmids into two hosts, which metabolize sorbitol: Lactobacillus plantarum and Lactobacillus casei. Upon growth in the presence of sorbitol, but not of glucose, only the presence of Pgut was required to support expression of mrfp in L. plantarum. In L. casei the presence of sorbitol in the growth medium and the pediococcal gutR or gutR plus gutM in the genome was required for Pgut functionality. This demonstrates that: (i) Pgut is required for expression of the gut cluster, (ii) Pgut is subjected to catabolic repression in lactobacilli, (iii) GutR is an activator, and (iv) in the presence of sorbitol, trans-complementation for activation of Pgut exists in L. plantarum but not in L. casei.

In this study, we investigated the multifunctionality (microbial starters and probiotics) of Lactobacillus plantarum WCFS1 and Lactobacillus plantarum CECT 8328 strains used as microbial starters for the production of yogurt in combination with Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. The ability of the probiotic strains to survive oro-gastrointestinal stresses was monitored by an in vitro assay simulating the human digestive tract. The transcriptional level of several genes involved in the immune response suggested that the probiotic strains may have a favorable influence on immunomodulation. Overall, this study revealed that the tested Lactobacilli exhibited suitable technological features for yogurt production and might be used to formulate novel food with immunomodulating effects.

Among the innovative trends in the wine sector, the continuous exploration of enological properties associated with wine microbial resources represents a cornerstone driver of quality improvement. Since the advent of starter cultures technology, the attention has been focused on intraspecific biodiversity within the primary species responsible for alcoholic fermentation (Saccharomyces cerevisiae) and, subsequently, for the so-called ‘malolactic fermentation’ (Oenococcus oeni). However, in the last decade, a relevant number of studies proposed the enological exploitation of an increasing number of species (e.g., non-Saccharomyces yeasts) associated with spontaneous fermentation in wine. These new species/strains may provide technological solutions to specific problems and/or improve sensory characteristics, such as complexity, mouth-feel and flavors. This review offers an overview of the available information on the enological/protechnological significance of microbial resources associated with winemaking, summarizing the opportunities and the benefits associated with the enological exploitation of this microbial potential. We discuss proposed solutions to improve quality and safety of wines (e.g., alternative starter cultures, multistrains starter cultures) and future perspectives.

In this study, we explored the diversity of yeasts and lactic acid bacteria (LAB) associated with six spontaneous sourdough fermentations from the northern part of the Apulian region (Italy). Bacterial and yeast isolates from sourdough were investigated by amplified ribosomal DNA restriction analysis (ARDRA) and restriction fragment length polymorphism (RFLP) analysis, respectively. The identification of the isolates was confirmed by sequencing bacterial 16S gene and yeast ITS1−5.8S−ITS2 rRNA gene amplicons. Microbiological analysis of all sourdough samples revealed that LAB and yeast counts ranged between 1.7 × 10⁵ and 6.5 × 10⁸ cfu/g, and 7.7 × 10⁵ and 2.5 × 10⁷ cfu/g, respectively. The molecular identification at species level revealed the occurrence of Lactobacillus plantarum as the dominant LAB and Saccharomyces cerevisiae as the dominant yeast species in all different sourdough samples. Then, the ability of all isolated strains to inhibit and/or reduce the growth of several selected fungi was valued through the overlay method. In light of their antifungal performances, ten LAB strains were inoculated, singularly and in combination, in subsequent bread-making trials. Overall, we confirmed the potential of LAB to extend the shelf life of bread through spoilage inhibition and, for the first time, we observed a synergistic effect due to the combination of several isolated LAB on the inhibition behavior against selected fungal spoilage strains. Our findings suggest the exploration of a LAB-based approach in order to extend the shelf life of bread, reducing, at the same time, the use of chemical agents for food preservation.

Climate change threatens food systems, with huge repercussions on food security and on the safety and quality of final products. We reviewed the potential of food microbiology as a source of biotechnological solutions to design climate-smart food systems, using wine as a model productive sector. Climate change entails considerable problems for the sustainability of oenology in several geographical regions, also placing at risk the wine typicity. The main weaknesses identified are: (i) The increased undesired microbial proliferation; (ii) the improved sugars and, consequently, ethanol content; (iii) the reduced acidity and increased pH; (iv) the imbalanced perceived sensory properties (e.g., colour, flavour); and (v) the intensified safety issues (e.g., mycotoxins, biogenic amines). In this paper, we offer an overview of the potential microbial-based strategies suitable to cope with the five challenges listed above. In terms of microbial diversity, our principal focus was on microorganisms isolated from grapes/musts/wines and on microbes belonging to the main categories with a recognized positive role in oenological processes, namely Saccharomyces spp. (e.g., Saccharomyces cerevisiae), non-Saccharomyces yeasts (e.g., Metschnikowia pulcherrima, Torulaspora delbrueckii, Lachancea thermotolerans, and Starmerella bacillaris), and malolactic bacteria (e.g., Oenococcus oeni, Lactobacillus plantarum).

For three consecutive years, an Italian winery in Apulia has dealt with sudden alcoholic stuck fermentation in the early stages of vinification process, i.e., typical defects addressable to bacterial spoilage. After a prescreening trial, we assessed, for the first time, the influence of the commercial fungicide preparation Ridomil Gold® (Combi Pepite), containing Metalaxyl-M (4.85%) and Folpet (40%) as active principles, on the growth of several yeasts (Saccharomyces cerevisiae and non-Saccharomyces spp.) and lactic acid bacteria of oenological interest. We also tested, separately and in combination, the effects of Metalaxyl-M and Folpet molecules on microbial growth both in culture media and in grape must. We recalled the attention on Folpet negative effect on yeasts, extending its inhibitory spectrum on non-Saccharomyces (e.g., Candida spp.). Moreover, we highlighted a synergic effect of Metalaxyl-M and Folpet used together and a possible inhibitory role of the fungicide excipients. Interestingly, we identified the autochthonous S. cerevisiae strain E4 as moderately resistant to the Folpet toxicity. Our findings clearly indicate the urgent need for integrating the screening procedures for admission of pesticides for use on wine grape with trials testing their effects on the physiology of protechnological microbes.

A graphical representation of the transdisciplinary pyramid, as a tool, helps us in the design of practical classroom activities that deal with transdisciplinarity, microbiology, and, more generally, with biology. As regard to classroom implementation, we propose two main practical approaches. On the one hand, we can concretize/realize transdisciplinarity through a new design of the lesson, which shall rely on a team of four teachers/professors representing disciplines from all the four levels of the pyramid. On the other hand, a class debate with a teacher–moderator shall be used as a Trojan horse to launch the insights into the newly proposed point of view. The presence of four teachers/professors provides a rigorous study of the topics together with a concretized high-quality transdisciplinarity. Conversely, the class discussion promotes student involvement and the development of critical thought.

Biocontrol strategies for the limitation of undesired microbial developments in foods and beverages represent a keystone toward the goal of more sustainable food systems. Brettanomyces bruxellensis is a wine spoilage microorganism that produces several compounds that are detrimental for the organoleptic quality of the wine, including some classes of volatile phenols. To control the proliferation of this yeast, sulfur dioxide is commonly employed, but the efficiency of this compound depends on the B. bruxellensis strain; and it is subject to wine composition and may induce the entrance in a viable, but nonculturable state of yeasts. Moreover, it can also elicit allergic reactions in humans. In recent years, biological alternatives to sulfur dioxide such as the use of yeasts and lactic acid bacteria starter cultures as biocontrol agents are being investigated. The controlled inoculation of starter cultures allows secure, fast and complete alcoholic and malolactic fermentations, limiting the residual nutrients that B. bruxellensis utilizes to survive and grow in wine. The current study is focused on the assessment of the effect of autochthonous yeasts and bacterial strains from the Apulia Region on the development of B. bruxellensis in wine, in terms of both growth and volatile phenols’ production. The investigation evidences the positive role of indigenous mixed cultures in the control of this spoilage yeast, either co-inoculating different strains of Saccharomyces cerevisiae, S. cerevisiae/non-Saccharomyces or co-inoculating S. cerevisiae/Oenococcus oeni. Our findings expand the existing knowledge of the application of protechnological microbial diversity and of non-Saccharomyces as a biocontrol agent in oenology. We report a further demonstration of the interest in selecting indigenous strains as a strategic tool for winemakers interested in the improvement of regional wines.

Bread is a staple food consumed worldwide on a daily basis. Fungal contamination of bread is a critical concern for producers since it is related to important economic losses and safety hazards due to the negative impact of sensorial quality and to the potential occurrence of mycotoxins. In this work, Lactobacillus plantarum UFG 121, a strain with characterized broad antifungal activity, was analyzed as a potential protective culture for bread production. Six different molds belonging to Aspergillus spp., Penicillium spp., and Fusarium culmorum were used to artificially contaminate bread produced with two experimental modes: (i) inoculation of the dough with a commercial Saccharomyces cerevisiae strain (control) and (ii) co-inoculation of the dough with the commercial S. cerevisiae strain and with L. plantarum UFG 121. L. plantarum strain completely inhibited the growth of F. culmorum after one week of storage. The lactic acid bacterium modulated the mold growth in samples contaminated with Aspergillus flavus, Penicillium chrysogenum, and Penicillium expansum, while no antagonistic effect was found against Aspergillus niger and Penicillium roqueforti. These results indicate the potential of L. plantarum UFG 121 as a biocontrol agent in bread production and suggest a species- or strain-depending sensitivity of the molds to the same microbial-based control strategy.

Oenococcus oeni is the bacterium most often associated with spontaneous malolactic fermentation (MLF) of wine. During MLF, malic acid is transformed into lactic acid and several metabolites are modified, modulating wine’s total acidity and improving its sensory properties. Previous works have suggested that certain genetic groups of O. oeni strains are associated to different kinds of products. In the present study we have spotted two groups of strains isolated mainly from Burgundy wines, one associated to red wines and the other to white wines. Sequencing 14 genomes of red and white wine strains revealed that they share a common ancestor that probably colonised two different substrates –red and white wine-associated environments–, diverging over time and disseminating to various regions. Their capacity to perform MLF and modify the volatile profile of wine was determined by fermenting a chardonnay wine and analysing its volatile fraction with a non-targeted metabolomics approach by GC-MS. The strains had a different impact on the volatile composition depending on their group of origin. These results show for the first time a correspondence between the product of origin of the strains and the volatile profile of the wines they produce. Furthermore, the genetic features that might be implied in these different phenotypes are examined.

Fermented foods and beverages are a heterogeneous class of products with a relevant worldwide significance for human economy, nutrition and health for millennia. A huge diversity of microorganisms is associated with the enormous variety in terms of raw materials, fermentative behavior and obtained products. In this wide microbiodiversity it is possible that the presence of microbial pathogens and toxic by-products of microbial origin, including mycotoxins, ethyl carbamate and biogenic amines, are aspects liable to reduce the safety of the consumed product. Together with other approaches (e.g., use of preservatives, respect of specific physico-chemical parameters), starter cultures technology has been conceived to successfully dominate indigenous microflora and to drive fermentation to foresee the desired attributes of the matrix, assuring quality and safety. Recent trends indicate a general return to spontaneous food fermentation. In this review, we point out the potential risks for human health associated with uncontrolled (uninoculated) food fermentation and we discuss biotechnological approaches susceptible to conciliate fermented food safety, with instances of an enhanced contribution of microbes associated to spontaneous fermentation.

Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.

In light of the increasing attention towards “green” solutions to improve food quality, the use of aromatic-enhancing microorganisms offers the advantage to be a natural and sustainable solution that did not negatively influence the list of ingredients. In this study, we characterize, for the first time, volatile organic compounds (VOCs) associated with aromatic bakery yeasts. Three commercial bakery starter cultures, respectively formulated with three Saccharomyces cerevisiae strains, isolated from white wine, red wine, and beer, were monitored by a proton-transfer-reaction time-of-flight mass spectrometer (PTR-ToF-MS), a direct injection analytical technique for detecting volatile organic compounds with high sensitivity (VOCs). Two ethanol-related peaks (m/z 65.059 and 75.080) described qualitative differences in fermentative performances. The release of compounds associated to the peaks at m/z 89.059, m/z 103.075, and m/z 117.093, tentatively identified as acetoin and esters, are coherent with claimed flavor properties of the investigated strains. We propose these mass peaks and their related fragments as biomarkers to optimize the aromatic performances of commercial preparations and for the rapid massive screening of yeast collections.