Novel Probiotic Candidates in Artisanal Feta-Type Kefalonian Cheese: Unveiling a Still-Undisclosed Biodiversity.

Autochthonous dairy lactic acid bacteria (LAB) isolates encompass a natural source of starter, adjunct, or probiotic candidates. In this context, traditionally manufactured, using exclusively animal rennet, Feta-type cheeses were collected from five farms located in different regions of Kefalonia island (Greece). The primary objective of this study was to isolate and characterize novel LAB, thereby exploring the unmapped microbial communities of Kefalonian Feta-type cheese and identifying new potential probiotics. The initial screening, included a preliminary gastrointestinal (GI) tolerance assessment (acidic conditions and bile salts), followed by their safety evaluation (hemolytic activity and antibiotic susceptibility). Based on the preliminary screening, selected strains underwent molecular identification and were further investigated for their probiotic attributes (lysozyme and phenol resistance, antimicrobial traits, antidiabetic aspects, cholesterol reduction and adhesion, adhesion to Caco-2 cells, and milk acidification potential). The results showed that 49, out of the 93 retrieved isolates, exhibited resistance to GI conditions, whereas 18 met the safety criteria. The molecular identification revealed strains belonging to the species Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and Lacticaseibacillus paracasei. The selected rod-shaped 14 isolates displayed a potential probiotic character. The best-performing isolates concerning cholesterol assimilation and adhesion, α-glucosidase inhibition, and epithelial adherence were Lpb. plantarum F89, F162, and F254 and Lcb. paracasei F214 and F216, whereas Lcb. paracasei F70 showed potential as a defined strain starter. The present study explores for the first time the biodiversity of traditionally fermented microbial communities in Kefalonian Feta-type cheese, revealing novel potential probiotic strains that can contribute to the development of innovative functional food products.

Novel Probiotic/Bacterial Cellulose Biocatalyst for the Development of Functional Dairy Beverage.

The development of innovative functional products with potential health benefits, under the concept of bio-economy, is flourishing. This study undertook an evaluation of non-dairy lactobacilli Lactiplantibacillus pentosus B329 and Lactiplantibacillus plantarum 820 as "ready to use" starter cultures. Lactic acid bacteria (LAB) cultures were evaluated for their fermentation efficiency, before and after freeze-drying, using cheese whey (CW) as a fermentation substrate and subsequent immobilization on bacteria cellulose (BC) to produce a novel biocatalyst. The biocatalyst was applied in functional sour milk production and compared with free cells via the assessment of physicochemical and microbiological properties and sensory evaluation. Evidently, LAB strains exhibited high fermentative activity before and after freeze-drying. Results of a 5-month storage stability test showed that viability was 19% enhanced by immobilization on BC, supporting the concept of "ready to use" cultures for the production of fermented beverages. Likewise, sour milk produced by the BC biocatalyst presented higher organoleptic scores, compared to the free cells case, whereas immobilization on BC enhanced probiotic viability during post-fermentation storage (4 °C, 28 days). The obtained high viability (>107 log cfu/g) demonstrated the efficacy of the proposed bioprocess for the production of functional/probiotic-rich beverages. Ultimately, this work presents a consolidated scheme that includes the advantages and the cooperative effect of probiotic LAB strains combined with a functional biopolymer (BC) towards the formulation of novel functional products that coincide with the pillars of food systems sustainability.

MALDI-TOF MS profiling of non-starter lactic acid bacteria from artisanal cheeses of the Greek island of Naxos.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), a culture based alternative for microbial diversity studies, is an attractive tool to dereplicate large numbers of isolates to a smaller set of representatives for downstream characterization. In the present study, MALDI-TOF MS, combined with a database of reference spectra compiled in previous studies, was applied to identify 88 non-starter lactic acid bacteria (NSLAB) isolated from 18 samples of four different artisanal cheeses produced in the Island of Naxos, Greece, from raw sheep and goat milk without the addition of starters. Eighty-four isolates (95.5%) could be identified directly via MALDI-TOF MS. Lactobacillus brevis and Lactobacillus plantarum were the dominant species, followed by Lactococcus lactis, Leuconostoc mesenteroides Lactobacillus paracasei, Lactobacillus rhamnosus, Pediococcus pentosaceus and Enterococcus faecium. The remaining four isolates represented species present in the database; however, within-species diversity was insufficiently covered. Additionally, pheS sequencing was applied to confirm identification.

Dual Transcriptional Profile of Aspergillus flavus during Co-Culture with Listeria monocytogenes and Aflatoxin B1 Production: A Pathogen-Pathogen Interaction.

The objective of this study was to investigate the effect of growth temperature and co-culture of Aspergillus flavus with Listeria monocytogenes on the production of Aflatoxin B1 (AFB1) and the transcriptional profile of associated regulatory and biosynthetic genes. The transcription of virulence- and homeostasis-associated genes of L. monocytogenes was also assessed. For this purpose, mono- and co-cultures of L. monocytogenes strain LQC 15257 and A. flavus strain 18.4 were inoculated into Malt Extract broth and allowed to grow for seven days at 25 °C and 30 °C. AFB1 quantification was performed by HPLC analysis and gene expression assessment by RT-qPCR. AFB1 production was lower at 30 °C compared to 25 °C during monoculture and also lower during co-cultures at both temperatures. This was accompanied by downregulation of aflM, aflR, aflP, and aflS during monoculture and aflM and aflS during co-culture at 30 °C. On the other hand, transcription of prfA, plcA, plcB, inlA, inlB, inlJ, murE, accA, acpP, as well as fapR, was not affected. sigB gene was downregulated after co-culture with the fungus at 25 °C and hly was downregulated after monoculture at 30 °C compared to 25 °C. In this work, the molecular interactions between A. flavus and L. monocytogenes were studied for the first time, offering a novel insight into their co-occurrence. Monitoring of their toxigenic and virulence potential at the molecular level revealed a complex dynamic in natural ecosystems.

Cheese Whey Processing: Integrated Biorefinery Concepts and Emerging Food Applications.

Cheese whey constitutes one of the most polluting by-products of the food industry, due to its high organic load. Thus, in order to mitigate the environmental concerns, a large number of valorization approaches have been reported; mainly targeting the recovery of whey proteins and whey lactose from cheese whey for further exploitation as renewable resources. Most studies are predominantly focused on the separate implementation, either of whey protein or lactose, to configure processes that will formulate value-added products. Likewise, approaches for cheese whey valorization, so far, do not exploit the full potential of cheese whey, particularly with respect to food applications. Nonetheless, within the concept of integrated biorefinery design and the transition to circular economy, it is imperative to develop consolidated bioprocesses that will foster a holistic exploitation of cheese whey. Therefore, the aim of this article is to elaborate on the recent advances regarding the conversion of whey to high value-added products, focusing on food applications. Moreover, novel integrated biorefining concepts are proposed, to inaugurate the complete exploitation of cheese whey to formulate novel products with diversified end applications. Within the context of circular economy, it is envisaged that high value-added products will be reintroduced in the food supply chain, thereby enhancing sustainability and creating "zero waste" processes.

Probiotics in Food Systems: Significance and Emerging Strategies Towards Improved Viability and Delivery of Enhanced Beneficial Value.

Preserving the efficacy of probiotic bacteria exhibits paramount challenges that need to be addressed during the development of functional food products. Several factors have been claimed to be responsible for reducing the viability of probiotics including matrix acidity, level of oxygen in products, presence of other lactic acid bacteria, and sensitivity to metabolites produced by other competing bacteria. Several approaches are undertaken to improve and sustain microbial cell viability, like strain selection, immobilization technologies, synbiotics development etc. Among them, cell immobilization in various carriers, including composite carrier matrix systems has recently attracted interest targeting to protect probiotics from different types of environmental stress (e.g., pH and heat treatments). Likewise, to successfully deliver the probiotics in the large intestine, cells must survive food processing and storage, and withstand the stress conditions encountered in the upper gastrointestinal tract. Hence, the appropriate selection of probiotics and their effective delivery remains a technological challenge with special focus on sustaining the viability of the probiotic culture in the formulated product. Development of synbiotic combinations exhibits another approach of functional food to stimulate the growth of probiotics. The aim of the current review is to summarize the strategies and the novel techniques adopted to enhance the viability of probiotics.

Control of Aspergillus carbonarius in grape berries by Lactobacillus plantarum: A phenotypic and gene transcription study.

The in vitro and in situ antifungal activity of Lactobacillus plantarum against the ochratoxigenic fungus Aspergillus carbonarius was investigated in this study. Four different fungal isolates from grape berries were co-cultured with four different strains of L. plantarum on Malt Extract Agar (MEA) plates at 30 °C. Bacterial strains inhibited fungal growth up to 88% and significantly reduced toxin production up to 100%. In addition, L. plantarum was evaluated as biocontrol agent against A. carbonarius growth and OTA production on table grapes. Temporal studies of bacterial antagonism were performed with two different grape cultivars. Artificially wounded and unwounded berries were pre-treated with 108 CFU/mL bacteria and inoculated with 106 spores/mL of A. carbonarius ochratoxigenic isolates. Biocontrol agents displayed high rate of colonization on grapes during 5 days of incubation at 30 °C. Scanning electron microscopy (SEM) also determined the presence of microorganisms on grape surface. Bacterial strains were effective in controlling fungal infection reaching up to 71% inhibition rates. However the presence of wounds on grape skin facilitated infection of berries by A. carbonarius, since unwounded berries showed lower levels of infection. Results also revealed significant reduction in mycotoxin production ranging between 32% and 92%. Transcriptome analysis following exposure to co-cultivation, exhibited differential expression for each gene studied of AcOTAnrps (Aspergillus carbonarius OTA nonribosomal), AcOTApks (Aspergillus carbonarius OTA polyketide synthase) and laeA, emphasizing the significance of strain variability. The genes AcOTAnrps and laeA were most influenced by the presence of L. plantarum. This work is a contribution for the potential biocontrol of toxigenic fungi in table grapes by lactic acid bacteria (LAB). The above findings underline the significance of bacterial strain variability on the effectiveness of biopreservative features of L. plantarum strains.

Evaluating the efficacy of turbimetric measurements as a rapid screening technique to assess fungal susceptibility to antimicrobial compounds as exemplified by the use of sodium metabisulfite.

The in vitro susceptibility to sodium metabisulphite (NaMBS) was investigated in 10 different food spoilage filamentous fungi, namely Aspergillus flavus, A. carbonarius, A. niger, A. ochraceus, A. tubingensis, A. westerdijkiae, Cladosporium cladosporioides, Fusarium oxysporum, Penicillium commune and P. expansum. The fungi were inoculated in sterile 96-well microtiter plates containing Yeast-extract Sucrose (YES) semi-solid agar supplemented with NaMBS in concentrations ranging from 2000 to 3.9 mg l-1 and incubated at 25 °C. Growth was monitored by absorbance measurements at 600 nm using a multi-spectrophotometer. The surface areas under the optical density (OD) vs. time growth curves obtained were used to calculate the fractional area f(a), from which the non-inhibitory (NIC) and minimum inhibitory concentrations (MIC) of the antifungal agent were calculated for each fungus using the Gompertz model for decay. Most Aspergillus species showed remarkable resistance to NaMBS, presenting NIC and MIC values higher than 250 and 2500 mg l-1, respectively. The most susceptible fungi were the two Penicillium species and A. carbonarius, which presented very low NIC (<100 mg l-1) and MIC (<1300 mg l-1) values, whereas C. cladosporioides and F. oxysporum presented intermediate values. The method has the advantage of good repeatability and accuracy, rapid results within 48-72 h, growth detection and susceptibility to the antifungal agent for several fungi at the same time, and optimal use of microbiological media by using small volumes of consumables.

Monitoring the Temporal Expression of Genes Involved in Ochratoxin A Production of Aspergillus carbonarius under the Influence of Temperature and Water Activity.

The objective of this study was to investigate the effect of environmental factors, namely temperature and water activity, on genes involved in the regulation of ochratoxin A (OTA) production over time. For this purpose, the previously characterized toxigenic Aspergilluscarbonarius Ac29 isolate from Greek vineyards and the A. carbonarius ITEM 5010 reference strain were subjected to combined temperature and water activity (aw) treatments to study OTA production and relative gene expression. The fungal isolates were grown on a synthetic grape juice liquid medium (SGM) under different temperature (20 °C, 25 °C and 30 °C) and aw (0.94 and 0.98) regimes. The expression of the AcOTApks, AcOTAnrps, and laeA OTA related genes was investigated using real time PCR. Gene expression was monitored at the same time points, along with fungal biomass and OTA accumulation at three, six and nine days of incubation. In gene expression analysis, stimulation of the biosynthetic genes was observed a few days before any toxin could be detected. This fact may underline a possible early indicator of potential toxin contamination of grapes. However, the transcript levels varied with respect to the different combinations of ecophysiological conditions and time, highlighting a complex regulation of OTA related gene expression of A. carbonarius in the specific medium.

Comparative study of growth responses and screening of inter-specific OTA production kinetics by A. carbonarius isolated from grapes.

The aim of this work was to assess OchratoxinA (OTA) production of different Aspergillus carbonarius isolates, evaluate their growth profile through different growth measurements, and reveal any underlying correlation between them. Ten different isolates of A. carbonarius isolated from Greek vineyards located in different geographical regions were examined in vitro for their OTA production potential after an incubation period of up to 11 days. All fungal isolates grew on a synthetic grape juice medium (SGM) similar to grape composition at optimum conditions of temperature and water activity (25°C and 0.98 aw). Samples for OTA determination were removed at 3, 5, 7, 9, and 11 days of growth and analyzed by HPLC. Based on OTA measurements the isolates were characterized by diverse OTA production ranging from 50 to 2000 ppb at day 11. The different fungal growth responses (colony diameter, colony area, biomass, biomass dry weight, and colony density) have been measured and correlated with toxin production by means of principal components analysis (PCA), confirming satisfactory correlation and explained over 99% of data variability. Leudeking-Piret model was also used to study OTA production with time, revealing a mixed-growth associated trend and pointing a fail-safe model with slightly better prediction through colony area. This approach contributes to the assessment of correlation between mycotoxin production and different methods of fungal growth determination in relation to time.