Gut Microbiota Modulation by Selenium and Zinc Enrichment Postbiotic on Dysbiosis Associated with Hypertension.

BACKGROUND: Targeting gut dysbiosis to treat chronic diseases or to alleviate the symptoms is a new direction for medical adjuvant therapies. Recently, postbiotics have received considerable attention as they are non-viable probiotic preparations that confer various health benefits to the host without the safety problems associated with using live microbial cells. OBJECTIVE: The aim of the study is to obtain selenium (Se) and zinc (Zn) enriched Saccharomyces boulardii postbiotic biomass and to analyze its modulation effect because these minerals play an important role in reducing gut dysbiosis linked to cardiovascular (CV) diseases. METHOD: The effect of the S. boulardii and Se/Zn enriched yeast postbiotics on CV microbial fingerprint was studied in vitro using the gastrointestinal system (GIS 1) and analyzed by microbiological, chemical, and qPCR methods. RESULT: There was a 2.2 log CFU/mL increase in the total bacterial load after SeZn postbiotic treatment and in the qPCR counts of Firmicutes phyla for both treatments. Beneficial taxa, Bifidobacterium spp. and Lactobacillus spp., as well as Bacteroides spp. were up to 1.5 log higher after mineral- enriched postbiotic application, while the acetic acid level increased. CONCLUSION: These preliminary studies highlight the therapeutic potential of using Se/Zn enriched yeast postbiotics as adjuvants for clinical treatments of CV diseases.

Improving the Efficiency of Viability-qPCR with Lactic Acid Enhancer for the Selective Detection of Live Pathogens in Foods.

Pathogenic Escherichia coli are the most prevalent foodborne bacteria, and their accurate detection in food samples is critical for ensuring food safety. Therefore, a quick technique named viability-qPCR (v-qPCR), which is based on the ability of a selective dye, such as propidium monoazide (PMA), to differentiate between alive and dead cells, has been developed. Despite diverse, successful applications, v-qPCR is impaired by some practical limitations, including the ability of PMA to penetrate the outer membrane of dead Gram-negative bacteria. The objective of this study is to evaluate the ability of lactic acid (LA) to improve PMA penetration and, thus, the efficiency of v-qPCR in detecting the live fraction of pathogens. The pre-treatment of E. coli ATCC 8739 cells with 10 mM LA greatly increased PMA penetration into dead cells compared to conventional PMA-qPCR assay, avoiding false positive results. The limit of detection when using LA-PMA qPCR is 1% viable cells in a mixture of dead and alive cells. The optimized LA-PMA qPCR method was reliably able to detect log 2 CFU/mL culturable E. coli in milk spiked with viable and non-viable bacteria. Lactic acid is cheap, has low toxicity, and can be used to improve the efficiency of the v-qPCR assay, which is economically interesting for larger-scale pathogen detection applications intended for food matrices.

In Vitro Evaluation of Some Endophytic Bacillus to Potentially Inhibit Grape and Grapevine Fungal Pathogens.

Romania has a long history of grapevine culturing and winemaking. However, like any agricultural sector, viticulture faces devastating biological threats. Fungi responsible for grapevine trunk diseases (GTDs) and grape spoilage lead to considerable yield losses and a decline in grapevine quality. In the actual context, many countries, including Romania, have reoriented their approaches to minimize chemical inputs, which have been proven to be toxic and to have negative impacts on the environment, and to replace them with sustainable biocontrol strategies for the wine-growing sector. Within biocontrol strategies, Bacillus spp. is a well-known plant-protective bacteria with antifungal properties. Within this paper, six endophytic bacteria from various plant sources were studied. The bacterial strains were identified as B. pumilus, B. subtilis, and B. velezensis by sequencing their 16S rDNA region. Regardless of the in vitro test methods (using living bacterial cells, bacterial-cell-free supernatant (CFS), and volatile active compounds (VOCs)), B. velezensis strains revealed strong and broad antifungal activity against grape and grapevine fungal pathogens such as Aspergillus spp., Botrytis cinerea, Penicillium expansum, Diplodia seriata, Eutypa lata, Fusarium spp., Clonostachys rosea, Neofusicoccum parvum, and Stereum hirsutum. The functional antifungal genes encoding for difficidin, fengycin, iturins, macrolactin, and mycosubtilin were molecularly detected, which could support the proven antifungal activity of the endophytic strains. Lytic enzymes involved in fungal growth inhibition, such as chitinase, cellulase, and proteases, were also revealed to be produced by some of these bacterial strains. Various other in vitro tests, such as phosphate and phytate solubilization, phytohormone synthesis, the production of enzymes involved in the polyamine biosynthetic pathway, and pH as well as temperature tolerance tests were carried out to reveal the plant-beneficial potential of these bacterial strains. These results revealed that the B. velezensis strains, especially BAHs1, are the most suitable endophytes for grapevine biologic control, which could lead to the future development of sustainable management strategies.

Functional properties of lactic acid bacteria isolated from Tilapia (Oreochromis niloticus) in Ivory Coast.

BACKGROUND: Probiotics have recently been applied in aquaculture as eco-friendly alternatives to antibiotics to improve fish health, simultaneously with the increase of production parameters. The present study aimed to investigate the functional potential of lactic acid bacteria (LAB) isolated from the gut of Tilapia (Oreochromis niloticus) originating from the aquaculture farm of Oceanologic Research Center in Ivory Coast. RESULTS: Twelve LAB strains were identified by 16 S rDNA gene sequence homology analysis belonging to two genera Pediococcus (P. acidilactici and P. pentosaceus) and Lactobacillus (L. plantarum) with a predominance of P. acidilactici. Several aspects including functional, storage, and safety characteristics were taken into consideration in the selection process of the native LAB isolates as potential probiotics. All LAB isolates showed high antagonistic activity against bacterial pathogens like Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, and Staphylococcus aureus. In addition, the LAB isolates exhibited different degrees of cell surface hydrophobicity in the presence of hexane, xylene, and chloroform as solvents and a good ability to form biofilm. The strong antioxidant activity expressed through the DPPH scavenging capacity of LAB intact cells and their cell-free supernatants was detected. LAB strains survived between 34.18% and 49.9% when exposed to low pH (1.5) and pepsin for 3 h. In presence of 0.3% bile salts, the growth rate ranged from 0.92 to 21.46%. Antibiotic susceptibility pattern of LAB isolates showed sensitivity or intermediate resistance to amoxicillin, cephalothin, chloramphenicol, imipenem, kanamycin, penicillin, rifampicin, streptomycin, tetracycline and resistance to oxacillin, gentamicin, and ciprofloxacin. No significant difference in antibiotic susceptibility pattern was observed between P. acidilactici and P. pentosaceus strains. The non-hemolytic activity was detected. Following the analysis of the enzyme profile, the ability of LAB isolates to produce either lipase or ß-galactosidase or both enzymes was highlighted. Furthermore, the efficacy of cryoprotective agents was proved to be isolate-dependent, with LAB isolates having a high affinity for D-sorbitol and sucrose. CONCLUSION: The explored LAB strains inhibited the growth of pathogens and survived after exposure to simulated gastrointestinal tract conditions. The safety and preservative properties are desirable attributes of these new probiotic strains hence recommended for future food and feed applications.

Promising Probiotic Properties of the Yeasts Isolated from Rabilé, a Traditionally Fermented Beer Produced in Burkina Faso.

In recent years, research on yeasts as probiotics has gained more and more interest, which will allow the development of "new" products in the probiotics market. In this context, seventeen yeast strains isolated from Rabilé, a traditional beer produced in Burkina Faso, were assessed for their probiotic attributes. The yeast identification was performed by molecular methods, including PCR-RFLP and 5.8S-ITS region sequencing. Saccharomyces cerevisiae (14 strains) was the predominantly identified species, followed by Pichia kudriavzevii (2 strains) and Rhodotorula mucilaginosa (1 strain). Except for R. mucilaginosa, all yeast strains grew well at human temperature. The yeast strains showed high resistance when they were exposed to simulated gastrointestinal conditions. Auto-aggregation ability was between 70.20 ± 10.53% and 91.82 ± 1.96%, while co-aggregation with E. coli ranged from 24.92 ± 3.96% to 80.68 ± 9.53% and with S. enterica serovar Typhimurium from 40.89 ± 8.18% to 74.06 ± 7.94%. Furthermore, the hydrophobicity of isolated strains toward n-hexane was in the range from 43.17 ± 5.07% to 70.73 ± 2.42%. All yeast strains displayed high antioxidant capabilities, and the strains did not show hemolysis halos, such that they can be considered safe. Additionally, S. cerevisiae strains strongly inhibited the growth of foodborne pathogens. This is the first preliminary study to identify and characterize the yeast strains isolated from Rabilé with interesting probiotic properties.

Chitosan-Based Edible Coatings Containing Essential Oils to Preserve the Shelf Life and Postharvest Quality Parameters of Organic Strawberries and Apples during Cold Storage.

Edible coatings and films have been researched for more than three decades due to their ability to be incorporated with different functional ingredients or compounds as an option to maintain the postharvest quality of fruits and vegetables. The aim of this study was to evaluate the effect of three types of chitosan-based (CH) edible coatings obtained from medium and high molecular weight chitosan, containing ascorbic or acetic acid and sea buckthorn or grape seed essential oils on the physical-chemical and microbiological properties of organic strawberries and apple slices during cold storage at 4 °C and 8 °C. Scanning electron microscope images showed both a smooth structure and a fracture and pore structure on strawberry coatings and a dense and smooth structure on the apple slices coatings. Further, the edible coatings managed to reduce the microbial load of yeasts and molds of the coated strawberries during the storage period. Overall, the treatments preserved the ascorbic acid, total polyphenol content, and antioxidant activity for all the tested samples compared to the control sample, throughout the storage period. In addition, the water activity (aw) of the coated samples presented lower values (0.96-0.98) than the control samples. The obtained results indicate that the developed chitosan-based edible coatings could maintain the postharvest parameters of the tested samples, also leading to their shelf-life prolongation.

In Vitro Assessment of Yeasts Strains with Probiotic Attributes for Aquaculture Use.

This study aimed to investigate in vitro the probiotic potential of three yeasts strains (BB06, OBT05, and MT07) isolated from agro-food natural sources. Screening was performed, including several functional, technological, and safety aspects of the yeast strains, in comparison to a reference Saccharomyces boulardii, to identify the ones with suitable probiotic attributes in aquaculture. The yeast strains were identified by 5.8S rDNA-ITS region sequencing as Metschnikowia pulcherrima OBT05, Saccharomyces cerevisiae BB06, and Torulaspora delbrueckii MT07. All yeast strains were tolerant to different temperatures, sodium chloride concentrations, and wide pH ranges. S. cerevisiae BB06 showed a strong and broad antagonistic activity. Moreover, the S. cerevisiae strain exhibited a high auto-aggregation ability (92.08 ± 1.49%) and good surface hydrophobicity to hexane as a solvent (53.43%). All of the yeast strains have excellent antioxidant properties (>55%). The high survival rate in the gastrointestinal tract (GIT) can promote yeast isolates as probiotics. All yeast strains presented a resistance pattern to the antibacterial antibiotics. Non-hemolytic activity was detected. Furthermore, freeze-drying with cryoprotective agents maintained a high survival rate of yeast strains, in the range of 74.95−97.85%. According to the results obtained, the S. cerevisiae BB06 strain was found to have valuable probiotic traits.

The Biotechnological Potential of Pediococcus spp. Isolated from Kombucha Microbial Consortium.

In the past decade, the probiotic market has grown rapidly, both for foods and supplements intended to enhance wellness in healthy individuals. Different lactic acid bacteria (LAB), especially Lactobacillus spp., of different origins have already been used to develop commercial probiotic products. Nowadays, LAB new alternative sources, such as non-dairy fermented food products, are being exploited. One such source is Kombucha, a fermented low-alcohol beverage made of tea leaves. In this regard, we tested seven Pediococcus spp. strains isolated from a local industrial Kombucha for their biotechnological potential. Two, out of the seven isolates, identified as Pediococcus pentosaceus (L3) and Pediococcus acidiliactici (L5), were selected as successful candidates for the food industry, due to their probiotic and technological properties. In regard to their resistance in the gastro-intestinal tract, both selected strains were tolerant to a pH of 3.5, presence of 0.3% pepsin, and 0.5% bile salt concentration. On the antagonistic side, the fresh suspension of selected isolates had high inhibitory activity against pathogenic bacteria, such as Salmonella enterica Typhimurium, Listeria monocytogenes, Listeria ivanovii, Bacillus cereus, Proteus hauseri, and methicillin resistant Staphylococcus aureus. In addition, moderate to high inhibitory activity was noticed against foodborne molds (e.g., Penicillium expansum and Penicillium digitatum). These safety issues were supported by their negative hemolytic activity and good antioxidant potential (56-58%). Selected isolates were sensitive to ampicillin, penicillin, erythromycin, and lincomycin, while a broad range of other antibiotics were not effective inhibitors. On the technological side, both strains tolerated 5% NaCl and, during the freeze-drying process, had a good survival rate (86-92%). The selected Pediococcus strains have proven properties to be used for further development of functional products.

Resistance and cross-resistance in Staphylococcus spp. strains following prolonged exposure to different antiseptics.

OBJECTIVES: This aim of this study was to evaluate the potential of staphylococci to develop resistance and cross-resistance to antibiotics following exposure to antiseptics. METHODS: The antibiotic susceptibility profile as well as the antiseptic minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) were determined for Staphylococcus epidermidis and Staphylococcus aureus clinical isolates and reference strains (methicillin resistant or not) before and after prolonged exposure to low concentrations of two antiseptics, namely chlorhexidine digluconate (CHG) and octenidine dihydrochloride (OCT). RESULTS: Resistance was observed to both tested antiseptics following exposure. CHG exposure led to increased MICs in five of the six tested strains of S. epidermidis and S. aureus and also led to clinically decreased susceptibility to gentamicin in S. aureus ATCC 43300 (MRSA) and to penicillin and tetracycline in S. aureus ATCC 25923 (MSSA). OCT exposure led to an increased MIC only in S. epidermidis ATCC 12228 (MSSE) and also led to a clinically decreased susceptibility to penicillin in S. aureus clinical strain SAL. One strain (MSSE) showed a four-fold increase in the MIC against CHG. Several strains showed a two-fold increase in the MIC against CHG and only one strain (MSSE) against OCT. CONCLUSION: These results support the urgent need to apply the same administration rules currently accepted for antibiotics to antiseptics in order to preserve the benefits both of antiseptics and antibiotics.

Molecular characterization by PCR-RFLP of indigenous fungal isolates from hypersaline stream water in România.

This is the first exhaustive report on the fungal community biodiversity in hypersaline water in România. A total of 27 fungal strains (19 molds and eight yeast) have been isolated from Lopatari hypersaline water, Buzau County. Based on classical investigation, these strains have been identified as belonging to the genera Aureobasidium, Alternaria, Aspergillus, Penicillium, and Fusarium. The molecular characterization of fungal isolates at species level was performed using PCR-RFLP analysis of the 5.8S-ITS region. PCR products were digested with different combinations of endonucleases. The most frequently isolated species were Aspergillus niger (14.81% of all isolates), A. versicolor, (14.81%) and Penicillium crustosum (14.81%). In addition, ribosomal restriction patterns which exhibited profiles specific to Aureobasidium pullulans were derived, and to discriminate between Aureobasidium isolates, the elongase-encoding gene (ELO) was chosen as a genetic marker followed by digestion with endonuclease HhaI. Five yeast isolates displayed restriction patterns corresponding to Aureobasidium melanogenum (18.52%) and three isolates to Aureobasidium pullulans (11.11%). In addition, the RFLP types of Aureobasidium pullulans varieties with HhaI are clearly distinguished and could be applied to assess the intraspecific variability.

Bee Collected Pollen with Enhanced Health Benefits, Produced by Fermentation with a Kombucha Consortium.

The bioavailability of pollen bioactive compounds for humans is limited. In this study, our aim was to enhance the health-related benefits of pollen by fermentation with a Kombucha/SCOBY (symbiotic culture of bacteria and yeasts) consortium. We performed the fermentation of pollen suspended from the beginning with SCOBY on sweetened green tea or on Kombucha vinegar, by adding pollen after 20 days of Kombucha fermentation. We analyzed: formation of bioactive compounds (anti-oxidant polyphenols, soluble silicon, hydroxy-acids, short chain fatty acids-SCFA); parameters related to Kombucha fermentation (dynamics of lactic acid bacteria-LAB, formation of organic acids, soluble sugar evolution on Kombucha vinegar); the influence of Kombucha fermentation on pollen morphology and ultrastructure; in vitro cytotoxic and antitumoral effects of the Kombucha fermented pollen. The pollen addition increases LAB proportion in the total number of SCOBY microbial strains. SEM images highlight the adhesion of the SCOBY bacteria to pollen. Ultrastructural analysis reveals the release of the pollen content. The content of bioactive compounds (polyphenols, soluble silicon species and SCFA) is higher in the fermented pollen and the product shows a moderate antitumoral effect on Caco-2 cells. The health benefits of pollen are enhanced by fermentation with a Kombucha consortium.

Non-Botrytis grape-rotting fungi responsible for earthy and moldy off-flavors and mycotoxins.

The grape microflora is complex and includes filamentous fungi, yeasts and bacteria with different physiological characteristics and effects on wine production. Most studies have focused on the wine microbiota, but a few studies have reported the ecology of grape microorganisms. Some of these organisms - such as non-Botrytis bunch rotting fungi, which greatly influence the safety or sensory quality of wine, due to the production of mycotoxins and off-flavors, respectively - are considered to be spoilage agents. We review here the diversity of filamentous fungi on grapes and the factors influencing their development, such as grape ripening stage, environmental factors (climate, rain and cultivation practices), grape variety and grape health status. We also discuss the pathways by which mycotoxins and off-flavors are produced, the control of the population, the metabolites responsible for wine spoilage and the methods for detecting and characterizing the microorganisms involved.