Identification and Selection of Prospective Probiotics for Enhancing Gastrointestinal Digestion: Application in Pharmaceutical Preparations and Dietary Supplements.

Our study investigated the effectiveness of 446 strains of lactic acid bacteria (LAB) belonging to different species and isolated from diverse sources (food, human, and animal) as potential probiotic candidates, with the perspective of producing dietary supplements or pharmacological formulations suitable for enhancing gastrointestinal digestion. The survival capability of all the isolates under harsh gastrointestinal tract conditions was evaluated, in which only 44 strains, named high-resistant, were selected for further food digestibility investigations. All 44 strains hydrolyzed raffinose and exhibited amino and iminopeptidase activities but at various extents, confirming species- and strain-specificity. After partial in vitro digestion mimicking oral and gastric digestive phases, food matrices were incubated with single strains for 24 h. Fermented partially digested matrices provided additional functional properties for some investigated strains by releasing peptides and increasing the release of highly bio-accessible free phenolic compounds. A scoring procedure was proposed as an effective tool to reduce data complexity and quantitively characterize the probiotic potential of each LAB strain, which could be more useful in the selection procedure of powerful probiotics.

How Microbiome Composition Correlates with Biochemical Changes during Sauerkraut Fermentation: a Focus on Neglected Bacterial Players and Functionalities.

This study provided a new perspective on the bacterial community succession during sauerkraut fermentation, and on resulting metabolic functions. While culture-dependent methods confirmed the key role of the well-known core microbiome species, metagenomic approach (shotgun) revealed Secundilactobacillus malefermentans as a species of the core microbiome, especially during the last weeks of fermentation. Although the potentiality of S. malefermentans has not yet fully explored, it held core functional genes usually attributed to others lactic acid bacteria driving sauerkraut fermentation. Based on our results it is arguable that S. malefermentans might have a key a role during sauerkraut fermentation carried out at low temperature. Under our experimental conditions, the profile of phenolic compounds changed throughout sauerkraut fermentation. The amount of free phenolics, including free phenolic acids, increased at the beginning of the fermentation, whereas the conversion of phenolic acids into microbial derivatives was consistent during the last part of the sauerkraut fermentation. We pioneered correlating changes in the phenolics profile to changes in the microbiome, although the framework presented is still fragmentary. Annotated genes linked to the phenolic compounds metabolism (VprA and padA) were found in many core species during the whole process. A high metabolic potential for phenolics bioconversion emerged for lactobacilli and Pediococcus spp. through correlation analysis between microbiome composition and phenolics profile. IMPORTANCE Our study was not limited to describe the succession pattern of the microbial community during sauerkraut fermentation, but also revealed how some neglected bacterial players belong to the core species during sauerkrauts processing, especially at low temperature. Such species might have a role as potential starters to optimize the fermentation processes and to obtain sauerkrauts with improved and standardized nutritional and sensory features. Furthermore, our correlations between microbiome composition and phenolics profile might also represent new references for sauerkraut biotechnology, aiming to identify new metabolic drivers of potential sauerkraut functionalities. Finally, sauerkraut ecosystem is a tractable model, although with high level of complexity, and resultant ecological information might be extended to other plant ecosystems.

Glutathione production by Saccharomyces cerevisiae: current state and perspectives.

Glutathione (L-γ-glutamyl-cysteinyl-glycine, GSH) is a tripeptide synthesized through consecutive enzymatic reactions. Among its several metabolic functions in cells, the main one is the potential to act as an endogenous antioxidant agent. GSH has been the focus of numerous studies not only due to its role in the redox status of biological systems but also due to its biotechnological characteristics. GSH is usually obtained by fermentation and shows a variety of applications by the pharmaceutical and food industry. Therefore, the search for new strategies to improve the production of GSH during fermentation is crucial. This mini review brings together recent papers regarding the principal parameters of the biotechnological production of GSH by Saccharomyces cerevisiae. In this context, aspects, such as the medium composition (amino acids, alternative raw materials) and the use of technological approaches (control of osmotic and pressure conditions, magnetic field (MF) application, fed-batch process) were considered, along with genetic engineering knowledge, trends, and challenges in viable GSH production. KEY POINTS: • Saccharomyces cerevisiae has shown potential for glutathione production. • Improved technological approaches increases glutathione production. • Genetic engineering in Saccharomyces cerevisiae improves glutathione production.

From the vineyard to the cellar: new insights of Starmerella bacillaris (synonym Candida zemplinina) technological properties and genomic perspective.

A large diversity of yeasts can be involved in alcoholic fermentation; however, Starmerella bacillaris strains have gained great attention due to their relevant and particular characteristics. S. bacillaris is commonly known as an osmotolerant, acidogenic, psychrotolerant, and fructophilic yeast. Most strains of this species are high producers of glycerol and show low ethanol production rates, being highlighted as promising alternatives to the manufacture of low-alcohol beverages. The increased production of high alcohols, such as benzyl alcohol that has antifungal and antibacterial properties, highlights S. bacillaris potential as a biocontrol agent. After harvest, antifungal yeasts become part of the must microbiota and may also improve the fermentation process. Moreover, during the fermentation, S. bacillaris releases important molecules with biotechnological properties, such as mannoproteins and glutathione. Considering the potential biotechnological properties of S. bacillaris strains, this review presents an overview of recent trends concerning the application of S. bacillaris in fermented beverages. KEY POINTS: •S. bacillaris as an alternative to the production of low-alcohol beverages. •S. bacillaris strains present biocontrol potential. •Molecules released by S. bacillaris may be of great biotechnological interest.

Chemoprevention of DMH-Induced Early Colon Carcinogenesis in Male BALB/c Mice by Administration of Lactobacillus Paracasei DTA81.

We evaluated the effects of the probiotic candidate Lactobacillus paracasei DTA81 (DTA81) on liver oxidative stress, colonic cytokine profile, and gut microbiota in mice with induced early colon carcinogenesis (CRC) by 1,2-dimethylhydrazine (DMH). Animals were divided into four different groups (n = 6) and received the following treatments via orogastric gavage for 8 weeks: Group skim milk (GSM): 300 mg/freeze-dried skim milk/day; Group L. paracasei DTA81 (DTA81): 3 × 109 colony-forming units (CFU)/day; Group Lactobacillus rhamnosus GG (LGG): 3 × 109 CFU/day; Group non-intervention (GNI): 0.1 mL/water/day. A single DMH dose (20 mg/kg body weight) was injected intraperitoneally (i.p), weekly, in all animals (seven applications in total). At the end of the experimental period, DTA81 intake reduced hepatic levels of carbonyl protein and malondialdehyde (MDA). Moreover, low levels of the pro-inflammatory cytokines Interleukin-6 (IL-6) and IL-17, as well as a reduced expression level of the proliferating cell nuclear antigen (PCNA) were observed in colonic homogenates. Lastly, animals who received DTA81 showed an intestinal enrichment of the genus Ruminiclostridium and increased concentrations of caecal acetic acid and total short-chain fatty acids. In conclusion, this study indicates that the administration of the probiotic candidate DTA81 can have beneficial effects on the initial stages of CRC development.

Complete Genome Sequence and Carbohydrates-Active EnZymes (CAZymes) Analysis of Lactobacillus paracasei DTA72, a Potential Probiotic Strain with Strong Capability to Use Inulin.

The whole genome sequence of Lactobacillus paracasei DTA72, isolated from healthy infant feces, is reported, along with the Carbohydrates-Active enZymes (CAZymes) analysis and an in silico safety assessment. Strain DTA72 had previously demonstrated some interesting potential probiotic features, such as a good resistance to gastrointestinal conditions and an anti-Listeria activity. The 3.1 Mb sequenced genome consists of 3116 protein-coding sequences distributed on 340 SEED subsystems. In the present study, we analyzed the fermentation capability of strain DTA72 on six different carbohydrate sources, namely, glucose, fructose, lactose, galactose, xylose, and inulin by using phenotypical and genomic approaches. Interestingly, L. paracasei DTA72 evidenced the best growth performances on inulin with a much shorter lag phase and higher number of cells at the stationary phase in comparison with all the sugars tested. The CAZyme analysis using the predicted amino acid sequences detected 80 enzymes, distributed into the five CAZymes classes. Moreover, the in silico analysis revealed the absence of blood hemolytic genes, transmissible antibiotic resistances, and plasmids in DTA72. The results described in this study, together with those previously reported and particularly the strong capability to utilize inulin as energy source, make DTA72 a very interesting potential probiotic strain to be considered for the production of synbiotic foods. The complete genome data have been deposited in GenBank under the accession number WUJH00000000.

Safety and Stability of Two Potentially Probiotic Lactobacillus Strains After In Vitro Gastrointestinal Transit.

According to FAO and WHO, probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Most probiotic bacteria used today belong to the genera Lactobacillus and Bifidobacterium and are of animal or human origin. The fundamental characteristic routinely evaluated in potential probiotics strains is their limited viability loss during gastrointestinal transit (GIT), but to date, no studies reported whether probiotics, besides viability, still also maintain their beneficial properties intact. To study this aspect, we considered two strains, Lactobacillus rhamnosus DTA 79 and L. paracasei DTA 83, previously characterised for the presence of some probiotic properties, isolated from faeces of 7- to 21-day-old babies. Here, we examined some additional properties, namely antibiotic resistance, resistance to lysozyme, presence of haemolytic activity and inhibition of pathogen biofilm formation. We then tested the effect of in vitro GIT on all these features and our results show evidence that this procedure had in some cases limited and in others no significant effects on them. Additionally, we examined the gastrointestinal resistance of the strains after skim milk fermentation and successive storage of the product for 20 and 40 days at refrigeration temperature, to see whether prolonged storage could weaken cell resistance to GIT. Our results demonstrate that a protracted refrigeration period before in vitro GIT did not affect or influenced very weakly this essential probiotic property.

Growth Parameters and Survivability of Saccharomyces boulardii for Probiotic Alcoholic Beverages Development.

The aim of this research was to optimize the growth parameters (pH, ethanol tolerance, initial cell concentration and temperature) for Saccharomyces boulardii and its tolerance to in vitro gastrointestinal conditions for probiotic alcoholic beverage development. Placket-Burman screening was used to select only statistically significant variables, and the polynomial mathematical model for yeast growth was obtained by central composite rotatable design. Confirmation experiments to determine the kinetic parameters for yeast growth were carried out by controlling the temperature and pH. Soon after, the survivability of yeast was tested under in vitro conditions mimicking the human upper gastrointestinal transit. S. boulardii had suitable resistance to alcohol and gastrointestinal conditions for probiotic alcoholic beverage development.

Potential use of Starmerella bacillaris as fermentation starter for the production of low-alcohol beverages obtained from unripe grapes.

To obtain beverages with reduced alcohol content, the use of unripe grapes, with low sugar and high malic acid concentration, was recently explored. Due to the low sugar, ethanol and glycerol production is limited during fermentation affecting important sensory aspects such as the palate fullness of these beverages. The high acidity influences their organoleptic quality, as well. So far, only S. cerevisiae starter, used in conventional fermentations, have been tested in this condition, and no selection has been performed to identify alternative yeasts suitable for unripe grape fermentation. S. bacillaris is known for the low ethanol tolerance, high glycerol and moderate volatile acidity production. Therefore, this non- Saccharomyces yeast have been investigated to improve the quality of low-alcohol beverages. Seven S. bacillaris strains were tested in synthetic musts with different sugar and malic acid levels, mimicking natural ripe and unripe grape musts. In all the tested conditions, S. bacillaris produced higher glycerol than S. cerevisiae. In single-strain fermentation at low sugar and high malic acid no S. bacillaris strains was able to transform all the sugars, although the produced ethanol was lower than that at high sugar condition. Therefore, sequential fermentations with S. cerevisiae were evaluated at low sugar and high malic acid. In this condition all the sugars were consumed and a significant glycerol increase was found. These results were confirmed when sequential fermentations were run in natural unripe grape must. Moreover, an increase in malic acid degradation, with respect to EC1118 single-strain fermentation, was observed.

Draft genome sequence data of Lactobacillus paracasei strain DTA83 isolated from infant stools.

Here the draft genome sequence of Lactobacillus paracasei strain DTA83, isolated from stools of healthy infants in Rio de Janeiro (Brazil), is reported. The 2.8-Mb genome possesses 2825 protein-coding sequences distributed on 330 SEED subsystems. This strain belongs to a set of potentially probiotic Lactobacillus spp. strains used to study genetic factors related to antibiotic resistance after stress conditions, such as simulated gastrointestinal conditions. The complete genome data have been deposited in GenBank under the accession number QRBH00000000, https://www.ncbi.nlm.nih.gov/nuccore/QRBH00000000.

Whole genome comparison of two Starmerella bacillaris strains with other wine yeasts uncovers genes involved in modulating important winemaking traits.

Starmerella bacillaris is an osmotolerant yeast with interesting winemaking traits such as low-ethanol and high-glycerol production, previously considered as wine spoilage and recently proposed to improve the sensory quality of wine. This is the first work performing a whole-genome analysis of the variants identified by comparing two S. bacillaris strains (PAS13 and FRI751). Additionally, an extensive search for orthologous genes against Saccharomyces and non-Saccharomyces yeasts produced a detailed reconstruction of the pan-genome for yeast species used in winemaking. Starmerella bacillaris PAS13 was able to produce 36% more glycerol than S. bacillaris FRI751 without increasing ethanol level over 5% (v/v). Orthologous genes revealed new insights in the response to osmotic stress determined by the mitogen-activated protein kinase (MAPK) from S. bacillaris strains. The comparison between the two S. bacillaris genomes revealed 33 771 high-quality variants that were ranked considering their predicted impact on gene functions. Furthermore, analysis of structural variations in the genome revealed five translocations. The absence of some transcriptional factors involved in the regulation of GPD (glycerol-3-phosphate dehydrogenase), like the protein kinases YpK1p and YpK2p, and the identification of a tandem duplication increasing the GPP1 (glycerol-3-phosphate phosphatase) gene copy number suggest a remarkably different regulation of the glycerol pathway for S. bacillaris in comparison to S. cerevisiae.

The Different Physical and Chemical Composition of Grape Juice and Marc Influence Saccharomyces cerevisiae Strains Distribution During Fermentation.

During white-grape winemaking, grape marc is separated from juice immediately after crushing. Both mark and juice are obtained from the same grapes, but they differ strongly for their physical and chemical properties. Marc is mainly composed of solid residues. Its pH is usually higher than that of the juice and Saccharomyces cerevisiae strains are largely present. Therefore, it can be considered as a potential alternative environment for the selection of industrial yeasts. In order to evaluate the effect of different pH and physical state of the two matrices on grapes yeast population composition, the isolation of S. cerevisiae, from both grape juice and marc during simultaneous fermentations, was performed. After yeast identification and genotyping, strains present at high frequencies were tested in fermentation at different pH values. Biofilm production was also tested to evaluate strain ability to develop on a solid matrix. Genotype analysis showed that high-frequency strains were always more abundant in one of the two environments, suggesting the existence of a selective effect. Generally, fermentations at different pH revealed that the best fermentation performance of each strain, in terms of CO2 production, was in the pH range of its original environment. Only one strain, mostly present in grape marc, produced a high biofilm level. Therefore, biofilm production does not seem to favor strain adaptation to grape marc condition. PRACTICAL APPLICATION: These results demonstrate that grape juice and marc represent two different environments able to influence yeast strains distribution. The pH level can be included among the selection factors acting on yeast strains distribution. Grape marc can be considered a yeasts reservoir and its fermentation can be used for the development and isolation of new strains, genetically and physiologically different from those present in the grape juice.

Lactobacillus paracasei probiotic properties and survivability under stress-induced by processing and storage of ice cream bar or ice-lolly / Propriedades probióticas e sobrevivência de Lactobacillus paracasei sobre estresse induzido pelo processamento e armazenamento em picolé a base de leite ou água

ABSTRACT: Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. The aim of this study was to identify genotypically lactobacilli strains isolated from newborn stools and selecting strain based on probiotic properties (gastrointestinal tolerance, antibiotic susceptibility, inhibition of pathogen biofilm formation, absence of alfa or gamma-blood hemolysis, and lysozyme sensibility) and technological properties of surviving either in ice cream bar or ice-lolly. Reduction of 1.2log cfu ml-1 of the Lactobacillus paracasei strain was observed after exposure through in vitro gastrointestinal conditions. It inhibited biofilms of Escherichia coli, Salmonella Typhimurium and Candida albicans by mechanisms of competition, exclusion and displacement, and was resistant up to 3000μg ml-1 of egg white lysozyme. It presented neither alfa nor gamma-hemolysis or was antibiotic resistant to usual antibiotics for human use. Microbial survivability in ice cream bar or ice-lolly was assessed up to 21 days of storage at -18°C. Viability was maintained in ice cream bar, but there was a reduction of almost 2.0logs in ice-lolly.

RESUMO: Probióticos são microrganismos vivos que, quando administrados em quantidade adequadas, conferem benefícios à saúde do hospedeiro. O objetivo desta pesquisa foi identificar genotipicamente lactobacilos isolados de material fecal de recém-nascidos, assim como selecionar uma cepa com base em suas propriedades probióticas (tolerância às condições gastrointestinais, susceptibilidade à antibióticos, inibição da formação de biofilmes por microrganismos patogênicos, ausência de hemólise alfa ou gama e sensibilidade à lisozima) e tecnológicas de sobrevivência em picolé à base de água ou leite. Houve redução de 1,2log ufc ml-1 do isolado identificado como Lactobacillus paracasei após passagem pelas condições gastrointestinais in vitro. Esta cepa inibiu a formação de biofilmes de Escherichia coli, Salmonella Typhimurium e Candida albicans pelos mecanismos de competição, exclusão e desacoplamento e apresentou resistência até 3000μg ml-1 frente a lisozima de ovo. Entre os aspectos de segurança, a cepa não apresentou hemólise alfa ou gama e não foi resistente a antibióticos comumente ministrados em humanos. A sobrevivência microbiana em picolé a base de água ou leite foi analisada ao longo de 21 dias de estocagem a -18°C. Houve manutenção da viabilidade no produto à base de leite, mas naquele à base de água, ocorreu redução de quase 2,0logs.

Whole-Genome Sequence of Starmerella bacillaris PAS13, a Nonconventional Enological Yeast with Antifungal Activity.

Starmerella bacillaris is a fermentative yeast commonly found in vineyards. Here, we present the draft genome sequence of S. bacillaris PAS13, a nonconventional enological yeast with a potential role as a biocontrol agent. This gene sequence will provide insights into the genetic basis of yeast activity against gray mold disease (Botrytis cinerea).

Draft Genome Sequence of the Yeast Starmerella bacillaris (syn., Candidazemplinina) FRI751 Isolated from Fermenting Must of Dried Raboso Grapes.

Starmerella bacillaris is an ascomycetous yeast commonly present in enological environments. Here, we report the first draft genome sequence of S. bacillaris FRI751, which will facilitate the study of the characteristics of this interesting enological yeast.

Corrigendum: Biocontrol Ability and Action Mechanism of Starmerella bacillaris (Synonym Candida zemplinina) Isolated from Wine Musts against Gray Mold Disease Agent Botrytis cinerea on Grape and Their Effects on Alcoholic Fermentation.

[This corrects the article on p. 1249 in vol. 7, PMID: 27574517.].