In vitro digestion and characterization of selenized Saccharomyces cerevisiae, Pichia fermentans and probiotic Saccharomyces boulardii.

BACKGROUND AND OBJECTIVE: Yeasts have the remarkable capability to transform and integrate inorganic selenium into their cellular structures, thereby enhancing its bioavailability and reducing its toxicity. In recent years, yeasts have attracted attention as potential alternative sources of protein. METHODS: This study explores the selenium accumulation potential of two less explored yeast strains, namely the probiotic Saccharomyces boulardii CCDM 2020 and Pichia fermentas CCDM 2012, in comparison to the extensively studied Saccharomyces cerevisiae CCDM 272. Our investigation encompassed diverse stress conditions. Subsequently, the selenized yeasts were subjected to an INFOGEST gastrointestinal model. The adherence and hydrophobicity were determined with undigested cells RESULTS: Stress conditions had an important role in influencing the quantity and size of selenium nanoparticles (SeNPs) generated by the tested yeasts. Remarkably, SeMet synthesis was limited to Pichia fermentas CCDM 2012 and S. boulardii CCDM 2020, with S. cerevisiae CCDM 272 not displaying SeMet production at all. Throughout the simulated gastrointestinal digestion, the most substantial release of SeCys2, SeMet, and SeNPs from the selenized yeasts occurred during the intestinal phase. Notably, exception was found in strain CCDM 272, where the majority of particles were released during the oral phase. CONCLUSION: The utilization of both traditional and non-traditional selenized yeast types, harnessed for their noted functional attributes, holds potential for expanding the range of products available while enhancing their nutritional value and health benefits.

Superoxide Dismutase Catalyzed Size-Adjustable Selenium Nanoparticles in Saccharomyces boulardii.

Selenium nanoparticles (SeNPs) are important and safe food and feed additives that can be used for dietary supplementation. In this study, a mutagenic strain of Saccharomyces boulardii was employed to obtain biologically synthesized SeNPs (BioSeNPs) with the desired particle size by controlling the dosage and duration of sodium selenite addition, and the average particle size achieved was 55.8 nm with protease A encapsulation. Transcriptomic analysis revealed that increased expression of superoxide dismutase 1 (SOD1) in the mutant strain effectively promoted the synthesis of BioSeNPs and the formation of smaller nanoparticles. Under sodium selenite stress, the mutant strain exhibited significantly increased expression of glutathione peroxidase 2 (GPx2), which was significantly greater in the mutant strain than in the wild type, facilitating the synthesis of glutathione selenol and providing abundant substrates for the production of BioSeNPs. Furthermore, based on the experimental results and transcriptomic analysis of relevant genes such as sod1, gpx2, the thioredoxin reductase 1 gene (trr1) and the thioredoxin reductase 2 gene (trr2), a yeast model for the size-controlled synthesis of BioSeNPs was constructed. This study provides an important theoretical and practical foundation for the green synthesis of controllable-sized BioSeNPs or other metal nanoparticles with potential applications in the fields of food, feed, and biomedicine.

New biomarkers underlying acetic acid tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii.

Evolutionary engineering experiments, in combination with omics technologies, revealed genetic markers underpinning the molecular mechanisms behind acetic acid stress tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii. Here, compared to the ancestral Ent strain, evolved yeast strains could quickly adapt to high acetic acid levels (7 g/L) and displayed a shorter lag phase of growth. Bioinformatic-aided whole-genome sequencing identified genetic changes associated with enhanced strain robustness to acetic acid: a duplicated sequence in the essential endocytotic PAN1 gene, mutations in a cell wall mannoprotein (dan4Thr192del), a lipid and fatty acid transcription factor (oaf1Ser57Pro) and a thiamine biosynthetic enzyme (thi13Thr332Ala). Induction of PAN1 and its associated endocytic complex SLA1 and END3 genes was observed following acetic acid treatment in the evolved-resistant strain when compared to the ancestral strain. Genome-wide transcriptomic analysis of the evolved Ent acid-resistant strain (Ent ev16) also revealed a dramatic rewiring of gene expression among genes associated with cellular transport, metabolism, oxidative stress response, biosynthesis/organization of the cell wall, and cell membrane. Some evolved strains also displayed better growth at high acetic acid concentrations and exhibited adaptive metabolic profiles with altered levels of secreted ethanol (4.0-6.4% decrease), glycerol (31.4-78.5% increase), and acetic acid (53.0-60.3% increase) when compared to the ancestral strain. Overall, duplication/mutations and transcriptional alterations are key mechanisms driving improved acetic acid tolerance in probiotic strains. We successfully used adaptive evolutionary engineering to rapidly and effectively elucidate the molecular mechanisms behind important industrial traits to obtain robust probiotic yeast strains for myriad biotechnological applications. KEY POINTS: •Acetic acid adaptation of evolutionary engineered robust probiotic yeast S. boulardii •Enterol ev16 with altered genetic and transcriptomic profiles survives in up to 7 g/L acetic acid •Improved acetic acid tolerance of S. boulardii ev16 with mutated PAN1, DAN4, OAF1, and THI13 genes.

Microbiome Engineering Using Probiotic Yeast: Saccharomyces boulardii and the Secreted Human Lysozyme Lead to Changes in the Gut Microbiome and Metabolome of Mice.

The probiotic yeast Saccharomyces boulardii has great potential for use as a chassis for microbiome engineering because of its high resistance to environmental stress, well-developed genetic tools, and the ability to secrete recombinant proteins in the intestine. As oral feeding of lysozyme has been reported to change the gut microbiome and fecal metabolites, we engineered S. boulardii to secrete human lysozyme, and investigated the changes in the microbiome and fecal metabolites in response to the administration of the engineered probiotic yeast into mice. Administration of S. boulardii changed the structure of the gut microbiome by promoting the growth of clostridia and increasing the diversity of strains. The human lysozyme secreted by S. boulardii in the intestine resulted in a unique gut microbiome structure through selective growth. In addition, the administration of probiotic yeast S. boulardii affected host energy metabolism and decreased blood urea and fructose levels, suggesting a mechanism of health benefits in mice. IMPORTANCE Our study identified changes in the microbiome by administering wild-type S. boulardii in mice to healthy mice based on long-read sequencing and demonstrated that a recombinant protein secreted by engineered S. boulardii in the intestine could change the microbiome. Our results provide valuable information for the development of therapeutics using engineered S. boulardii that changes the gut microbiome and host physiology.

Improving therapeutic protein secretion in the probiotic yeast Saccharomyces boulardii using a multifactorial engineering approach.

The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.

Antigenotoxicity and Cytotoxic Potentials of Cell-Free Supernatants Derived from Saccharomyces cerevisiae var. boulardii on HT-29 Human Colon Cancer Cell Lines.

Microbial-derived postbiotics are of interest recently due to their lower side effects than chemotherapy for cancer treatment and prevention. This study aimed to investigate the potential antigenotoxic and cytotoxic effects of cell-free-supernatant (CFS) postbiotics derived from Saccharomyces boulardii by applying SOS chromotest and MTT assay on HT-29 cell lines. Also, further cellular pathway-related assays such as cell cycle, DAPI, and annexin V-FITC/PI staining were performed. Real-time PCR was utilized to assess the expression levels of some genes involved in apoptosis. Based on the outcomes, the CFSs of S. boulardii showed significant antigenotoxic effects (20-60%, P < 0.05), decreased cell viability (with the significant IC50 values of 33.82, 22.68, and 27.67 µg/mL after 24, 48, and 72 h respectively), suppressed the initial (G0/G1) phase of the cell's division, influenced the nucleus of the treated cells, induced apoptosis, and increased the expression of Caspas3 and PTEN genes after 48 h, while the RelA and Bcl-XL genes indicated diminished expression in treated HT-29 cells. Consequently, CFS postbiotics of S. boulardii exhibited significant antigenotoxic and cytotoxic effects and induced apoptosis responses in HT-29 cancer cells. The results of this investigation lead us to recommend that the CFS postbiotics generated from Saccharomyces cerevisiae var. boulardii be taken into consideration as a potential anticancer agent or in the design of supplementary medications to treat and prevent colon cancers.

PRKN/parkin-mediated mitophagy is induced by the probiotics Saccharomyces boulardii and Lactococcus lactis.

Mitochondrial impairment is a hallmark feature of neurodegenerative disorders, such as Parkinson disease, and PRKN/parkin-mediated mitophagy serves to remove unhealthy mitochondria from cells. Notably, probiotics are used to alleviate several symptoms of Parkinson disease including impaired locomotion and neurodegeneration in preclinical studies and constipation in clinical trials. There is some evidence to suggest that probiotics can modulate mitochondrial quality control pathways. In this study, we screened 49 probiotic strains and tested distinct stages of mitophagy to determine whether probiotic treatment could upregulate mitophagy in cells undergoing mitochondrial stress. We found two probiotics, Saccharomyces boulardii and Lactococcus lactis, that upregulated mitochondrial PRKN recruitment, phospho-ubiquitination, and MFN degradation in our cellular assays. Administration of these strains to Drosophila that were exposed to paraquat, a mitochondrial toxin, resulted in improved longevity and motor function. Further, we directly observed increased lysosomal degradation of dysfunctional mitochondria in the treated Drosophila brains. These effects were replicated in vitro and in vivo with supra-physiological concentrations of exogenous soluble factors that are released by probiotics in cultures grown under laboratory conditions. We identified methyl-isoquinoline-6-carboxylate as one candidate molecule, which upregulates mitochondrial PRKN recruitment, phospho-ubiquitination, MFN degradation, and lysosomal degradation of damaged mitochondria. Addition of methyl-isoquinoline-6-carboxylate to the fly food restored motor function to paraquat-treated Drosophila. These data suggest a novel mechanism that is facilitated by probiotics to stimulate mitophagy through a PRKN-dependent pathway, which could explain the potential therapeutic benefit of probiotic administration to patients with Parkinson disease.

Health-promoting properties of Saccharomyces cerevisiae var. boulardii as a probiotic; characteristics, isolation, and applications in dairy products.

Saccharomyces cerevisiae var. boulardii (S. boulardii) has been isolated from lychee (Litchi chinensis), mangosteen fruit, kombucha, and dairy products like kefir. Dairy products containing S. boulardii have been revealed to possess potential probiotic activities owing to their ability to produce organic acids, essential enzymes, vitamins, and other important metabolites such as vanillic acid, phenyl ethyl alcohol, and erythromycin. S. boulardii has a wide spectrum of anti-carcinogenic, antibacterial antiviral, and antioxidant activity, and is known to reduce serum cholesterol levels. However, this yeast has mainly been prescribed for prophylaxis treatment of gastrointestinal infectious diseases, and stimulating the immune system in a number of commercially available products. The present comprehensive review article reviews the properties of S. boulardii related to their use in fermented dairy foods as a probiotic microorganism or starter culture. Technical aspects regarding the integration of this yeast into the dairy foods matrix its health advantages, therapeutic functions, microencapsulation, and viability in harsh conditions, and safety aspects are highlighted.

Unique Probiotic Properties and Bioactive Metabolites of Saccharomyces boulardii.

Saccharomyces boulardii (S. boulardii) is a probiotic and is widely used to improve the nutritional and functional value of food. This study aimed to compare the probiotic properties of S. boulardii and Saccharomyces cerevisiae. A series of in vitro probiotic experiments was performed, including simulated gastrointestinal digestion, bile salt tolerance, hydrophobicity, self-aggregation, and antioxidant and antibacterial properties. Self-aggregation and hydrophobic properties of S. boulardii were relatively poor, but they showed high tolerance, antioxidant properties, and broad antibacterial properties. In addition, non-targeted metabolomics was used to comprehensively analyze the active metabolites of S. boulardii and the metabolic differences between S. boulardii and S. cerevisiae were compared. Saccharomyces boulardii produced many bioactive metabolites, which generally showed antioxidant, antibacterial, antitumor, anti-inflammatory, and other properties. In contrast to S. cerevisiae, S. boulardii produced phenyllactic acid and 2-hydroxyisocaproic acid. There were also significant differences in their metabolic pathways. These results may be of great significance in the medical and food industries and provide a basis for understanding the metabolism of S. boulardii. It also shows that metabolomics is an effective and novel method for screening microbial functional metabolites and identifying functional differences between similar microorganisms.

Strain engineering and metabolic flux analysis of a probiotic yeast Saccharomyces boulardii for metabolizing L-fucose, a mammalian mucin component.

BACKGROUND: Saccharomyces boulardii is a probiotic yeast that exhibits antimicrobial and anti-toxin activities. Although S. boulardii has been clinically used for decades to treat gastrointestinal disorders, several studies have reported weak or no beneficial effects of S. boulardii administration in some cases. These conflicting results of S. boulardii efficacity may be due to nutrient deficiencies in the intestine that make it difficult for S. boulardii to maintain its metabolic activity. RESULTS: To enable S. boulardii to overcome any nutritional deficiencies in the intestine, we constructed a S. boulardii strain that could metabolize L-fucose, a major component of mucin in the gut epithelium. The fucU, fucI, fucK, and fucA from Escherichia coli and HXT4 from S. cerevisiae were overexpressed in S. boulardii. The engineered S. boulardii metabolized L-fucose and produced 1,2-propanediol under aerobic and anaerobic conditions. It also produced large amounts of 1,2-propanediol under strict anaerobic conditions. An in silico genome-scale metabolic model analysis was performed to simulate the growth of S. boulardii on L-fucose, and elementary flux modes were calculated to identify critical metabolic reactions for assimilating L-fucose. As a result, we found that the engineered S. boulardii consumes L-fucose via (S)-lactaldehyde-(S)-lactate-pyruvate pathway, which is highly oxygen dependent. CONCLUSION: To the best of our knowledge, this is the first study in which S. cerevisiae and S. boulardii strains capable of metabolizing L-fucose have been constructed. This strategy could be used to enhance the metabolic activity of S. boulardii and other probiotic microorganisms in the gut.

Saccharomyces boulardii, a yeast probiotic, inhibits gut motility through upregulating intestinal serotonin transporter and modulating gut microbiota.

Saccharomyces boulardii (Sb) is a widely used fungal probiotic in treating various digestive diseases, including irritable bowel syndrome (IBS). However, the specific mechanisms of Sb relieving IBS remain unclear. The abnormal serotonin transporter (SERT) / 5-hydroxytryptamine (5-HT) system could cause disordered gastrointestinal sensation and motility, which closely related to IBS pathogenesis. The aim of this study was to explore the effects and mechanisms of Sb on regulating gut motility. Sb supernatant (SbS) was administered to intestinal epithelial cells and mice. SbS upregulated SERT expression via enhancing heparin-binding epidermal growth factor (HB-EGF) release to activate epidermal growth factor receptor (EGFR). EGFR kinase inhibitor treatment or HB-EGF siRNA transfection in cells blocked SbS upregulating SERT. Consistently, SbS-treated mice presented inhibited gut motility, and EGFR activation and SERT upregulation were found. Moreover, 16 S rDNA sequence presented an evident decrease in Firmicutes / Bacteroidetes ratio in SbS group. In genus level, SbS reduced Escherichia_Shigella, Alistipes, Clostridium XlVa, and Saccharibacteria_genera_incertae_sedis, meanwhile, increased Parasutterella. The abundance of Saccharibacteria_genera_incertae_sedis positively correlated with defecation parameters and intestinal 5-HT content. Fecal microbiota transplantation showed that SbS could modulate gut microbiota to influence gut motility. Interestingly, elimination of gut microbiota with antibiotic cocktail did not entirely block SbS regulating gut motility. Furthermore, SbS administration to IBS-D mice significantly upregulated SERT and inhibited gut motility. In conclusion, SbS could upregulate SERT by EGFR activation, and modulate gut microbiota to inhibit gut motility. This finding would provide more evidence for the application of this yeast probiotic in IBS and other diarrheal disorders.

Saccharomyces boulardii exerts renoprotection by modulating oxidative stress, renin angiotensin system and uropathogenic microbiota in a murine model of diabetes.

AIMS: We aimed to investigate whether Saccharomyces boulardii strain might exert renoprotective effects by modulating renal renin angiotensin system, oxidative stress and intestinal microbiota in streptozotocin-diabetic mice. MAIN METHODS: Thirty-six C57BL/6 male mice were divided into four groups: control (C), control + probiotic (CP), diabetes (D), diabetes + probiotic (DP). Diabetes was induced by one intraperitoneal injection of streptozotocin and Saccharomyces boulardii was administered by oral gavage for 8 weeks. Blood glucose, albuminuria and urinary volume were measured. Renal levels of angiotensin peptides (angiotensin I, II and 1-7) and the activities of angiotensin-converting enzyme (ACE) and ACE2 were determined, besides that, renal morphology, serotonin and dopamine levels and also microbiota composition were analyzed. KEY FINDINGS: Probiotics significantly increased C-peptide secretion and reduced blood glucose of diabetic animals. Saccharomyces boulardii also improved renal antioxidant defense, restored serotonin and dopamine concentration, and activated the renin-angiotensin system (RAS) vasodilator and antifibrotic axis. The modulation of these markers was associated with a beneficial impact on glomerular structure and renal function of diabetic treated animals. The phenotypic changes induced by Saccharomyces boulardii were also related to modulation of intestinal microbiota, evidenced by the decreased abundance of Proteus and Escherichia-Shigella, considered diabetic nephropathy biomarkers. SIGNIFICANCE: Therefore, probiotic administration to streptozotocin-induced diabetic mice improves kidney structure and function in a murine model and might represent a reasonable strategy to counteract nephropathy-associated maladaptive responses in diabetes.

Saccharomyces boulardii attenuates lipopolysaccharide-induced anxiety-like behaviors in rats.

Anxiety is the brain's response to dangerous or stressful situations. Exposure to stressors can cause gut microbiota dysbiosis and activate the hypothalamic-pituitary-adrenal (HPA) axis, leading to the secretion of glucocorticoids associated with anxiety. Recent studies have reported that probiotics can attenuate anxiety-like behaviors by modulation of the gut microbiome composition. The present study aimed to investigate the effects of Saccharomyces boulardii (Sb) administration on anxiety-like behaviors induced by lipopolysaccharide (LPS) in rats. The animals were randomly divided into four groups (Control, LPS, Sb + LPS, and Sb). All animals were orally treated with saline or S. boulardii (1010 CFU/ml/rat) for 28 days. They were also injected with saline or LPS (250 µg/kg/day) intraperitoneally from day 14 until day 22. Anxiety-like behaviors were assessed using the elevated plus-maze and open-field tests. Besides, the serum levels of cortisol, corticosterone, serotonin, and brain-derived neurotrophic factor (BDNF) were measured. The results revealed that S. boulardii could attenuate LPS-induced anxiety-like behaviors. The findings also showed that oral administration of S. boulardii significantly attenuated the elevated levels of cortisol and corticosterone in the LPS-induced model. Moreover, S. boulardii alleviated the decremental effect of LPS on the serum serotonin and BDNF levels. According to the present findings, S. boulardii can prevent LPS-induced anxiety-like behaviors, probably through modulation of the HPA axis and the gut microbiome.

A Tunable and Expandable Transactivation System in Probiotic Yeast Saccharomyces boulardii.

Precise transcriptional modulation is a key requirement for developing synthetic probiotics with predictably tunable functionalities. In this study, an expandable and tunable transactivation system was constructed and validated in probiotic yeast Saccharomyces boulardii. The use of nuclease-null Cas9 and scaffold RNA (scRNA) directed regulation enabled transactivation under the control of a synthetic promoter in S. boulardii. A synthetic promoter consisting of the scRNA target sequence and the core GAL7 promoter region restricted interference from the native galactose regulon. The system was readily expanded by introducing new target sequences to the promoter and scRNA. Complementarity between the promoter and scRNA, and binding specificity between scRNA and transcriptional activator, served as two layers of orthogonality of the transactivation. In addition, activator expression under the control of an inducible promoter enabled control of the transactivation via chemical inducer. The described system has the potential to enable engineering of probiotic yeast to more precisely perform therapeutic functions.

Production of neoagarooligosaccharides by probiotic yeast Saccharomyces cerevisiae var. boulardii engineered as a microbial cell factory.

BACKGROUND: Saccharomyces cerevisiae var. boulardii is a representative probiotic yeast that has been widely used in the food and pharmaceutical industries. However, S. boulardii has not been studied as a microbial cell factory for producing useful substances. Agarose, a major component of red macroalgae, can be depolymerized into neoagarooligosaccharides (NAOSs) by an endo-type ß-agarase. NAOSs, including neoagarotetraose (NeoDP4), are known to be health-benefiting substances owing to their prebiotic effect. Thus, NAOS production in the gut is required. In this study, the probiotic yeast S. boulardii was engineered to produce NAOSs by expressing an endo-type ß-agarase, BpGH16A, derived from a human gut bacterium Bacteroides plebeius. RESULTS: In total, four different signal peptides were compared in S. boulardii for protein (BpGH16A) secretion for the first time. The SED1 signal peptide derived from Saccharomyces cerevisiae was selected as optimal for extracellular production of NeoDP4 from agarose. Expression of BpGH16A was performed in two ways using the plasmid vector system and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. The production of NeoDP4 by engineered S. boulardii was verified and quantified. NeoDP4 was produced by S. boulardii engineered using the plasmid vector system and CRISPR-Cas9 at 1.86 and 0.80 g/L in a 72-h fermentation, respectively. CONCLUSIONS: This is the first report on NAOS production using the probiotic yeast S. boulardii. Our results suggest that S. boulardii can be considered a microbial cell factory to produce health-beneficial substances in the human gut.

Growth, survival, and metabolic activities of probiotics Lactobacillus rhamnosus GG and Saccharomyces cerevisiae var. boulardii CNCM-I745 in fermented coffee brews.

Amidst rising demand for non-dairy probiotic foods, and growing interest in coffees with added functionalities, it would be opportune to ferment coffee brews with probiotics. However, challenges exist in maintaining probiotic viability in high-moisture food products. Here, we aimed to enhance the viability of the probiotic bacteria, Lactobacillus rhamnosus GG, in coffee brews by co-culturing with the probiotic yeast, Saccharomyces cerevisiae var. boulardii CNCM-I745. The yeast significantly enhanced the viability of L. rhamnosus GG, as bacterial populations beyond 7 Log CFU/mL were maintained throughout 14 weeks of storage at 4 and 25 °C. In contrast, the single culture of L. rhamnosus GG suffered viability losses below 6 Log CFU/mL within 10 weeks at 4 °C, and 3 weeks at 25 °C. Growth and survival of S. boulardii CNCM-I745 remained unaffected by the presence of L. rhamnosus GG. Volatile profiles of coffee brews were altered by probiotic metabolic activities, but co-culturing led to suppressed generation of diacetyl and ethanol compared to single cultures. Probiotic fermentation did not alter principal coffee bioactive compounds and antioxidant capacities; however, declines in peroxyl radical scavenging capacities were observed after ambient storage. Overall, we illustrate that yeasts are effective in enhancing probiotic bacterial viability in coffee brews, which may be useful in developing shelf stable probiotic food products.

In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii.

Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (ß-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled ß-carotene synthesis (194 µg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional ß-carotene recovered in feces was 56-fold higher than the ß-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.

Production of a synbiotic composed of galacto-oligosaccharides and Saccharomyces boulardii using enzymatic-fermentative method.

Motivated by the search for healthy alimentation and sustainable technological processes, this study aimed to produce a synbiotic composed of the prebiotic galacto-oligosaccharides (GOS) and the probiotic yeast Saccharomyces boulardii, simultaneously, using cheese whey permeate as substrate by enzymatic-fermentative method. A central composite rotatable design with center point was used to evaluate the influence of temperature and enzyme concentration in the GOS and S. boulardii production. The best condition to obtain the prebiotic was at 32 °C and enzyme concentration of 0.175% (w/w), providing 56.84 g L-1 of GOS concentration and Ln(3.59) 107 viable cells mL-1 of S. boulardii production. However, the condition that would favor the simultaneous production of GOS and S. boulardii studied through desirability function is 29.5 °C and 0.14% (w/w) of enzyme concentration. The simultaneous enzymatic-fermentative method showed promising results considering industrial application and can be easily incorporated into dairy production lines as functional food.

Carob kibbles as an alternative raw material for production of kvass with probiotic potential.

BACKGROUND: Non-diary beverages with probiotic properties are of great interest nowadays. In this research, we evaluated the suitability of carob kibbles in the manufacture of kvass. Kvass is a low-alcohol drink popular in Central and Eastern Europe and indicated as a potential non-diary beverage with probiotic properties. Therefore, the viability of probiotic strains of Lactobacillus plantarum and Saccharomyces boulardii during 4 weeks' storage in manufactured beverages was tested. RESULTS: Carob kibbles introduced significant amounts of phenolic compounds into kvasses, especially gallic acid (up to 117.45 ± 10.56 mg L-1 ), and improved antiradical activity up to 78% after fermentation. Moreover, fermentation efficiently reduced furfural and hydroxymethylfurfural content in samples up to 12.9% and 29.9%, respectively. Kvasses with rye malt extract possessed coffee-like, chocolate-like, roasted and caramel-like odours and a more bitter taste. Whereas kvass with carob kibbles was characterized by fruit-like odour and sweeter taste. Fermentation contributed to a creation as well as degradation of volatiles. L. plantarum exhibited higher general mortality during storage, whereas, in the case of S. boulardii, the viability was significantly higher regardless of the sample composition. CONCLUSION: This is the first study reporting the use of carob kibbles for kvass production. The obtained results showed that carob kibbles can replace rye malt extract, at least partially, in the production of kvass, giving to the product added health benefits. Moreover, S. boulardii is a better choice for production of kvass with probiotic properties. © 2021 Society of Chemical Industry.

Heterologous Expression of the Leuconostoc Bacteriocin Leucocin C in Probiotic Yeast Saccharomyces boulardii.

The yeast Saccharomyces boulardii is well known for its probiotic effects such as treating or preventing gastrointestinal diseases. Due to its ability to survive in stomach and intestine, S. boulardii could be applied as a vehicle for producing and delivering bioactive substances of interest to human gut. In this study, we cloned the gene lecC encoding the antilisterial peptide leucocin C from lactic acid bacterium Leuconostoc carnosum in S. boulardii. The constructed S. boulardii strain secreted a peptide, which had molecular weight corresponding to leucocin C in SDS-PAGE. The peptide band inhibited Listeria monocytogenes in gel overlay assay. Likewise, concentrated S. boulardii culture supernatant inhibited the growth of L. monocytogenes. The growth profile and acid tolerance of the leucocin C secreting S. boulardii were similar as those of the strain carrying the empty vector. We further demonstrated that the cells of the leucocin C producing S. boulardii efficiently killed L. monocytogenes, also without antibiotic selection pressure. These results showed that antilisterial activity could be added to the arsenal of probiotic activities of S. boulardii, demonstrating its potential as a carrier for therapeutics delivery.