Evaluation of probiotic efficacy of indigenous yeast strain, Saccharomyces cerevisiae Y-89 isolated from a traditional fermented beverage of West Bengal, India having protective effect against DSS-induced colitis in experimental mice.

Increasing awareness regarding health promotion and disease prevention has driven inclusion of fermented foods and beverages in the daily diet. These are the enormous sources of beneficial microbes, probiotics. This study aims to isolate yeast strains having probiotic potential and effectivity against colitis. Initially, ninety-two yeast strains were isolated from Haria, an ethnic fermented beverage of West Bengal, India. Primary screening was done by their acid (pH 4) and bile salt (0.3%) tolerance ability. Four potent isolates were selected and found effective against Entamoeba histolytica, as this human pathogen is responsible to cause colitis. They were identified as Saccharomyces cerevisiae. They showed luxurious growth even at 37 oC, tolerance up to 5% of NaCl, resistance to gastric juice and high bile salt (2.0%) and oro-gastrointestinal transit tolerance. They exhibited good auto-aggregation and co-aggregation ability and strong hydrophobicity. Finally, heat map and principal component analysis revealed that strain Y-89 was the best candidate. It was further characterised and found to have significant protective effects against DSS-induced colitis in experimental mice model. It includes improvement in colon length, body weight and organ indices; reduction in disease activity index; reduction in cholesterol, LDL, SGPT, SGOT, urea and creatinine levels; improvement in HDL, ALP, total protein and albumin levels; decrease in coliform count and restoration of tissue damage. This study demonstrates that the S. cerevisiae strain Y-89 possesses remarkable probiotic traits and can be used as a potential bio-therapeutic candidate for the prevention of colitis.

Alcoholic and non-alcoholic rosé wines made with Saccharomyces cerevisiae var. boulardii probiotic yeast.

The production of alcoholic and non-alcoholic rosé wines using Saccharomyces cerevisiae var. boulardii probiotic yeast is described in this study for the first time. Before and after fermentation and distillation, the volatile acidity, lactic, and malic acid levels were evaluated for S. cerevisiae var. boulardii. These contents were compared to those obtained with a standard S. cerevisiae EC-1118 yeast. We measured the levels of gluconic acid and free amino nitrogen in the musts. After fermentation and distillation, yeast viability was assessed as a function of time (0, 15 days, 3 months, and 6 months), both at ambient temperature (25 ± 0.5 °C) and refrigerator temperature (4 ± 0.5 °C). The outcomes revealed that the rosé wine made with S. cerevisiae var. boulardii had the same values and preliminary sensory characteristics as other commercial wines made with S. cerevisiae EC-1118. The S. cerevisiae var. boulardii yeast successfully survived the high alcohol level produced during fermentation and vacuum distillation. The study also revealed that this unique rosé wine retains its probiotic viability for at least 6 months when stored at room temperature or in the refrigerator, making it a suitable candidate for large-scale production where long storage intervals are required by both producers and consumers.

Exploring Extracellular Vesicles of Probiotic Yeast as Carriers of Biologically Active Molecules Transferred to Human Intestinal Cells.

Extracellular vesicles (EVs) are nanoparticles containing various bioactive cargos-e.g., proteins, RNAs, and lipids-that are released into the environment by all cell types. They are involved in, amongst other functions, intercellular communication. This article presents studies on EVs produced by the probiotic yeast Saccharomyces boulardii CNCM I-745. The size distribution and concentration of EVs in the liquid culture of yeast were estimated. Moreover, the vesicles of S. boulardii were tested for their cytotoxicity against three model human intestinal cell lines. This study did not show any significant negative effect of yeast EVs on these cells under tested conditions. In addition, EVs of S. boulardii were verified for their ability to internalize in vitro with human cells and transfer their cargo. The yeast vesicles were loaded with doxorubicin, an anticancer agent, and added to the cellular cultures. Subsequently, microscopic observations revealed that these EVs transferred the compound to human intestinal cell lines. A cytotoxicity test confirmed the activity of the transferred doxorubicin. Detailed information about the proteins present in EVs might be important in terms of exploring yeast EVs as carriers of active molecules. Thus, proteomic analysis of the EV content was also conducted within the present study, and it allowed the identification of 541 proteins after matching them to the Saccharomyces Genome Database (SGD). Altogether, this study provides strong evidence that the EVs of the probiotic CNCM I-745 strain could be considered a drug delivery system.

Potential assessment of probiotic Cystobasidium benthicum LR192 strain in mice.

Antibiotic bacterial resistant is a huge concern worldwide and probiotics offer an alternative to mitigate it. This study explores Cystobasidium benthicum LR192 as possible probiotic through microbiological and immunological analyses in mouse model. C. benthicum LR192 was isolated from lichens in a hyperarid environment in Baja California Sur, Mexico. First, microbiological analysis was assessed using 1 × 105 CFU/mL in YM broth: resistance to 1% of bile salts and pH of 2, 3 and 5 (control). Then, yeast capacity to adhere onto the intestinal mucosa and safety to mouse splenocytes were tested. Finally, immunological parameters (phagocytic ability, respiratory burst and myeloperoxidase activities, nitric oxide and IgG production) and immune-associated gene expression (IL-1ß, IL-6 and INF-γ) were determined in daily supplemented mice with the yeast (1 × 108 CFU) at days 10 and 15. The results indicate that C. benthicum LR192 has medium resistance to bile salts and low pH, can adhere to the intestine and did not cause cytotoxicity in splenocytes. Immune parameters and immune-related gene expression indicated immunomodulation at day 10 and 15, specially in leucocytes challenged with Escherichia coli. In conclusion, C. benthicum LR192 showed safe potential probiotic properties, but further studies should be performed to confirm it as a probiotic prospect for humans.

Transcriptome-wide differences between Saccharomyces cerevisiae and Saccharomyces cerevisiae var. boulardii: Clues on host survival and probiotic activity based on promoter sequence variability.

Although Saccharomyces cerevisiae and S. cerevisiae var. boulardii share more than 95% genome sequence homology, only S. cerevisiae var. boulardii displays probiotic activity. In this study, the transcriptomic differences exhibited by S. cerevisiae and S. cerevisiae var. boulardii in intestinal like medium were evaluated. S. cerevisiae was found to display stress response overexpression, consistent with higher ability of S. cerevisiae var. boulardii to survive within the human host, while S. cerevisiae var. boulardii exhibited transcriptional patterns associated with probiotic activity, suggesting increased acetate biosynthesis. Resorting to the creation of a S. cerevisiae var. boulardii genomic database within Yeastract+, a possible correlation between loss or gain of transcription factor binding sites in S. cerevisiae var. boulardii promoters and the transcriptomic pattern is discussed. This study suggests that S. cerevisiae var. boulardii probiotic activity, when compared to S. cerevisiae, relies, at least partially, on differential expression regulation, based on promoter variability.

Effect of the Addition of Buckwheat Sprouts Modified with the Addition of Saccharomyces cerevisiae var. boulardii to an Atherogenic Diet on the Metabolism of Sterols, Stanols and Fatty Acids in Rats.

The aim of the study was to evaluate the effect of the addition of Fagopyrum esculentum Moench buckwheat sprouts modified with the addition of Saccharomyces cerevisiae var. boulardii to an atherogenic diet on the metabolism of sterols and fatty acids in rats. It was noticed in the study that the group fed with modified sprouts (HFDPRS) had a greater amount of sterols by 75.2%, compared to the group fed on an atherogenic diet (HFD). The content of cholesterol in the liver and feces was lower in the HFDPRS group than the HFD group. In the serum of the HFDPRS group, a more significant amount of the following acids was observed: C18:2 (increase by 13.5%), C20:4 (increase by 15.1%), and C22:6 (increase by 13.1%), compared to the HFDCS group. Regarding the biochemical parameters, it was noted that the group fed the diet with the addition of probiotic-rich sprouts diet had lower non-HDL, LDL-C and CRP ratios compared to the group fed the high-fat diet. The obtained results indicate that adding modified buckwheat sprouts to the diet by adding the probiotic strain of the yeast may have a significant impact on the metabolism of the indicated components in the organism.

Dietary supplementation of marine yeast Yarrowia lipolytica modulates immune response in Litopenaeus vannamei.

The immunostimulatory potential of the marine yeast Yarrowia lipolytica (D1 and N6 strains) administered orally was evaluated in the white shrimp Litopenaeus vannamei. Yeasts and commercial glucans were mixed with a commercial feed to formulate diets with a 1.1% concentration of immunostimulants. The shrimp were fed daily for a period of 21 days. Weekly determinations were performed for immunological parameters in hemolymph, such as total hemocyte count (THC), lysozyme activity (LYZ), prophenoloxidase activity, antioxidant enzymatic activities (superoxide dismutase [SOD], catalase [CAT], and peroxidases), and bactericidal activity against Vibrio parahaemolyticus. Expression profiles of penaeidin (PEN), lysozyme (LYZ), and prophenoloxidase (proPO) immune genes were evaluated in hemocytes. In general, an increase in the immune parameters was observed in shrimp fed yeast diet compared to glucan and the control diets. Yarrowia lipolytica, especially strain N6, provided maximum immunostimulatory effects evidenced by the increase of immune parameters (THC, LYZ, SOD, CAT) and gene expression profile. In conclusion, this study demonstrated that Y. lipolytica had immunostimulatory effects and increased bactericidal activity in L. vannamei hemocytes against V. parahaemolyticus. These findings open the path for the potential application of Y. lipolytica-based immunostimulant for shrimp aquaculture.

Evaluation of sensory acceptance, purchase intention and color parameters of potentially probiotic mead with Saccharomyces boulardii.

Mead is a fermented alcoholic beverage produced by yeast action on a diluted solution of honey. In this study, for the first time, sensory acceptance, purchase intention and color parameters of potentially probiotic mead with Saccharomyces boulardii were evaluated. The mead with S. boulardii presented yeast counts higher than 106 CFU/mL, being considered potentially probiotic, and tended to be yellow in color. About 160 tasters participated in the sensory evaluation, and 69.38% knew mead, but only 35.62% had tried the beverage. In terms of acceptance, the mead were within the acceptable range (above 5), and F2 (with initial soluble solids of 30° Brix and S. boulardii concentration of 0.030 g/L) was the most accepted, with an overall average of 7.63 ± 1.42 on the nine-point hedonic scale. In addition, F2 presented the highest purchase intention. In conclusion, the mead showed a tendency towards the color yellow and good sensory acceptance.

Assessment of the Probiotic Properties of Yarrowia lipolytica Isolated from Cold-Pressed Olive Oil.

This research aimed to identify the probiotic features of Yarrowia lipolytica strains isolated from olive oils in Turkey. The in vitro survival capabilities of Y. lipolytica strains in gastric and pancreatic solutions were assessed. The hydrophobicity of Y. lipolytica strains was determined to be between 25.8% and 46.08% for xylene, 22.5% and 45.85% for chloroform, and 14.83% and 37.09% for ethyl acetate. In addition, auto-aggregation values were measured as 11.07-60.35%; 16.28-67.70% and 42.89-85.21% after 2, 4 and 24 h of incubation, respectively. The Y. lipolytica strains tested in this study demonstrated aggregation ability against the pathogens Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 7644. Antibiotic resistance and hemolytic activities were also checked to ensure the safety of the Y. lipolytica. Cholesterol removal by Y. lipolytica strains ranged from 12.30% to 47.42%, and their free radical scavenging activity varied between 2.85% and 39.10%. Out of 13 Y. lipolytica samples from 10 different olive oil sources, Y. lipolytica Y6, Y7, and Y11 exhibited the best strains with probiotic potential properties. This study discovered that Y. lipolytica with probiotic properties can be isolated in olive oil samples, a finding that has not been previously documented in the literature and may have potential industrial applications.

Production of iron enriched Saccharomyces boulardii: impact of process variables.

About half of the 1.62 billion cases of anemia are because of poor diet and iron deficiency. Currently, the use of iron-enriched yeasts can be used as the most effective and possible way to prevent and treat anemia due to the ability of biotransformation of mineral compounds into the organic form. In this research, for the first time, Saccharomyces (S.) boulardii was used for iron enrichment with the aim that the probiotic properties of yeast provide a potential iron supplement besides improving the bioavailability of iron. Also, due to its higher resistance than other Saccharomyces strains against stresses, it can protect iron against processing temperatures and stomach acidic-enzymatic conditions. So, the effect of three important variables, including concentration of iron, molasses and KH2PO4 on the growth and biotransformation of yeast was investigated by the Box-Behnken design (BBD). The best conditions occurred in 3 g/l KH2PO4, 20 g/l molasses and 12 mg/l FeSO4 with the highest biotransformation 27 mg Fe/g dry cell weight (DCW) and 6 g/l biomass weight. Such yeast can improve fermented products, provide potential supplement, and restore the lost iron of bread, which is a useful iron source, even for vegetarians-vegans and play an important role in manage with anemia. It is recommended that in future researches, attention should be paid to increasing the iron enrichment of yeast through permeabilizing the membrane and overcoming the structural barrier of the cell wall.

Postbiotic characterization of a potential probiotic yeast isolate, and its microencapsulation in alginate beads coated layer-by-layer with chitosan.

Considering biosafety concerns and survivability limitations of probiotics (PRO) under different stresses, application of postbiotics and encapsulated PRO has received considerable attentions. Accordingly, the objective of the present study was to investigate the postbiotic capabilities of a potential PRO yeast isolate and the effect of encapsulation with alginate (Alg) and chitosan (Ch) on its survival under SGI conditions. Sequencing results of the PCR products led to the identification of Saccharomyces cerevisiae as the selected potential PRO yeast isolated from wheat germ sourdough. High survival of the isolate under simulated gastrointestinal (SGI) conditions (95.74%), its proper adhesion abilities, as well as its potent inhibitory activity against Listeria monocytogenes (75.84%) and Aspergillus niger (77.35%) were approved. Interestingly, the yeast cell-free supernatant (CFS) showed the highest antioxidant (84.35%) and phytate-degrading (56.19%) activities compared to the viable and heat-dead cells of the isolate. According to the results of the HPLC-based assay, anti-ochratoxin A (OTA) capability of the dead cells was also significantly (P < 0.05) higher than that of the viable cell. Meanwhile, the yeast CFS had no anti-OTA and antimicrobial activities against the foodborne bacteria and fungi tested. Further, microencapsulation of the yeast isolate in Alg beads coated layer-by-layer with Ch (with 77.02% encapsulation efficacy and diameter of 1059 µm based on the field emission scanning electron microscopy analysis) significantly enhanced its survivability under SGI conditions in comparison with the free cells. In addition, electrostatic cross-linking between negatively charged carboxylic groups of Alg and positively charged amino groups of Ch was verified in accordance with Fourier transform infrared and zeta potential data. Human and/or industrial food trials in future are needed for practical applications of these emerging ingredients.

Investigation of impact of siderophore and process variables on production of iron enriched Saccharomyces boulardii by Plackett-Burman design.

The primary cause of anemia worldwide is due to poor diet and iron deficiency. Iron (Fe) enriched yeast can be the most effective way to manage anemia because of the capability for biotransformation of mineral to organic and bioavailable iron. To overcome the low richness of yeast, the use of siderophore as cellular iron carriers is a new approach. In this research, for the first time the potential of siderophore in increasing the Fe enrichment of Saccharomyces boulardii (S. boulardii), which is important because of its probiotic properties and resistance to different stresses, has been investigated to produce of potential iron supplements. For this purpose, siderophore was produced by Pseudomonas aeruginosa (P. aeruginosa). Siderophore impact, along with ten other independent process variables, has been studied on the efficiency of iron biotransformation by the Plackett-Burman design (PBD). The results showed that the highest biotransformation yield was 17.77 mg Fe/g dry cell weight (DCW) in the highest biomass weight of 9 g/l. Iron concentration is the most important variable, with contributions of 46% and 70.79% for biomass weight and biotransformation, respectively, followed by fermentation time, agitation speed, and KH2PO4 concentration. But increasing the level of siderophore and zinc led to a significant negative effect. siderophore inefficiency may be attributed to the absence of membrane receptors for pyoverdine (Pvd) and pyochelin (Pch) siderophores. Also, the steric hindrance of the cell wall mannan, the stickiness and sediment ability of the yeast, can create limitations in the absorption of elements. Such yeast can be used as a potential source of iron even for vegetarians and vegans in the form of medicinal and fortified food products to improve the treatment of anemia. It is recommended that further research be focused on increasing the iron enrichment of yeast by overcoming the structural barrier of the cell wall, investigating factors affecting membrane permeability and iron transport potential of other types of siderophores.

In vitro assessment for the probiotic potential of Pichia kudriavzevii.

It is of interest to isolate the probiotic yeast Pichia kudriavzevii based on its probiotic characteristics and enzyme production. The isolate was able to withstand high acid, bile concentration and showed a high viability. Additionally, it showed auto aggregation ability that increases with time and hydrophobicity with xylene. It was resistant to different antibiotics and showed no hemolytic activity. The isolate was also capable of producing phytase that can break down phytate. Overall, the characteristics of P. kudriavzevii suggest that it could potentially have probiotic properties, and its ability to produce phytase could also make it useful in feed and animal industries.

Chitosan-glucose derivative as effective wall material for probiotic yeasts microencapsulation.

A chitosan-glucose derivative (ChG) with lower antimicrobial activity against whey native probiotic yeast K. marxianus VM004 was synthesized by the Maillard reaction. The ChG derivative was characterized by FT-IR, 1H NMR, and SLS to determine the structure, deacetylation degree (DD), and molecular weight (Mw). In addition, we evaluated the antioxidant, cytotoxic, and antimicrobial activities of ChG. ChG was then used for microencapsulation of K. marxianus VM004 by spray drying. The microcapsules were characterized by evaluating their encapsulation yield, encapsulation efficiency, morphology, tolerance to the gastrointestinal tract, and viability during storage. The results indicated that a non-cytotoxic product with lower MW and DD and higher antioxidant activity than native chitosan was obtained by the Maillard reaction. The yeast ChG microcapsules exhibited an encapsulation efficiency >57 %, improved resistance to gastrointestinal conditions, and enhanced stability during storage. These results demonstrate that ChG may be a promising wall material for the microencapsulation of probiotic yeasts.

Probiotic Properties and Safety Evaluation in the Invertebrate Model Host Galleria mellonella of the Pichia kudriavzevii YGM091 Strain Isolated from Fermented Goat Milk.

Potential probiotic yeast strains isolated from fermented food need to meet safe and beneficial conditions for the host's health. The Pichia kudriavzevii YGM091 strain isolated from fermented goat milk has outstanding probiotic characteristics, including: the high survival percentage in digestive system conditions (reaching up 247.13 ± 0.12 and 145.03 ± 0.06% at pH 3.0 and bile salt 0.5%, respectively); good tolerance to temperature, salt, phenol, ethanol; good surface properties such as high hydrophobicity percentage (> 60%), the high auto-aggregation percentage rate (66.56 ± 1.45% after 45 min of incubation) and the high co-aggregation percentage rate with pathogenic bacteria in a short time (> 40% after 2 h of incubation); biofilm forming after 24 h of incubation on abiotic surfaces; antioxidant activity reached excellent level after only 24 h of incubation (The percentage free radical scavenging and the Trolox equivalent reaching up 79.86 ± 0.70% and 92.09 ± 0.75 µg/mL after 72 h of incubation); extracellular enzymes production protease and cellulase with high activity, amylase and pectinase with moderate activity and non-lipase activity. Simultaneously, the YGM091 strain is the in vitro safety yeast: insensitive to antibiotics and fluconazole, negative for gelatinase, phospholipase, coagulase, and non-hemolysis activities. Furthermore, this strain is in vivo safety yeast with the dosages below 106 CFU/larva in the Galleria mellonella model with over 90% survival larvae and the yeast density reduced to just 102-103 CFU/larva after 72 h post-injection. Research results have demonstrated that the Pichia kudriavzevii YGM091 strain is a safe potential probiotic yeast and could become a candidate probiotic food to be used in the future.

Isolation and characterization of potential probiotic yeasts from Ethiopian injera sourdough.

This study aimed to isolate and characterize potential probiotic yeasts from Ethiopian injera sourdough and the study was conducted by collecting samples from Gondar and Bahir Dar cities, Ethiopia. The potential yeasts were isolated and identified using morphological, physiological, biochemical and molecular based analysis. Promising isolates were selected to further investigate their in vitro probiotic properties, including survival at different temperatures (25, 30, 37, and 42 °C), acidic pH (2, 3, 4 and 5), bile salt (0.1, 0.3, and 0.5%), and osmotolerance (20, 30, 40, and 50% glucose concentration), antimicrobial activities, proteolytic and lipolytic activities as well as resistance to four antibiotics. From 20 samples, 38 isolates were obtained. Among these, 10 produced low or non-hydrogen sulfide and were selected for further work. Further screening tests revealed that five isolates (G1N1, G2N4, G3N1, G8N1, and B6N3) were able tolerate and grow at 37 °C, with harsh conditions of the human digestive tract like low pH, bile salt, and higher osmotic effect. The maximum growth OD values were recorded at 37 °C by isolate G4N1 (OD value (0.6667), while G3N1 exhibited a maximum growth OD value of 0.4227 at pH 2. On the other hand, G2N4 gave a maximum OD value of 0.8800 at 0.3% bile salt concentration. The promising isolates were sequenced and identified to species level. Based on phylogenetic tree analysis, all the five probiotic yeast isolates had one common ancestor and belonging to Saccharomyces cerevisiae (G1N1 and G2N4), Candida humilis (G3N1 and B6N3), and Pichia kudriavzevii (G8N1). This study revealed that Ethiopian injera sourdough could be potential source of different probiotic yeast strains. Strong emphasis should be given about the use of probiotic yeasts that are isolated from Ethiopian fermented foods.

Biocontainment strategies for in vivo applications of Saccharomyces boulardii.

The human gastrointestinal tract is a complex and dynamic environment, playing a crucial role in human health. Microorganisms engineered to express a therapeutic activity have emerged as a novel modality to manage numerous diseases. Such advanced microbiome therapeutics (AMTs) must be contained within the treated individual. Hence safe and robust biocontainment strategies are required to prevent the proliferation of microbes outside the treated individual. Here we present the first biocontainment strategy for a probiotic yeast, demonstrating a multi-layered strategy combining an auxotrophic and environmental-sensitive strategy. We knocked out the genes THI6 and BTS1, causing thiamine auxotrophy and increased sensitivity to cold, respectively. The biocontained Saccharomyces boulardii showed restricted growth in the absence of thiamine above 1 ng/ml and exhibited a severe growth defect at temperatures below 20°C. The biocontained strain was well tolerated and viable in mice and demonstrated equal efficiency in peptide production as the ancestral non-biocontained strain. In combination, the data support that thi6∆ and bts1∆ enable biocontainment of S. boulardii, which could be a relevant chassis for future yeast-based AMTs.

Engineering of a probiotic yeast for the production and secretion of medium-chain fatty acids antagonistic to an opportunistic pathogen Candida albicans.

Candida albicans is an opportunistic pathogen, with its infection as one of the causes of morbidity or mortality. Notably, the probiotic yeast Saccharomyces cerevisiae var. boulardii has shown the potential to fight against Candida infections. In this study, we aimed to engineer a commercial boulardii strain to produce medium-chain fatty acids (MCFAs) with antagonistic effects against C. albicans. First, we identified and characterized a boulardii strain and created its auxotrophic strain Δura3. Next, we constructed and expressed a heterologous MCFA biosynthetic pathway under the control of inducible and constitutive promoters. Aside from examining MCFA production and secretion, we confirmed MCFAs' effects on C. albicans' anti-biofilm and anti-hyphal formations and the immunomodulatory effect of MCFA-containing supernatants on Caco-2 cells. We found that under constitutive promoters, the engineered boulardii strain constitutively produced and secreted a mixture of C6:0, C8:0, and C10:0. The secreted MCFAs then reduced biofilm and hyphal formations in C. albicans SC5314. We also confirmed that MCFAs upregulated the expression of virulence-related genes in SC5314. Furthermore, we found that the constitutively produced MCFAs in the supernatant induced the upregulation of immune response genes in Caco-2 cells co-cultured with SC5314, indicating MCFAs' roles in immunomodulation. Overall, the engineered boulardii strain produced and secreted MCFAs, as well as demonstrated antagonistic effects against C. albicans SC5314 and immune-modulatory effects in Caco-2. To our knowledge, this represents the first study tackling the metabolic engineering of a commercial probiotic yeast strain to constitutively produce and secrete MCFAs showing anti-Candida effects. Our study forms the basis of the potential development of a live biotherapeutics probiotic yeast against Candida infections through metabolic engineering strategies.

Heme Oxygenase-1 (HMX1) Loss of Function Increases the In-Host Fitness of the Saccharomyces 'boulardii' Probiotic Yeast in a Mouse Fungemia Model.

The use of yeast-containing probiotics is on the rise; however, these products occasionally cause fungal infections and possibly even fungemia among susceptible probiotic-treated patients. The incidence of such cases is probably underestimated, which is why it is important to delve deeper into the pathomechanism and the adaptive features of S. 'boulardii'. Here in this study, the potential role of the gene heme oxygenase-1 (HMX1) in probiotic yeast bloodstream-derived infections was studied by generating marker-free HMX1 deletion mutants with CRISPR/Cas9 technology from both commercial and clinical S. 'boulardii' isolates. The six commercial and clinical yeasts used here represented closely related but different genetic backgrounds as revealed by comparative genomic analysis. We compared the wild-type isolates against deletion mutants for their tolerance of iron starvation, hemolytic activity, as well as kidney burden in immunosuppressed BALB/c mice after lateral tail vein injection. Our results reveal that the lack of HMX1 in S. 'boulardii' significantly (p < 0.0001) increases the kidney burden of the mice in most genetic backgrounds, while at the same time causes decreased growth in iron-deprived media in vitro. These findings indicate that even a single-gene loss-of-function mutation can, surprisingly, cause elevated fitness in the host during an opportunistic systemic infection. Our findings indicate that the safety assessment of S. 'boulardii' strains should not only take strain-to-strain variation into account, but also avoid extrapolating in vitro results to in vivo virulence factor determination.

The Probiotic Effects of the Saccharomyces cerevisiae 28-7 Strain Isolated from Nuruk in a DSS-Induced Colitis Mouse Model.

Probiotics are microorganisms that can benefit host health when ingested in a live state, and lactic acid bacteria are the most common type. Among fungi, Saccharomyces boulardii (SB) is the only strain known to have a probiotic function with beneficial effects on colitis; however, information on other probiotic yeast strains is limited. Therefore, this study aimed to discover yeast strains expressing intestinal anti-inflammatory activities by exhibiting probiotic properties in dextran sodium sulfate (DSS)-induced colitis mice model. Nuruk (Korean traditional fermentation starter) containing various microbial strains was used as a source for yeast strains, and S. cerevisiae 28-7 (SC28-7) strain was selected with in vitro and in vivo characteristics to enable survival in the intestines. After 14 days of pretreatment with the yeast strains, DSS was co-administered for six days to induce colitis in mice. The results revealed that the disease activity index score was lowered by SC28-7 treatment compared to the DSS group, and the colon length and weight/length ratio were recovered in a pattern similar to that of the normal group. SC28-7 administration significantly reduced the secretion of pro-inflammatory cytokines in the serum and modified the mRNA expression of inflammatory cytokines (interleukin-1ß, transforming growth factor-ß, and interferon-γ) and proteins involved in gut barrier functions (mucin 2, mucin 3, zonula occludens-1, and occludin) in colon tissues. These results indicate that SC28-7 attenuates DSS-induced colon damage and inflammation, supporting its future use as a probiotic yeast for treating and preventing intestinal inflammatory diseases such as inflammatory bowel disease.