Genetic basis for probiotic yeast phenotypes revealed by nanopore sequencing.

Probiotic yeasts are emerging as preventative and therapeutic solutions for disease. Often ingested via cultured foods and beverages, they can survive the harsh conditions of the gastrointestinal tract and adhere to it, where they provide nutrients and inhibit pathogens like Candida albicans. Yet, little is known of the genomic determinants of these beneficial traits. To this end, we have sequenced 2 food-derived probiotic yeast isolates that mitigate fungal infections. We find that the first strain, KTP, is a strain of Saccharomyces cerevisiae within a small clade that lacks any apparent ancestry from common European/wine S. cerevisiae strains. Significantly, we show that S. cerevisiae KTP genes involved in general stress, pH tolerance, and adherence are markedly different from S. cerevisiae S288C but are similar to the commercial probiotic yeast species S. boulardii. This suggests that even though S. cerevisiae KTP and S. boulardii are from different clades, they may achieve probiotic effect through similar genetic mechanisms. We find that the second strain, ApC, is a strain of Issatchenkia occidentalis, one of the few of this family of yeasts to be sequenced. Because of the dissimilarity of its genome structure and gene organization, we infer that I. occidentalis ApC likely achieves a probiotic effect through a different mechanism than the Saccharomyces strains. Therefore, this work establishes a strong genetic link among probiotic Saccharomycetes, advances the genomics of Issatchenkia yeasts, and indicates that probiotic activity is not monophyletic and complimentary mixtures of probiotics could enhance health benefits beyond a single species.

Yeasts originating from fermented foods, their potential as probiotics and therapeutic implication for human health and disease.

Yeasts derived from fermented foods have historically been known for their organoleptic properties, enriching nutritional values, and producing bioactive metabolites with therapeutic potential. In this review, we discuss the yeast flora in fermented foods, their functional aspects in fermentation, as well as their probiotic and biotherapeutic properties. These yeasts have numerous physical and biochemical characteristics, such as larger cells as compared to bacteria, a rigid cell wall composed primarily of glucans and mannans, natural resistance to antibiotics, and the secretion of secondary metabolites that are both pleasing to the consumer and beneficial to the host's health and well-being. The review also focused on therapeutic applications of probiotic yeasts derived from fermented foods on infections associated with Candida species. These potential probiotic yeasts present an additional avenue to treat dysbiosis of the gut microbiota and prevent health complications that arise from opportunistic fungal colonization, especially drug-resistant superbugs, which are highlighted in this review.

Secondary Metabolites from Food-Derived Yeasts Inhibit Virulence of Candida albicans.

A sparse number of available antifungal drugs, therapeutic side effects, and drug resistance are major challenges in current antifungal therapy to treat Candida albicans-associated infections. Here, we describe two food-derived yeasts, Saccharomyces cerevisiae and Issatchenkia occidentalis, that inhibit virulence traits of C. albicans, including hyphal morphogenesis, biofilm formation, and adhesion to intestinal epithelial cells. These yeasts also protect the model host Caenorhabditis elegans from C. albicans infection. We demonstrate that the protective activity is primarily retained in the secretome of the beneficial yeasts, and the protection they provide as a physical barrier is negligible. S. cerevisiae aro8 aro9 mutant analysis demonstrate that phenylethanol and tryptophol are necessary for protection, and experiments with commercially procured compounds indicate that they are sufficient to inhibit C. albicans virulence. We propose food-derived yeasts as an alternative or combination therapy to conventional antifungal therapy for C. albicans infection. IMPORTANCE The gut microbiome, primarily established by food, is complex and contributes to the health of the host. Molecular mechanisms that regulate microbial interactions and host health remain unclear. Here, we show that the pathogen C. albicans interacts with food-derived beneficial yeasts in the gut of the microscopic worm, C. elegans, forming a simple microbiome. C. albicans can colonize the worm gut, compromising the worm's health, and exposure to the food-derived yeasts ameliorates this effect protecting the nematode host. We identify small molecules from food-derived yeasts that are necessary and sufficient to inhibit multiple virulence traits of C. albicans and protect the nematode host. The nematode gut faithfully recapitulates a mammalian intestine. This could be an effective alternative or combination therapy for C. albicans infection.

Utilization of coffee pulp waste for rapid recovery of pectin and polyphenols for sustainable material recycle.

Coffee pulp is one of the major underutilized byproduct of coffee processing in farm level. Disposal of this agro-industrial waste has become one of the most challenging tasks for coffee planters. However, most of the efforts are towards the management of coffee pulp as an effluent, and not-on re-use. The problem is compounded due to the large volumes produced in diluted forms, which makes it expensive to reuse. The preliminary proximate analysis of coffee pulp indicated it to be rich in pectin and polyphenols. The efficacy of various chemicals like ethanol, sulfuric acid, hydrochloric acid, nitric acid, ammonium oxalate and metal salts for effective precipitation of pectin from coffee pulp was evaluated. HPLC characterization of the extracted and concentrated polyphenols fractions was analyzed. The maximum extraction of pectin was achieved by using metal salts and ethanol with 6.0% and 6.7% on wet weight basis respectively. The equivalent weight of extracted pectin (1180.5 mg/g) was found to be higher than that of commercial pectin (724.8 mg/g). The methoxyl content of the commercial pectin and crude pectin were 9.3 and 5.6% respectively. Gallic, vanillin, catechin, ethyl catechol, coumaric, Caffeic, and ferulic acid were the major polyphenols as quantified by the HPLC. The polyphenol fraction showed a good antioxidant activity with phosphomolybdate, FRAP, DPPH, and ABTS radicals respectively. The sustainable utilization of coffee pulp as a source of pectin and polyphenols with good antioxidant activities could help to solve the problem of waste generated in coffee processing in farm level.

Identification and characterization of novel transglycosylating α-glucosidase from Aspergillus neoniger.

AIM: Aspergillus niger is well established for secreting α-glucosidase having transglycosylation activity, which is used as processing aid for synthesis of isomaltooligosaccharides. The present study focuses on identification and characterization of a non-niger Aspergillus isolate and its gene conferring strong transglycosylation activity. METHODS AND RESULTS: The soil isolate was identified as Aspergillus neoniger belonging to Aspergillus section Nigri using ITS (internal transcribed spacer) and ß-tubulin analysis. The sequence analysis of gene responsible for α-glucosidase synthesis revealed significant nucleotide variations when compared to other Aspergillus species. Molecular docking studies using the homology model revealed the presence of threonine at 694 subsite position instead of asparagine as in case of A. niger's α-glucosidase. The enzyme was purified to several fold using DEAE Sepharose-CL6B column and on SDS-PAGE analysis, it was found to be 145 kDa. MS/MS analysis of the purified enzyme validated the presence of threonine at 694 position. Commercial α-glucosidase (Transglucosidase L 'Amano') derived from A. niger and the α-glucosidase from isolate were compared for transglycosylation activity using constant test conditions. α-glucosidase from the isolate produced 27·4% higher panose when compared to that of commercial enzyme. Moreover, the rate of secondary hydrolysis of panose is much lower in case of the isolate's enzyme. CONCLUSIONS: Fungal isolate A. neoniger was characterized, and its gene conferring α-glucosidase activity was established for strong transglycosylation activity having higher panose yields. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report to establish a variant of α-glucosidase having strong transglycosylation activity from A. neoniger strain. We have demonstrated that this enzyme when used as processing aid could improve panose significantly, which is a potential prebiotic. Also, the sequence analysis established in our studies could provide pointers for directed evolution of this enzyme to further improve transglycosylation activity.

Probiotic Yeasts Inhibit Virulence of Non-albicans Candida Species.

Systemic infections of Candida species pose a significant threat to public health. Toxicity associated with current therapies and emergence of resistant strains present major therapeutic challenges. Here, we report exploitation of the probiotic properties of two novel, food-derived yeasts, Saccharomyces cerevisiae (strain KTP) and Issatchenkia occidentalis (strain ApC), as an alternative approach to combat widespread opportunistic fungal infections. Both yeasts inhibit virulence traits such as adhesion, filamentation, and biofilm formation of several non-albicans Candida species, including Candida tropicalis, Candida krusei, Candida glabrata, and Candida parapsilosis as well as the recently identified multidrug-resistant species Candida auris They inhibit adhesion to abiotic surfaces as well as cultured colon epithelial cells. Furthermore, probiotic treatment blocks the formation of biofilms of individual non-albicans Candida strains as well as mixed-culture biofilms of each non-albicans Candida strain in combination with Candida albicans The probiotic yeasts attenuated non-albicans Candida infections in a live animal. In vivo studies using Caenorhabditis elegans suggest that exposure to probiotic yeasts protects nematodes from infection with non-albicans Candida strains compared to worms that were not exposed to the probiotic yeasts. Furthermore, application of probiotic yeasts postinfection with non-albicans Candida alleviated pathogenic colonization of the nematode gut. The probiotic properties of these novel yeasts are better than or comparable to those of the commercially available probiotic yeast Saccharomyces boulardii, which was used as a reference strain throughout this study. These results indicate that yeasts derived from food sources could serve as an effective alternative to antifungal therapy against emerging pathogenic Candida species.IMPORTANCE Non-albicans Candida-associated infections have emerged as a major risk factor in the hospitalized and immunecompromised patients. Besides, antifungal-associated complications occur more frequently with these non-albicans Candida species than with C. albicans Therefore, as an alternative approach to combat these widespread non-albicans Candida-associated infections, here we showed the probiotic effect of two yeasts, Saccharomyces cerevisiae (strain KTP) and Issatchenkia occidentalis (ApC), in preventing adhesion and biofilm formation of five non-albicans Candida strains, Candida tropicalis, Candida krusei, Candida glabrata, Candida parapsilosis, and Candida auris The result would influence the current trend of the conversion of conventional antimicrobial therapy into beneficial probiotic microbe-associated antimicrobial treatment.

Diverse physiological and metabolic adaptations by Lactobacillus plantarum and Oenococcus oeni in response to the phenolic stress during wine fermentation.

Understanding the mechanisms of combating phenolic stress in wine is important for optimization of starter culture for improved competitiveness. The cellular membrane adaptations, as well as metabolic transformations driven by malo-lactic starter cultures, i.e. Lactobacillus plantarum (Lp2565) and Oenococcus oeni (Oo2219) with response to wine phenolic compounds were studied. The morphological changes based on scanning electron microscopy indicated the higher tolerance of Oo2219 to phenolic stress than Lp2565. Further, the fatty acid profiling suggested that the membrane fluidization in Lp2565 was attributed to higher unsaturated fatty acids whereas the rigidification in Oo2219 was by incorporation of saturated fatty acid in the membrane. The metabolic transformation of phenolic compounds suggested that Lp2565 has more versatile phenolic detoxification enzyme systems compared to Oo2219. The metabolic conversions resulted in degradation of phenolic compounds into volatile phenols, aromatic alcohol, and phenyl propionic acids, thus indicating the possible involvement of oxidoreductases, decarboxylases, and demethylases.

Antagonistic effect of Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC on hyphal development and adhesion of Candida albicans.

The morphological transition from yeast to a hyphal form, as well as the adhesion capability to the gastrointestinal tract, are implicated virulent determinant in Candida albicans and could be potential targets for prevention of the opportunistic pathogen. Based on this rationale, two yeast strains Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC along with reference strain Saccharomyces boulardii NCDC 363 were screened for the probiotic potential. Characters like pH, temperature, bile, simulated gastrointestinal juice tolerance tests, and Caco-2 cell line adhesion assay were determined in the present study. Further, the evaluation of its impact on C. albicans morphological transition and adhesion was assessed using microtitre germ tube test. In terms of probiotic characteristics, both the strains were tolerant to pH 2.5 and the presence of bile (0.3 to 0.6%) with an optimum growth temperature of 37°C. The strain KTP was also resistant to simulated gastric and intestinal juices as compared to control (13% and 41%, respectively) and NCDC 363 (55% and 35%, respectively). In contrast, both the yeasts had reduced adhesiveness to Caco-2 monolayer. Candida virulence in in vitro systems indicated that treatment of live probiotic yeast cells (108 ml) effectively reduced the filamentation and adhesion of C. albicans. The S. cerevisiae KTP had a profound effect on the hyphal development and adhesion when compared to the ApC and NCDC 363. The strain significantly reduced (P < .05) the hyphal growth in co-cultivated (93% and 94%, respectively) and pre-existing hyphae (54% and 68%) of strains C. albicans 183 and 1151. Isolates KTP and ApC also reduced the adhesion (≈ 22% and 41%, respectively) and transition of blastoconidia at two hours of incubation in abiotic surface. This study provides knowledge on the effect of potential probiotic yeasts such as Saccharomyces and non- Saccharomyces strains against virulence characteristic of Candida albicans.

Augmentation of chemical and organoleptic properties in Syzygium cumini wine by incorporation of grape seeds during vinification.

The role of grape seed tannins on improving organoleptic properties and its involvement in color stabilization in red wine are well established. The addition of grape seeds as the source of condensed tannins in fruit wine may provide a solution for its color instability and improvement of sensory attributes. Syzgium cumini is traditionally known for its therapeutic properties. In the current study, the influence of yeasts and grape seed addition during fermentation on the chromatic, phenolic and sensory attributes of the wine was accessed. Grape seed addition improved the color characteristics of wine and increased overall phenolic composition. Analysis by HPLC revealed 6 major anthocyanins, among which 3, 5-diglucoside form of delphidin and petunidin was found to be the major components. Cluster and PLSR analysis explained the impact of seed addition on the yeasts, as well as on the perception of panelists, with bitterness and astringency as the dominating attributes.

Detection system for Saccharomyces cerevisiae with phenyl acrylic acid decarboxylase gene (PAD1) and sulphur efflux gene (SSU1) by multiplex PCR.

Production of wine with selected yeast strains is a common enological practice followed for the production of wine with desirable organoleptic properties and to guarantee the homogeneity of successive vintages. Sulphur dioxide tolerance and phenyl acrylic acid resistance are the enological traits essential for the survivability of the yeast during fermentation. The present study describes the detection of S. cerevisiae with enological traits, such as phenyl acrylic acid resistance and sulphur dioxide tolerance in a single test. Phenyl acrylic acid decarboxylase (PAD1) and sulphite efflux genes (SSU1) were detected by multiplex PCR, thus confirming the specificity of primers. A single cocktail of all reagents required for the simultaneous detection of both these genes was designed. The ready-to-use formulation optimized was stable at 4 and - 20 °C for 6 months. The amplification of phenyl acrylic acid decarboxylase and sulphite efflux genes, validated the suitability of the ready-to-use formulation for the detection of S. cerevisiae in food samples. The ready-to-use formulation optimized, minimizes the end user requirements for the detection of S. cerevisiae. Thus, the method was suitable for the identification of S. cerevisiae strains from a mixture of yeast prior to the sequencing analysis, thereby reducing the cost and time of screening.

Antiaflatoxigenic and Antimicrobial Activities of Schiff Bases of 2-Hydroxy-4-methoxybenzaldehyde, Cinnamaldehyde, and Similar Aldehydes.

2-Hydroxy-4-methoxybenzaldehyde (HMBA) is a nontoxic phenolic flavor from dietary source Decalipus hamiltonii and Hemidesmus indicus. HMBA is an excellent antimicrobial agent with additional antiaflatoxigenic potency. On the other hand, cinnamaldehyde from cinnamon is a widely employed flavor with significant antiaflatoxigenic activity. We have attempted the enhancement of antiaflatoxigenic and antimicrobial properties of HMBA, cinnamaldehyde, and similar molecules via Schiff base formation accomplished from condensation reaction with amino sugar (d-glucamine). HMBA derived Schiff bases exhibited commendable antiaflatoxigenic activity at the concentration 0.1 mg/mL resulting in 9.6 ± 1.9% growth of Aspergillus flavus and subsequent 91.4 ± 3.9% reduction of aflatoxin B1 with respect to control.

Seed incorporation during vinification and its impact on chemical and organoleptic properties in Syzygium cumini wine.

Syzgium cumini (Jamun) is an evergreen tropical tree, its various parts are known for many therapeutic properties. The present work represents the production of wines from jamun fruits using two different native isolates (Saccharomyces cerevisiae - KF551990 and Pichia gummiguttae - MCC 1273) and influence of jamun seeds on the physico-chemical parameters, chromatic properties, phenolic components and sensory attributes of wine. Wine produced was bottle aged for one year. On aging there was a reduction in bitterness and astringency. Aging lead to reduction in monomeric anthocyanin with an increase in co-pigmented and polymeric anthocynins thus affecting the wine color. Anthocyanin analysis in jamun wine indicated petunidin 3,5-diglucoside as the principal anthocyanin. PCA analysis of wine revealed association of young jamun wine with anthocyanin components. PLS analysis exhibited both positive and negative correlation between various attributes indicating sensory perception of jamun wine is affected by overall composition of the wine.

Correlation between ethanol stress and cellular fatty acid composition of alcohol producing non-Saccharomyces in comparison with Saccharomyces cerevisiae by multivariate techniques.

Wine production is a complex process both from biochemical and microbiological point of view in which yeast plays a central role. The use of the wine yeast Saccharomyces cerevisiae and non- Saccharomyces yeasts as mixed starter cultures for wine fermentations is of increasing interest to enhance the quality of wine.The most common stress, yeast cells encounter during wine fermentation is the increase in ethanol concentration.To enhance ethanol tolerance, alteration in the cellular lipid composition is one of its defence mechanism. Ethanol tolerance and cellular fatty acid composition of alcohol producing non Saccharomyces forms were compared with enological strains of Sacccharomyces cerevisiae. Saccharomyces cerevisiae used for the study, tolerated 15 % of ethanol and the non Saccharomyces strains such as, Issatchenkia occidentalis and Issatchenkia orientalis tolerated 10 % of ethanol. On exposure of Saccharomyces cerevisiae to ethanol stress, the proportion of monounsaturated fatty acids increased with concomitant decrease in saturated fatty acids. Decrease in monounsaturated fatty acids, exhibited by non-Saccharomyces yeasts when exposed to ethanol stress, could be one of the reasons for their inability to withstand more than 10 % of alcohol. Multivariate techniques of data analysis - principal component analysis and linear discriminant analysis were employed in order to establish differentiation criteria as function of yeast strains, alcohol stress and their fatty acid profile. Based on the data, Chemometrics, such as principal component analysis and discriminant function analysis, can be successfully applied to fatty acid data to categorize the yeast.

Production of bioethanol from fermented sugars of sugarcane bagasse produced by lignocellulolytic enzymes of Exiguobacterium sp. VSG-1.

Exiguobacterium sp. VSG-1 was isolated from the soil sample and characterized for the production of lignocellulolytic enzymes. Production of these enzymes by the strain VSG-1 was carried out using steam-exploded sugarcane bagasse (SCB) and found to secrete cellulase, pectinase, mannanase, xylanase, and tannase. The growth and enzyme production were found to be optimum at pH 9.0 and 37 °C. Upon steam explosion of SCB, the cellulose increased by 42 %, whereas hemicelluloses and lignin decreased by 40 and 62 %, respectively. Enzymatic hydrolysis of steam-exploded SCB yielded 640 g/l of total sugars. Fermentation of sugars produced from pretreated SCB was carried out by using Saccharomyces cerevisiae at pH 5.0 and 30 °C. The alcohol produced was calculated and found to be 62.24 g/l corresponding to 78 % of the theoretical yield of ethanol. Hence, the strain VSG-1 has an industrial importance for the production of fermentable sugars for biofuels.