Immunostimulatory activity of Pichia kluyveri, Hanseniaspora uvarum, Candida intermedia, Saccharomyces boulardii and their derivatives on RAW 264.7 macrophages

Yeasts are a group of microorganisms with structural and metabolic characteristics that influence their recognition by immune cells resulting in a species-specific response. Although Saccharomyces boulardii is a widely studied probiotic yeast, immunostimulation by non-Saccharomyces yeasts still underexplored. Therefore, the aim of this study was to characterize the response induced in macrophages stimulated by yeasts Pichia kluyveri, Hanseniaspora uvarum, Candida intermedia and their derivatives: heat-killed cells, supernatant and DNA. RAW 264.7 murine macrophages were stimulated in vitro for 24 h and the response generated was evaluated by analyzing mRNA transcription of cytokines (IL2, IL4, IL10, IL13, IL23, TNF-α), transcription factors (Bcl6, NFκβ, STAT3), Toll-like receptor 2 (TLR2) and YM1 protein. Viable and heat-killed cells of P. kluyveri and H. uvarum were responsible for high levels of relative mRNA transcription of transcription factors and TLR2 (between 2 – 8-fold increase), however were able to induce only low transcription levels for analyzed cytokines (≤ 2-fold increase). Viable cells of C. intermedia were able to stimulate a significant transcription of IL4 (7.6-fold increase) and Bcl6 (4-fold increase), while heat-killed cells stimulated the highest level of TNF-α (2.4-fold increase) among yeasts and their derivatives. Furthermore, supernatant from C. intermedia culture induced significant (p < 0.05) levels of TLR2 (4.4-fold increase), being the only one among supernatants to present high levels of relative mRNA transcription of TLR2. Data found in this work arouse interest in further studies on interaction between non-Saccharomyces yeasts and immune system cells, mainly referring to immunomodulatory capacity.

Brewing and Probiotic Potential Activity of Wild Yeasts Hanseniaspora Uvarum PIT001, Pichia Kluyveri LAR001 and Candida Intermedia ORQ001

Non-conventional yeasts can be isolated from a wide range of environmental sources, often found in beverage industry in mixed fermentations, in which the microorganisms’ inoculum usually is not fully known. It is important to know starter cultures, since in addition to favoring reproducibility, other properties can be discovered. Thus, the objective of this work was to identify and characterize yeasts isolated from environment, evaluating their probiotic potential and possible use in brewery. Isolates were obtained from flowers, fruits, leaves and mixed-fermentation beers, being identified by PCR. Yeasts with promising activity were evaluated regarding their growth under different pHs, temperature and presence of organic acids. To explore probiotic potential, in vitro tests were performed of antimicrobial activity and co-aggregation with food pathogens, auto-aggregation, and survival in simulated gastrointestinal tract conditions. In our study, Pichia kluyveri (LAR001), Hanseniaspora uvarum (PIT001) and Candida intermedia (ORQ001) were selected among 20 isolates. P. kluyveri was the only one that tolerated pH 2.5. Lactic acid was not inhibitory, while acetic acid and incubation at 37 °C had a partially inhibitory effect on yeasts growth. All yeasts tolerated α-acids from hops and NaCl up to 1%. It is suggested that isolates are able to adhere to intestinal cells and influence positively the organism in combating pathogens, as they showed auto-aggregation rates above 99% and antagonistic activity to pathogenic bacteria. The yeasts tolerated gastric environment conditions, however were more sensitive to pancreatic conditions. We conclude that isolated non-conventional yeasts showed probiotic potential and promising application in beer fermentation.

Candida duobushaemulonii: An Old But Unreported Pathogen

Candidiasis caused by species of the Candida haemulonii complex (Candida haemulonii and Candida duobushaemulonii) and closely related species, Candida auris and Candida pseudohaemulonii are increasing. These species often show reduced susceptibility to antifungal drugs, such as azoles and amphotericin B or, less frequently, echinocandins. However, conventional phenotypic identification methods are unable to accurately differentiate these species and, therefore, their prevalence may have been underestimated. In this study, 150 isolates that were probably misidentified were reanalyzed using two novel PCR approaches. We found that one isolate previously identified in 1996 as Candida intermedia was C. duobushaemulonii, being one of the oldest isolates of this species described to date. We also found that this isolate had reduced susceptibility to fluconazole, itraconazole, and amphotericin B.

Candida intermedia CBS 141442: A Novel Glucose/Xylose Co-Fermenting Isolate for Lignocellulosic Bioethanol Production

The present study describes the isolation of the novel strain Candida intermedia CBS 141442 and investigates the potential of this microorganism for the conversion of lignocellulosic streams. Different C. intermedia clones were isolated during an adaptive laboratory evolution experiment under the selection pressure of lignocellulosic hydrolysate and in strong competition with industrial, xylose-fermenting Saccharomyces cerevisiae cells. Isolates showed different but stable colony and cell morphologies when growing in a solid agar medium (smooth, intermediate and complex morphology) and liquid medium (unicellular, aggregates and pseudohyphal morphology). Clones of the same morphology showed similar fermentation patterns, and the C. intermedia clone I5 (CBS 141442) was selected for further testing due to its superior capacity for xylose consumption (90% of the initial xylose concentration within 72 h) and the highest ethanol yields (0.25 ± 0.02 g ethanol/g sugars consumed). Compared to the well-known yeast Scheffersomyces stipitis, the selected strain showed slightly higher tolerance to the lignocellulosic-derived inhibitors when fermenting a wheat straw hydrolysate. Furthermore, its higher glucose consumption rates (compared to S. stipitis) and its capacity for glucose and xylose co-fermentation makes C. intermedia CBS 141442 an attractive microorganism for the conversion of lignocellulosic substrates, as demonstrated in simultaneous saccharification and fermentation processes.

Effect of Candida intermedia LAMAP1790 Antimicrobial Peptides against Wine-Spoilage Yeasts Brettanomyces bruxellensis and Pichia guilliermondii

Wine spoilage yeasts are one of the main issues in the winemaking industry, and the control of the Brettanomyces and Pichia genus is an important goal to reduce economic loses from undesired aromatic profiles. Previous studies have demonstrated that Candida intermedia LAMAP1790 produces antimicrobial peptides of molecular mass under 10 kDa with fungicide activity against Brettanomyces bruxellensis, without affecting the yeast Saccharomyces cerevisiae. So far, it has not been determined whether these peptides show biocontroller effect in this yeast or other spoilage yeasts, such as Pichia guilliermondii. In this work, we determined that the exposure of B. bruxellensis to the low-mass peptides contained in the culture supernatant of C. intermedia LAMAP1790 produces a continuous rise of reactive oxygen species (ROS) in this yeast, without presenting a significant effect on membrane damage. These observations can give an approach to the antifungal mechanism. In addition, we described a fungicide activity of these peptides fraction against two strains of P. guilliermondii in a laboratory medium. However, carrying out assays on synthetic must, peptides must show an effect on the growth of B. bruxellensis. Moreover, these results can be considered as a start to develop new strategies for the biocontrol of spoilage yeast.

Genomic and transcriptomic analysis of Candida intermedia reveals the genetic determinants for its xylose-converting capacity

Background An economically viable production of biofuels and biochemicals from lignocellulose requires microorganisms that can readily convert both the cellulosic and hemicellulosic fractions into product. The yeast Candida intermedia displays a high capacity for uptake and conversion of several lignocellulosic sugars including the abundant pentose d-xylose, an underutilized carbon source since most industrially relevant microorganisms cannot naturally ferment it. Thus, C. intermedia constitutes an important source of knowledge and genetic information that could be transferred to industrial microorganisms such as Saccharomyces cerevisiae to improve their capacity to ferment lignocellulose-derived xylose. Results To understand the genetic determinants that underlie the metabolic properties of C. intermedia, we sequenced the genomes of both the in-house-isolated strain CBS 141442 and the reference strain PYCC 4715. De novo genome assembly and subsequent analysis revealed C. intermedia to be a haploid species belonging to the CTG clade of ascomycetous yeasts. The two strains have highly similar genome sizes and number of protein-encoding genes, but they differ on the chromosomal level due to numerous translocations of large and small genomic segments. The transcriptional profiles for CBS 141442 grown in medium with either high or low concentrations of glucose and xylose were determined through RNA-sequencing analysis, revealing distinct clusters of co-regulated genes in response to different specific growth rates, carbon sources and osmotic stress. Analysis of the genomic and transcriptomic data also identified multiple xylose reductases, one of which displayed dual NADH/NADPH co-factor specificity that likely plays an important role for co-factor recycling during xylose fermentation. Conclusions In the present study, we performed the first genomic and transcriptomic analysis of C. intermedia and identified several novel genes for conversion of xylose. Together the results provide insights into the mechanisms underlying saccharide utilization in C. intermedia and reveal potential target genes to aid in xylose fermentation in S. cerevisiae.