Search Results (125)

Milk beer, a modern Chinese dairy beverage, is usually fermented by the co-culture of lactic acid bacteria (LAB) and Kluyveromyces marxianus (K. marxianus), with the latter known for its ability to produce aroma compounds. However, the accumulation of lactic acid produced by LAB can inhibit the growth of K. marxianus, which inevitably hinders the diversity and intensity of flavor compounds in milk beer. In this study, adaptive laboratory evolution (ALE) was applied to the parental strain Kluyveromyces marxianus CICC1953 (Km-P) under different concentrations of lactic acid to obtain an evolved strain Km-ALE-X20 with enhanced acid tolerance and increased titer of phenylethyl alcohol, which has a floral, rose-like aroma. Km-ALE-X20 demonstrated a 16-fold increase in OD₆₀₀ and a 28-fold increase in phenylethyl alcohol production compared with Km-P in chemically defined medium (CDM) containing 20 g/L lactic acid. Comparative genomics analysis suggested that mutated genes CTA1, TSL1, ERG2 were related to enhanced acid tolerance, while ARO8, ARO9, FKS2 were related to increased production of aroma compounds. Furthermore, Km-ALE-X20-fermented milk beer showed 33.87% and 32.43% higher production in alcohol and ester compounds than that of Km-P-fermented milk beer. Interestingly, sensory analysis showed that while Km-ALE-X20-fermented milk beer had higher sensory scores for rose and fruity aroma attributes, Km-P-fermented milk beer possessed a more balanced aroma profile. This paper highlights the first application of ALE to enhance the signature rose aroma of K. marxianus-fermented milk beer and provides an efficient framework for ALE-based breeding of aroma-producing food microorganisms. Full article

The aim of this work was to identify the microbial agent(s) responsible for a structural defect in Provolone Valpadana, Protected Designation of Origin (PDO) cheese, and to establish their spread along the production line. Repeated sampling of defective cheeses and analyses of processing intermediates following two inspections at the cheese factory identified yeasts as the main causative agents. Microbiological analysis highlighted an almost constant presence of yeasts, which dominate over the other microbial groups. Forty yeast isolates from defective cheeses were identified by sequencing the D1/D2 region of the 26S rRNA gene. Saccharomyces cerevisiae, Kluyveromyces marxianus, and Debaryomyces hansenii dominated in all sampled cheeses, followed by D. tyrocola, Pichia kudriavzevii, and Torulaspora delbrueckii. Yeast and bacterial metabarcoding on three cheeses with a yeast count > log 4.0 CFU/mL indicated D. hansenii as the dominant yeast taxon and confirmed the absence of gas-producing bacterial taxa. RAPD-PCR analysis suggested the presence of yeast biofilms in the dairy environment or along the production line, as confirmed by the repeated isolation of specific genotypes of S. cerevisiae, K. marxianus, and D. hansenii in different defective cheeses sampled between April and August 2023, as well as in samples taken following two inspections at the production site, during cheese processing and ripening. Full article

Compound yeast culture (CYC) is known to enhance animal health, but its effects on hepatic immune function are unclear. This study systematically examined CYC’s regulatory effects on the liver of weaned lambs using transcriptomics and integrative bioinformatics. Ten lambs were randomly assigned to a control diet or a basal diet supplemented with 30 g/d per head of Saccharomyces cerevisiae and Kluyveromyces marxianus co-culture (CYC group) for 42 days. Histological analysis showed that CYC improved hepatocyte arrangement and sinusoidal integrity, suggesting enhanced hepatic tissue stability. Cytokine analysis revealed CYC significantly increased IL-6 and IL-1β while reducing IL-10, TGF-β1, TNF-α, and CXCL9, indicating a bidirectional modulation of the immune response. Additionally, CYC enhanced antioxidant defenses by increasing T-SOD, GSH-Px, and T-AOC activities and decreasing MDA content. Transcriptomic sequencing indicated that CYC reshaped hepatic gene expression. Upregulated genes were enriched in immune-regulatory and structural pathways, including PI3K-AKT signaling, ECM–receptor interactions, Toll-like receptor pathways, and cell adhesion molecules. Protein-level validation further confirmed activation of PI3K and AKTAKT phosphorylation with limited engagement of NF-κB signaling. Conversely, downregulated genes were mainly associated with oxidative stress and energy metabolism, such as ROS-related pathways and MAPK signaling. WGCNA identified key hub genes (PTPRC, CD86, and ITGAV), which correlate with pro-inflammatory factors and participate in immune recognition, T-cell activation, and cell adhesion. These data suggest that CYC promotes hepatic immune homeostasis by enhancing immune signaling, stabilizing tissue architecture, and modulating oxidative stress/metabolic processes. This study provides mechanistic insights into CYC’s regulation of liver immune function and supports its targeted application as a functional feed additive for ruminants. Full article

Winemaking is facing significant challenges caused by industrialization of the process, climate change, and increased consumer awareness regarding the use of chemical preservatives. Although several solutions have been proposed, the utilization of non-Saccharomyces species seems to be the most efficient one. Several non-Saccharomyces species have been employed for this purpose, with Hanseniaspora uvarum, H. vineae, Kluyveromyces marxianus, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia fermentans, P. kluyveri, Schizosaccharomyces pombe, Starmerella bacillaris, Torulaspora delbrueckii, and Wickerhamomyces anomalus being the most promising ones. However, only a restricted amount of metabolic activities can be reliably attributed to the species level, while most of them are characterized by strain variability and are also affected by the Saccharomyces cerevisiae strains used to carry out alcoholic fermentation, as well as the efficient supply of precursor molecules by the grape varieties and the conditions for their effective bioconversion. This variability necessitates the application of optimization strategies, taking into consideration all these parameters. This review article aims to assist in this direction by collecting the data referring to the winemaking practice of the most interesting non-Saccharomyces species, presenting clearly and comprehensively their most relevant features, and highlighting the effect of strain diversity. Full article

Brewer’s spent grain (BSG), the main lignocellulosic by-product of the beer industry, represents an abundant yet underutilized resource with high potential for valorization. This study presents an integrated biorefinery approach to convert BSG into second-generation (2G) ethanol, bioactive vinasse for plant growth promotion, and fungal biomass as a potential mycoprotein source. The biomass was first subjected to biological delignification using the white-rot fungus Ganoderma lucidum, after which two valorization routes were explored: (i) evaluation of the fungal biomass as a mycoprotein candidate and (ii) alcoholic fermentation for ethanol production. For the latter, three pretreatment strategies were assessed (diluted sulfuric acid and two deep eutectic solvents (DESs) based on choline chloride combined with either glycerol or lactic acid) followed by a one-pot enzymatic saccharification and fermentation using Kluyveromyces marxianus SLP1. The highest ethanol yield on substrate (Y_P/S) was achieved with [Ch]Cl:lactic acid pretreatment (0.46 g/g, 89.32% of theoretical). Vinasse, recovered after distillation, was characterized for organic acid content and tested on Solanum lycopersicum seed germination, showing promising biostimulant activity. Overall, this work highlights the potential of BSG as a sustainable feedstock within circular economy models, enabling the production of multiple bio-based products from a single residue. Full article

Suaeda salsa, an annual herb belonging to the genus Suaeda within the Chenopodiaceae family, is highly salt-tolerant and can thrive in large quantities on saline and alkaline soils. This study presents a novel fermentation technique to produce Suaeda tea, utilizing a synergistic blend of microbial agents: Kluyveromyces marxianus, Komagataeibacter europaeus, and Acetobacter schutzenbachii. The resulting tea demonstrates a potent antioxidant capacity, with a hydroxyl radical scavenging rate of 64.2% and an exceptional 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity of 83.3%, along with increased ferric ion reduction/antioxidant power (FRAP) reducing power (1.82), indicating its superior antioxidant profile. Through the comparison of different microbial strain combinations under varying process parameters such as fermentation temperature and duration, the experiment revealed that fermentation at 37 °C for 24 h results in the highest concentrations of tea polyphenols (TPs) (≥10.87 mg/mL) and free amino acids (26.32 mg/100 mL). The quality of the fermented Suaeda tea meets the stringent GB/T 21733-2008 standards for tea beverages, exhibiting excellent physicochemical indices and sensory attributes. The antioxidant efficacy of the fermented Suaeda tea persists significantly throughout a 180-day duration. The optimization of the fermentation process for Suaeda tea not only provides a theoretical framework for large-scale production but also establishes a foundation for Suaeda salsa in the tea beverage sector. This innovation enriches the market with a diverse range of health-promoting teas, catering to the growing consumer demand for nutritious and beneficial beverages. Full article

The development of non-alcoholic beer (NAB) with health benefits, using non-conventional potential probiotic yeasts, offers an interesting alternative to standard NAB brewing strategies. In this study, potential probiotic non-Saccharomyces yeasts Pichia manshurica, Kluyveromyces lactis, and Kluyveromyces marxianus, along with commercial probiotic yeast Saccharomyces boulardii, were characterised and tested for functional NAB production, whereas P. manshurica was used in NAB production for the first time. Growth and viability were assessed across a range of temperatures, pH, and iso-α-bitter acids. The tested yeasts withstood conditions typical of the beer matrix and human digestive tract and had a positive phenolic off-flavour phenotype. Two strains, K. lactis and K. marxianus, showed strong β-glucosidase activity, which may enhance beverage aroma complexity. Ethanol levels in beers fermented with non-Saccharomyces yeasts remained below the NAB limit (≤0.5% v/v). An analysis of volatile organic compound profiles revealed the potential of these yeasts to produce higher alcohols and esters valuable from a brewer’s perspective. This study provides valuable insight into novel probiotic fermentations and the potential application of unconventional yeasts in functional, aromatic, and health-oriented non-alcoholic beverages. Full article

Kluyveromyces marxianus exhibits advantages such as strong thermotolerance, rapid growth rate, and high safety, making it an excellent host cell for the production of bio-based products. In this study, two exopolysaccharides, KE1 and KE2, were isolated from the fermentation broth of the strain K. marxianus KM-502, and their hydroxyl radical scavenging, DPPH scavenging, and Fe²⁺-reducing activities were evaluated. In vitro antioxidant assays demonstrated that exopolysaccharide KE2 exhibited superior antioxidant activity compared to KE1. The fermentation conditions were optimized using single-factor experiments followed by response surface methodology (RSM). The optimized fermentation process revealed that the most suitable fermentation medium consisted of 8% sucrose, 1.99% peptone, and 0.13% CaCl₂, while the optimal fermentation conditions were a medium volume of 74 mL in a 300 mL flask, pH 6.7, an inoculum size of 1.99%, a temperature of 30 °C, a shaking speed of 160 r/min, and a cultivation time of 96 h. After optimizing the fermentation conditions of K. marxianus KM-502, the exopolysaccharide (EPS) yield reached 5842.42 mg/L, representing a 22.77-fold increase compared to the yield before optimization. In summary, this study isolated exopolysaccharides KE1 and KE2 from K. marxianus KM-502. These exopolysaccharides demonstrated significant antioxidant activities, and the fermentation conditions for exopolysaccharide production were optimized. The findings of this study will facilitate the further development and utilization of exopolysaccharides from K. marxianus. Full article

Coffee cherry pulp (CCP), a coffee by-product rich in pectin and phenolic compounds, serves as a valuable substrate for microbial enzyme production, improving the nutritional and antioxidant properties of poultry feed. This study evaluated the potential of Kluyveromyces marxianus ST5 to produce pectin-degrading enzymes using CCP. Under unoptimized conditions, the pectin lyase (PL) and polygalacturonase (PG) activities were 3.29 ± 0.22 and 6.32 ± 0.13 U/mL, respectively. Optimization using a central composite design (CCD) identified optimal conditions at 16.81% (w/v) CCP, 5.87% (v/v) inoculum size, pH 5.24, and 30 °C for 48 h, resulting in PL and PG activities of 9.17 ± 0.20 and 15.78 ± 0.14 U/mL, representing increases of 178.7% and 149.7% over unoptimized conditions. Fermented CCP was further evaluated using an in vitro chicken gastrointestinal digestion model. Peptide release increased by 66.2% compared with unfermented CCP. Antioxidant capacity also improved, with significant increases observed in DPPH (32.4%), ABTS (45.0%), and FRAP (42.3%) assays, along with an 11.1% increase in total phenolic content. These results demonstrate that CCP bioconversion by K. marxianus ST5 enhances digestibility and antioxidant properties, supporting its potential as a sustainable poultry feed additive and contributing to the valorization of agro-industrial waste. Full article

This study provides a comprehensive analysis of the microbial dynamics involved in the fermentation process of traditional Musalais wine, an intangible cultural heritage of Xinjiang. Utilizing metagenomic sequencing, we identified 2894 microbial species, of which 494 persisted throughout the fermentation process. Saccharomyces cerevisiae was the dominant species, with its prevalence increasing from 97.35% in the early phase to 99.38% in the mid phase, before slightly decreasing to 98.79% in the late phase. Additionally, 24 non-Saccharomyces yeast species, including Hanseniaspora uvarum, Lachancea thermotolerans, and Torulaspora delbrueckii, were detected. Common species associated with other fermented foods, including Wickerhamomyces anomalus, Kluyveromyces marxianus, Saccharomyces eubayanus, and Zygosaccharomyces parabailii, were also identified. Notably, species not previously used in food fermentation, such as Saccharomyces jurei, Sodiomyces alkalinus, Vanrija pseudolonga, and Moesziomyces antarcticus, were also identified in this study. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KO) and Gene Ontology (GO) revealed notable variations in metabolic pathways and enriched functional genes. In addition, a total of 82 volatile compounds were detected in the final product, with higher alcohols (60.12%), esters (37.80%), and organic acids (1.80%) being the most prevalent. These results offer important insights into microbial interactions and their influence on Musalais wine quality, laying the groundwork for optimizing the fermentation process. Full article

Biocontrol is one of the most promising alternatives to chemical preservatives for food preservation. This study investigated the biocontrol potential of yeasts isolated from raw milk cheese against spoilage moulds. Eighty-four native yeast strains were screened for antagonistic activity against Penicillium commune, Fusarium verticillioides, and Mucor plumbeus/racemosus via confrontation using a milk-based culture medium. Fifteen strains from the species Pichia jadinii, Kluyveromyces lactis, Kluyveromyces marxianus, and Geotrichum candidum exhibited significant antagonistic activity (inhibition zone > 2 mm) against M. plumbeus/racemosus and F. verticillioides. The modelling of the impact of ripening conditions revealed that temperature was the primary factor influencing yeast antagonism. In addition, notable variability at both species and strain levels was found. The antagonist activity was associated with different mechanisms depending on the species and strains. K. lactis stood out for its proteolytic activity and competition for iron and manganese. Additionally, two strains of this species (KL890 and KL904) were found to produce volatile organic compounds with antifungal properties (phenylethyl alcohol and 1-butanol-3-methyl propionate). G. candidum GC663 exhibited strong competition for space, as well as the ability to parasitise hyphae linked to its pectinase and β-glucanase activity. The latter enzymatic activity was detected in all P. jadinii strains, with P. jadinii PJ433 standing out due to its proteolytic activity. In a cheese matrix, the efficacy of eight yeast strains against three target moulds was assessed, highlighting the potential of G. candidum GC663 and P. jadinii PJ433 as biocontrol agents, exhibiting high and moderate efficacy, respectively, in controlling the growth of F. verticillioides and M. plumbeus/racemosus. Nonetheless, further research is necessary to elucidate their full spectrum of antifungal mechanisms and to validate their performance under industrial-scale conditions, including their impact on cheese quality. Full article

Fermentation is widely recognized for enhancing the sensory attributes and nutritional value in foods, with recent research focusing on non-alcoholic and root-based functional beverages. In this study, the genomic and fermentation characteristics of Kluyveromyces marxianus LRCC8279 (KM8279) and Saccharomyces cerevisiae LRCC8293 (SC8293) were analyzed, specifically for their application in root extract-based low-alcohol fermentations. Whole-genome sequencing revealed that both strains harbored key genes involved in glucose, fructose, and sucrose metabolism and genes implicated in ethanol production. Although SC8293 harbored maltose-metabolizing genes, including MAL13 and MAL31, these genes were absent in KM8279. This genetic difference was evident in the fermentation performance, manifesting as distinct variations in alcohol production depending on the carbohydrate source. A further investigation of fermentation conditions demonstrated that both strains maintained low alcohol levels and exhibited a consistent growth at 15–20 °C within 72 h. Fermentation using extracts from Pueraria lobata, Arctium lappa (AL), Zingiber officinale (ZO), and Platycodon grandifloras revealed that KM8279 markedly increased the production of volatile compounds, contributing to floral and fruity sensory attributes in ZO and AL, whereas SC8293 contributed to a more complex flavor profile in AL. Notably, KM8279-ZO and KM8279-AL fermentations maintained alcohol contents below 1%, indicating their potential application in non-alcoholic beverages. Future studies are needed to investigate the relationship between the key volatile compound production and associated genetic characteristics, along with sensory evaluations, to develop optimized flavor modulation strategies. Full article

The vinasse from Agave americana L. (blue cabuya) liquor has a high concentration of organic matter, nutrients with high chemical oxygen demand (COD), and low pH, properties that give it the potential to generate adverse impact on the environment if improperly disposed of. In other countries, studies have been conducted on yeast strain combinations in vinasses to produce biomass and reduce organic load, but there are no studies of the aforementioned yeast consortium in blue cabuya liqueur vinasses to produce biomass and reduce COD. Given this problem, the objective of the research was to reduce this adverse environmental impact through aerobic fermentation of this vinasse with the yeast consortium Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae (D 47-Lalvin). As a result, biomass production and COD reduction were achieved. The study evaluated temperature variables of 28 °C, 30 °C, and 32 °C, and pH values of 3, 4, and 5 under conditions of consortium and nutrient diammonium phosphate (DAP) concentrations of 1.32 g/L and 1.5 g/L, respectively, in a bioreactor with automatic control of temperature, time, stirring speed of 100 RPM, and air flow of 1 VVM. The result was a biomass yield of 93.4% and a COD reduction of 33.3%. It is concluded that the aerobic fermentation process of blue cabuya liquor vinasse with the yeast consortium employed produces a high biomass yield, which can be used for its protein value as an animal feed supplement and, due to its low COD value, as an agricultural fertilizer. Full article

With rising multidrug-resistant yeast pathogens, conventional antifungals are becoming less effective, urging the need for adjuvants that enhance their activity at lower doses. This study evaluated the synergistic activity of antimicrobial peptidomimetics (TM8 and RK758) or colistin sulphate in combination with conventional antifungals against Candida albicans, C. tropicalis, C. parapsilosis, Meyerozyma guilliermondii, Nakaseomyces glabratus, Pichia kudriavzevii and Kluyveromyces marxianus, and Candidozyma auris using the checkerboard microdilution test. RK758 was synergistic with fluconazole in 78% of isolates, with the remaining 22% of isolates still showing partial synergy; it showed synergy with amphotericin B in 56% of isolates, and with caspofungin, 78% of isolates exhibited either synergy or partial synergy. TM8 showed synergy with fluconazole in 44% (with partial synergy in another 44%) of isolates, with amphotericin B in 67% of isolates, and with caspofungin in 44% (with partial synergy in another 44%) of isolates. Colistin with fluconazole or caspofungin exhibited synergy or partial synergy in 56% of the isolates. No antagonism was observed in any of the combinations. Additionally, a time-kill assay further demonstrated synergistic activity between fluconazole and TM8 or RK758. The effects of these peptidomimetics on cell membrane integrity were demonstrated in an ergosterol binding assay, supported by SYTOX Green and cellular leakage assays, both indicating a lytic effect. These results suggest that peptidomimetics can synergise with conventional antifungals, offering a potential strategy for combination therapy against yeast infections. The membrane lytic activity of the peptidomimetics likely plays a role in their synergistic interaction with antifungals, thereby enhancing the antimicrobial activities of both compounds at sub-MIC levels. Full article

Fermentation at high temperatures may be a viable alternative for ethanol production, especially in tropical climate regions. This work describes the evaluation of ethanol production through extractive fermentation at high temperatures using thermotolerant Kluyveromyces marxianus. An experimental design was applied to assess the effect of temperature on the ethanol removal process by CO₂ stripping. Subsequently, kinetic modeling of conventional batch ethanol fermentation at high temperatures was performed, and the hybrid Andrews−Levenspiel model was found to be suitable for describing the kinetics of this process. Experiments were conducted to evaluate ethanol production at high temperatures using thermotolerant yeast, specifically evaluating the effects of different specific CO₂ flow rates (ϕ = 1.0, 1.5, and 2.0 vvm) on ethanol stripping. The results indicated that in all the extractive fermentations conducted with K. marxianus, there was faster substrate uptake and earlier substrate exhaustion compared to conventional fermentation. Significant ethanol removal by stripping was achieved using a CO₂ flow rate of 1.0 vvm (EF_HT1), and complete substrate consumption was observed by the end of 12 h of fermentation. This result highlights the positive effect of temperature on ethanol entrainment. In addition, integrating the CO₂ stripping technique with high-temperature fermentation (T = 40 °C) improves process efficiency with a lower gas flow rate. This is advantageous, especially for industrial-scale applications, as it can reduce equipment costs associated with the CO₂ feed. Full article