In Vitro Interaction Between Yeast Extracellular Vesicles and Human Monocyte-Derived Dendritic Cells.

Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and signaling. Recent literature reported that pathogenic or commensal yeast strains can produce EVs targeting the host's immune system and exerting immunomodulatory actions. In humans, yeast EVs can be endocytosed by dendritic cells (DCs), characterized by phagocyting and migrating capabilities with the role of capturing antigens to present to T lymphocytes, triggering the immune response. Physiological or disease-associated immunosenescence impairs both DC functionality and gut microbiota; thus investigating the interaction between commensal microorganisms and the host's immune system would help elucidate the impact of aging on the immune system-microbiota interplay. We hereby present a protocol for the incubation of in vitro-generated human monocyte-derived DCs with EVs purified from different yeast strains isolated from fermented milk. The protocol includes flow cytometry analysis on DC activation markers and endocytosis assay.

Enhancing nutritional composition and aroma characteristics of kiwifruit wines through indigenous non-Saccharomyces yeast extracellular extract treatment.

To unlock the potential of strains for further enhancing the aromatic complexity of kiwifruit wines while avoiding undesirable flavors, indigenous non-Saccharomyces yeast extracellular extract treatment for fermentation was established. The extracellular extract from Zygosaccharomyces rouxii, Pichia kudriavzevii, and Meyerozyma guilliermondii were prepared and supplemented individually or in pairs to the kiwifruit wine fermentation system. Subsequently, the changes in physicochemical properties, antioxidants, and volatile characteristics of kiwifruit wines produced by different protocols were comprehensively evaluated, and the major aroma descriptors affecting sensory acceptability were analyzed by sensory evaluation and partial least squares regression. The results showed that extracellular extract treatment significantly improved the organic acids and monomeric phenols content, antioxidant capacity, and volatiles of kiwifruit wines. Compared to Sc, the increase in esters and alcohols, along with the decrease in aldehydes and acids in Pk-Zr and Mg-Zr, enhanced the aromatic complexity while reduce grassy and fungal flavors, resulting in higher sensory acceptability.

Golden Gate Cloning for Efficient Biosynthesis of Lycopene in Synthetic Yeast.

Golden Gate Assembly (GGA) represents a versatile method for assembling multiple DNA fragments into a single molecule, which is widely used in rapid construction of complex expression cassettes for metabolic engineering. Here we describe the GGA method for facile construction and optimization of lycopene biosynthesis pathway by the combinatorial assembly of different transcriptional units (TUs). Furthermore, we report the method for characterizing and improving lycopene production in the synthetic yeast chassis.

The antidepressant effect of Komagataella pastoris KM 71 H in maternal separation mice model mediated by the microbiota-gut-brain axis.

BACKGROUND: The intestinal microbiota plays a fundamental role in maintaining host health, especially during childhood, a critical period for its establishment. Early life stress can lead to shifts in gut microbiota composition, thus increasing the risk of major depressive disorder (MDD) in adulthood. The supplementation with probiotics restores intestinal permeability and the health of gut microbial communities, therefore being potential study targets for the treatment of MDD. In this sense, the yeast Komagataella pastoris was reported as a promising probiotic with antidepressant effect. METHODS: Hence, the present study aims to investigate this effect in mice submitted to maternal separation (MS) 3 h per day from PND2 to PND14. Adult mice and mothers were treated with K. pastoris KM71H (8 log UFC.g-1/per animal, i.g.) or PBS (500 µl, i.g.) for 14 days. After behavioral tests, the animals were euthanized, followed by hippocampi and intestines removal for biochemical analysis. RESULTS: On behavioral tests, K. pastoris KM71H treatment reduced the immobility time in TST of adult mice and increased the grooming activity in splash test of adult mice and mothers induced by MS. The probiotic treatment restored plasma corticosterone levels and glucocorticoid receptor expression in hippocampi, alongside nitrate/nitrite levels and superoxide dismutase activity in intestine, in addition to reducing reactive species levels in both structures. Moreover, it also normalized the fecal pH and water content of feces. CONCLUSION: Thus, we conclude that K. pastoris KM71H is a promising therapeutic strategy for the treatment of MDD.

Fermentative and metabolic screening of candidate yeast strains hybridisable with Saccharomyces cerevisiae for beer production optimisation.

Yeast optimisation has been crucial in improving the quality and efficiency of beer production, one of the world's most widely consumed beverages. In this context, rare mating hybridisation is a promising technique for yeast optimization to generate novel and improved non-GMO strains. The limitation of this technique is the lack of knowledge and comparable data on yeast strains hybridisable to Saccharomyces cerevisiae, probably the most important yeast species in beer production. Yeast from the genera Saccharomyces, Naumovozyma, Nakaseomyces and Kazachstania have been described to be able to form hybrids with S. cerevisiae. In the present study, 242 yeast strains were analysed under brewing conditions, including Saccharomyces species (S. cerevisiae, S. kudriavzevii, S. uvarum, S. eubayanus, S. paradoxus, S. mikatae, S. jurei and S. arboricola) and non-Saccharomyces species (Naumovozyma, Nakaseomyces and Kazaschtania), representing the full genetic variability (species and subpopulations) described up to the start of the study. The fermentation profile was analysed by monitoring weight loss during fermentation to determine kinetic parameters and CO2 production. Metabolic analysis was performed to determine the concentration of sugars (maltotriose, maltose and glucose), alcohols (ethanol, glycerol and 2,3-butanediol) and organic acids (malic acid, succinic acid and acetic acid). Maltose and maltotriose are the predominant sugars in beer wort. The ability to consume these sugars determines the characteristics of the final product. Dataset comparisons were then made at species, subpopulation and isolation source level. The results obtained in this study demonstrate the great phenotypic variability that exists within the genus Saccharomyces and within each species of this genus, which could be useful in the generation of optimised brewing hybrids. Yeasts with different fermentative capacities and fermentative behaviours can be found under brewing conditions. S. cerevisiae, S. uvarum and S. eubayanus are the species that contain strains with similar fermentation performance to commercial strains.

Impact of fermentation by Saccharomyces Cerevisiae on the macronutrient and in vitro digestion characteristics of Chinese noodles.

This study examined the impact of live bread yeast (Saccharomyces cerevisiae) on the nutritional characteristics of Asian dried noodles. Micronutrient analysis of fermented noodles revealed a 6.9% increase in the overall amino acid content, a 37.1% increase in the vitamin B content and a 63.0% decrease in the phytic acid level. Molecular weight analysis of starch and protein contents revealed moderate decrease in the fermented noodles. The in vitro digestion of fermented noodles showed a slightly faster initial acidification, four-fold decrease in the initial shear viscosity (from 8.85 to 1.94 Pa·s). The initial large food particle count (>2 mm diameter) was 19.5% lower in the fermented noodles. The fermented noodles contained slightly higher free sugar content (73.5 mg g-1 noodle) during the gastric digestion phase. The overall nutrition and digestion results indicate nutritional improvement and digestion-easing attributes in the fermented noodles.

Formation and resuscitation of viable but non-culturable (VBNC) yeast in the food industry: A review.

The viable but non-culturable (VBNC) state is a survival strategy adopted by microorganisms in response to unfavorable conditions in the environment. VBNC cells are unable to form colonies but still maintain a low level of activity, posing a potential threat to food safety and public health. Therefore, the development of effective strategies to prevent the formation and resuscitation of VBNC cells of microorganisms is a key challenge in food science and microbiology research. However, current research on VBNC cells has primarily focused on bacteria, with relatively limited reports on fungi. This paper provides a comprehensive and systematic review of yeast in the VBNC state, discussing various factors that induce and facilitate resuscitation, along with detection methods and formation and recovery mechanisms. A comprehensive understanding of the induction and resuscitation of yeast in the VBNC state and exploration of its molecular mechanism hold significant implications for food safety and public health. It is imperative to enhance our comprehension of the underlying mechanisms and contributory factors pertaining to VBNC yeast, thereby facilitating the efficient management of the food fermentation process and ensuring the integrity of food quality and safety.

Assessing the physiological properties of baker's yeast based on single-cell Raman spectrum technology.

With rapid progress in the yeast fermentation industry, a comprehensive commercial yeast quality assessment approach integrating efficiency, accuracy, sensitivity, and cost-effectiveness is required. In this study, a new yeast quality assessment method based on single-cell Raman technology was developed and contrasted with traditional methods. The findings demonstrated significant associations (Pearson correlation coefficient of 0.933 on average) between the two methods in measuring physiological indicators, including cell viability and intracellular trehalose content, demonstrating the credibility of the Raman method compared to the traditional method. Furthermore, the sensitivity of the Raman method in viable but non-culturable cells was higher in measuring yeast cell viability (17.9 % more sensitive). According to the accurate quantitative analysis of metabolic activity level (MAL) of yeast cells, the cell vitality was accurately quantified at population and single-cell levels, offering a more comprehensive assessment of yeast fermentation performance. Overall, the single-cell Raman method integrates credibility, feasibility, accuracy, and sensitivity in yeast quality assessment, offering a new technological framework for quality assessments of live-cell yeast products.

Evaluation of the nutritional quality of yeast protein in comparison to animal and plant proteins using growing rats and INFOGEST model.

Yeast, identified as a microorganism, boasts a considerable protein content, positioning yeast protein as a highly promising alternative in the quest for sustainable protein sources. The primary aim of this study is to evaluate the protein quality of yeast protein and compare it with animal proteins (whey concentrate/isolate proteins) and plant proteins (soy, wheat, pea proteins). Notably, yeast protein exhibits the highest ratio of indispensable/dispensable amino acids (IAAs/DAAs, 0.91). However, in both in vivo and in vitro digestion experiments, yeast protein demonstrated lower true protein digestibility (TPD) and true ileal digestibility (TID) compared to other proteins. Despite this, the yeast protein's amino acid score (AAS, 1.37 for >3 years), protein digestibility-corrected amino acid score (PDCAAS, 100 % for >3 years), and digestibility-corrected amino acid score (DIAAS, 82.42 % for >3 years) of yeast protein surpassed those of plant proteins, yet remained lower than animal proteins primarily due to its lower digestibility.

Screening and identification of novel umami peptides from yeast proteins: Insights into their mechanism of action on receptors T1R1/T1R3.

This study aimed to unravel the peptide profiles of six distinct yeast protein samples and identify novel umami peptides within them. Peptide characteristics analysis support the proposition that yeast protein peptide pools represent exceptional reservoirs of umami peptides. Nine potential umami peptides were screened using the iUmami_SCM, UMPred-FRL, Umami_YYDS, Umami-MRNN, Innovagen, Expasy-ProtParam, and ToxinPred tools. Peptides AGVEDVY, LFEQHPEYRK, AFDVQ, GPTVEEVD, NVVAGSDLR, ATNGSR, and VEVVALND (1 mg/mL) were confirmed to possess umami taste, and the first five peptides exhibited significant umami-enhancing effects on 0.35 % monosodium glutamate. Molecular docking indicated that peptide residues His, Arg, Tyr, Asp, Gln, Thr, Ser, and Glu primarily bound to His71, Ser107/109/148, Asp147/218, and Arg277 of T1R1 and Ser104/146, His145, Asp216, Tyr218, and Ala302 of T1R3 through hydrogen bonds. This study enriches the umami peptide repository for potential food additive use and establishes a theoretical foundation for exploring taste compounds in yeast proteins and their broader applications.

Protein Kinase R (PKR) as a Novel dsRNA Sensor in Antiviral Innate Immunity.

Protein kinase R (PKR), a key double-stranded RNA (dsRNA)-activated sensor, is pivotal for cellular responses to diverse stimuli. This protocol delineates a comprehensive methodological framework employing single luciferase assays, yeast assays, immunoblot assays, and quantitative PCR (qPCR) to discern and validate PKR activities and their downstream impacts on NF-κB-activating signaling pathways. These methodologies furnish a systematic approach to unraveling the role of PKR as a dsRNA sensor and effector in antiviral innate immunity, enabling in-depth analyses of dsRNA sensor activities.

Yeast Two-Hybrid Assay for Investigating Antiviral Innate Immunity.

Yeast two-hybrid (YTH) technology is a powerful tool for studying protein interactions and has been widely used in various fields of molecular biology, including the study of antiviral innate immunity. This chapter presents detailed information and experimental procedures for identifying virus-host protein interactions involved in immune regulation using yeast two-hybrid technology.

Characterization and biocontrol potential of Wickerhamomyces subpelliculosus yeasts isolated from dates: Volatile compounds-mediated antifungal activity against mycotoxigenic Penicillium strains.

Seven yeast strains were isolated from Tunisian dates. The strains were identified by sequence analysis of the D1/D2 domain of the nuclear large subunit (LSU) rRNA gene. Based on this all strains in the study were almost identical with that of the type strain of Wickerhamomyces subpelliculosus (CBS 5767) indicating that they belong to this species. All strains were characterized physiologically and biochemically. All strains grew in the presence of 50 % sucrose, 10 % sodium chloride and at 42 °C. The potential of these yeasts as biocontrol agent against mycotoxigenic Penicillium species inhabiting date, was evaluated. All yeast strains inhibited the growth of P. citrinum P10 and P. chrysogenum C17 previously isolated from dates, with inhibition percentages ranging between 43.6 % and 70.3 % on dual culture plate assays. Moreover, the volatile compounds (VCs) produced by these yeasts inhibited the mycelial growth rate and sporulation of both fungus strains, up to 76.5 and 100 %, respectively, on inverted culture plate assay. The VCs of W. subpelliculosus strains Y4 and Y24, which exhibit strong inhibitory activity against toxigenic Penicillium, were determined by head-space solid-phase microextraction (HS-SPME) combined with gas chromatography coupled with mass spectrometry (GC-MS) analysis. Results revealed significant levels of alcohols (27.36 % for Y4 and 23.35 % for Y24) and esters (66.19 % for Y4 and 75.82 % for Y24). Their significant bioactivity, along with the lack of reported adverse effects on consumer health or the environment, makes them a sustainable and effective alternative to synthetic fungicides for the biocontrol of mycotoxigenic Penicillium affecting stored dates.

Endophytic bacteria in Camellia reticulata pedicels: isolation, screening and analysis of antagonistic activity against nectar yeasts.

Camellia reticulata, an ancient plant species endemic to Yunnan Province, China, remains underexplored in terms of its endophytic bacterial communities. The plant tissue pedicel serves as the connection between the flower and the stem, not only delivers nutrients but also transmits metabolic substances from endophytic bacteria to the nectar during long-term microbial colonization and probably improves the antagonistic activity of nectar against yeast. Hence, 138 isolates of endophytic bacteria have been isolated in this study from the pedicels of 12- and 60-year-old C. reticulata. Comparative analysis revealed significantly higher density of endophytic bacteria in older trees. Among these isolates, 29 exhibited inhibitory effects against nectar yeasts. Most of the isolates displayed positive results for Gram staining, catalase reaction, gelatin liquefaction, and motility. Additionally, the isolates demonstrated the ability to utilize diverse substrates, such as glucose, nitrate, and starch. Based on 16S rRNA molecular biology analysis, these isolates were identified to be 11 different species of 6 genera, with the majority belonging to Bacillus genus. Notably, C1 isolate, identified as Bacillus spizizenii, exhibited strongest antagonistic effect against three yeasts, i.e., Metschnikowia reukaufii, Cryptococcus laurentii, and Rhodotorula glutinis, with minimum inhibitory concentration values below 250 µg/mL. Major metabolites of B. spizizenii were aminoglycosides, beta-lactams, and quinolones, which possess antimicrobial activities. Furthermore, KEGG enrichment pathways primarily included the synthesis of plant secondary metabolites, phenylpropanoids, amino acids, alkaloids, flavonoids, neomycin, kanamycin, and gentamicin. Therefore, antagonistic activity of B. spizizenii against yeasts could be attributed to these antibiotics. The findings highlight the diverse endophytic bacteria associated with C. reticulata, indicating their potential as a valuable resource of bioactive metabolites. Additionally, this study provides new insights into the role of endophytic bacteria of pedicels in enhancing nectar resistance against yeasts.

A yeast surface display platform for screening of non-enzymatic protein secretion in Kluyveromyces lactis.

Enhancing the secretion of recombinant proteins, particularly non-enzymatic proteins that predominate in food and pharmaceutic protein products, remains a significant challenge due to limitations in high-throughput screening methods. This study addresses this bottleneck by establishing a yeast surface display system in the food-grade microorganism Kluyveromyces lactis, enabling efficient display of model target proteins on the yeast cell surface. To assess its potential as a universal high-throughput screening tool for enhanced non-enzymatic protein secretion, we evaluated the consistency between protein display levels and secretion efficiency under the influence of various genetic factors. Our results revealed a strong correlation between these two properties. Furthermore, screening in a random mutagenesis library successfully identified a mutant with improved secretion. These findings demonstrate the potential of the K. lactis surface display system as a powerful and universal tool for high-throughput screening of strains with superior non-enzymatic protein secretion capacity. We believe this study could pave the way for efficient large-scale production of heterologous food and therapeutic proteins in industries. KEY POINTS: • A YSD (yeast surface display) system was established in Kluyveromyces lactis • This system enables high-throughput screening of non-enzymatic protein secretion • This technology assists industrial production of food and therapeutic proteins.

Pediococcus pentosaceus RC007 and Saccharomyces boulardii RC009 as antibiotic alternatives for gut health in post-weaning pigs.

AIMS: The aim of the present study was to evaluate a novel probiotic Pediococcus pentosaceus RC007 used alone and convined with Saccharomyces cerevisiae var. boulardii RC009, as in-feed additives to substitute the non-therapeutic use of antibiotics, and evaluate the different structural characteristics of intestinal bacterial populations between groups, correlated with pig production performance. METHODS AND RESULTS: The in vivo study was conducted on post-weaning pigs, from 21 to 56 days-old. Three dietary treatments were included: T1- basal diet (BD - Control group); T2- BD with P. pentosaceus RC007; and T3- BD with a mix of P. pentosaceus RC007 and S. boulardii RC009. The weight gain increase of pigs consuming non-therapeutic antibiotics was similar to those that did not consume antibiotics during the study (p=0.0234), but had better health indicators. The use of a probiotic combination increased carcass weight and significantly reduced the lumbar fat thickness. In terms of taxonomic composition, there was a tendency to modify the abundance of Proteobacteria, Cyanobacteria, Enterobacteriaceae and Lactobacillaceae in pigs that consumed the additives. The genus Butyricicoccus, Collinsella and Ruminococcus tended to be more abundant in the microbiota of pigs at T3. CONCLUSIONS: For the first time, the results of the present study indicate that P. pentosaceus RC007 and S. boulardii RC009, a probiotic combination, could be a good substitute for antibiotics in improving pig production performance, while also contributing to a healthier gut microbiota, especially with the reduced abundance of Proteobacteria and Cyanobacteria.

Probiotic yeast characterization and fungal amplicon metagenomics analysis of fermented bamboo shoot products from Arunachal Pradesh, northeast India.

This study investigates the diverse fungal community and their probiotic functions present in ethnic fermented bamboo shoots of Arunachal Pradesh. Among 95 yeast isolates, 13 demonstrated notable probiotic attributes. These included growth at pH 3, bile tolerance, autoaggregation, co-aggregation, hydrophobicity, lysozyme tolerance and antimicrobial activity. Confirmation of some of the probiotic properties through specific primers enabled the detection of genes associated with acid and bile tolerance, antimicrobial activity, and adhesion. Probiotic yeasts were finally identified based on D1 and D2 sequences of large ribosomal subunit as Meyerozyma guilliermondii (BEP1, KGM1_3, NHR3), Meyerozyma caribbica (GEP7), Candida orthopsilopsis (ES1_2, EB1_2, EEGM2_4, GEP2, NEK9), Candida parasilopsis (HD1_1), Pichia kudriavzevii (NHR12), Pichia fermentans (BEP2), and Saccharomyces cerevisiae (NEP2). Fungal amplicon sequencing highlighted the predominance of Ascomycetes, particularly Pestalotiopsis and Penicillium genera. In this study we have perfomed a culture dependent isolation and probiotic study of yeasts and culture independent analysis of the fungal community present during the fermentation of bamboo shoots of Arunachal Pradesh which provides information about the beneficial properties of bamboo shoots as the reservoir of probiotic microorganisms.

Impact of corn shredlage and crabtree-negative yeast on silage quality and rumen fermentation characteristics.

The physical attributes of corn silage are enhanced by shredlage (SHR), while there is a rising interest in boosting its biological performance. This study aimed to assess and compare the impact of both the chopping method and different yeast strains on ensilage quality including the in vitro evaluation of corn silage. Both types of corn, including chopped and shredded, were harvested on the same day from the same field where the same corn hybrid (Suwan 5) was grown. Subsequently, whole-corn plants were fermented with additives. A 2 × 5 Factorial completely randomized design was employed, where factor A represents corn chopped (CON) and corn shredded (SHR), and factor B represents the additives: no additives, molasses + urea (M + U), M + U + Candida tropicalis KKU20, M + U + Pichia kudriavzevii KKU20, and M + U + saccharomyces cerevisiae. The results demonstrated that SHR fermentation with M + U and yeast significantly increased in vitro dry matter degradability (IVDMD) and organic matter degradability (IVOMD). Specifically, at 4 h post-incubation, the addition of Crabtree-negative yeast led to a 5.8% increase in total volatile fatty acids (TVFA) compared to the Crabtree-positive yeast group (P < 0.01). The C2 (acetic acid) + C4 (normal butyric acid and isobutyric acid): C3 (propionic acid) ratio showed a significant decrease without additives, but P. kudriavzevii KKU20 led to the highest ratio and methane production (P < 0.01). Based on this study, it could be concluded that SHR harvesting led to higher digestion efficiency in the rumen. The use of M + U + yeast also demonstrated uncertain effects on rumen fermentation efficiency, and the inclusion of P. kudriazevii KKU20 may potentially reduce rumen fermentation efficiency when used with corn silage.

Improved properties of dough fermented with rice wine prepared by mixed Saccharomycopsis fibuligera SF7 and Saccharomyces cerevisiae SC1.

In view of the special aptitudes of the yeast population in both rice wine production and dough fermentation, in this study, the characteristics of dough fermented with rice wine prepared from mixed Saccharomycopsis fibuligera SF7 and Saccharomyces cerevisiae SC1 (JFC) were evaluated and compared with those of dough fermented directly with the two yeast co-cultures (FC). Dough inoculated with JFC showed higher acidity, reducing sugar content and leavening activity than dough fermented with FC. The water distribution pattern and pasting properties of the JFC-fermented dough changed dramatically after fermentation, and the dough microstructure and extensibility were improved. The steamed bread made with JFC-fermented dough had improved specific volume and showed similar volatile compound profiles to that made with commercial Jiuqu. These results indicated that mixed S. fibuligera SF7 and S. cerevisiae SC1 could be used as defined starter cultures in rice wine preparation for steamed bread production. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-024-01598-w.

Screening using loop-mediated isothermal amplification (LAMP) assay and breeding of a Saccharomyces cerevisiae strain isolated from Muramatsu Park, Japan, for sake brewing.

Sake is a Japanese alcoholic beverage produced by fermenting steamed rice and koji (a culture of Aspergillus oryzae on steamed rice) with sake yeast, a strain of Saccharomyces cerevisiae. Sake yeast strains are important for maintaining product quality and process efficiency. In this study, a S. cerevisiae strain from Muramatsu Park, Gosen City, Niigata Prefecture was isolated using a loop-mediated isothermal amplification (LAMP) assay. The yeast strain was cultured using the mass spore-cell/cell-cell mating method with a sake yeast haploid. The resultant hybrid yeast strain, HG-3-F2, exhibited superior efficiency in alcoholic fermentation compared with the HG-3 strain. Our findings support the applicability of these original and mating strains in sake brewing.