Yeast Surface Display Methodology for the Characterization of Food Allergens In Situ.

High throughput allergen characterization is often based on phage display technique which is limited by the constraints of a prokaryotic expression system such as potential loss of conformational epitopes and lack of post-translational modifications. Replacing the phage display platform with a yeast surface display system could accelerate the immunological characterization of complex structured allergens. Yeast surface display is a powerful technique that allows faster immunochemical characterization of allergens in situ without the need for protein purification. Yeast surface display offers an alternative that could lead to the improvement of standard immunodiagnostic and immunotherapeutic approaches. In this chapter, we describe a protocol on yeast surface display for the characterization of plant-derived food allergens using actinidin (Act d 1), a major kiwifruit allergen, as a model system.

Recombinant Production of Food Allergens in Yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris (P. pastoris) is one of the expression systems widely used to produce recombinant heterologous proteins. In this chapter, the methodology to produce recombinant food allergens in P. pastoris is described. The methodology begins with the preparation of competent P. pastoris cells followed by the transformation of the competent cells by electroporation as well as the preparation of plasmid DNA for transformation. Moreover, the screening of yeast transformants by direct PCR to ensure integration of allergen DNA followed by small-scale expression of recombinant allergen in yeast cells is also described.

Label-free quantitative proteomics to exploit the impact of sourdough fermentation on reducing wheat allergenic fractions.

The microbial consortia of lactic acid bacteria and yeast of sourdough can partially degrade gluten subunits associated with wheat-related diseases. This study evaluated how sourdough fermentation interferes with wheat protein profiles and if it can be related to the reduction expression of allergenic proteins. Samples from five bread doughs (Saccharomyces cerevisiae -C1; chemical acidification -C2, and three sourdoughs formulations -S1, S2, and S3) were sequentially extracted, digested, and submitted to shotgun label-free proteomic analysis. Eight-five proteins were identified as allergenic, mainly belonging to gliadin fraction, including seven containing the 33-mer peptide sequence. The highest immunogenic potential was found in dough C1 and S3, while the least reactive group consisted of S1 and C2. The two folds down expression of an α-gliadin containing the 33-mer sequence corroborates this. This finding may indicate the role of organic acids produced by the microbiota sourdough type II during fermentation in changing the protein profile.

Fermentation-promoting effect of three salt-tolerant Staphylococcus and their co-fermentation flavor characteristics with Zygosaccharomyces rouxii in soy sauce brewing.

Staphylococcus is the dominant genus in the fermentation process of soy sauce, but its effect on the flavor of soy sauce has not been clearly established. In order to investigate the role of this genus in soy sauce fermentation, individual fermentation with Staphylococcus spp. screened from the moromi and their co-fermentation with an ester-producing yeast of Zygosaccharomyces rouxii were designed. Through the analysis of physicochemical properties, organic acid composition, volatile flavor compounds (VFCs) and sensory characteristics during fermentation, Staphylococcus was confirmed as a contributor to the acidity, ester aroma and alcohol aroma of soy sauce. In their co-fermentation with yeast, the ester aroma of soy sauce was further enhanced. Moreover, pathway enrichment analysis and network construction of key VFCs also revealed potential metabolic networks for formation of characteristic flavor compounds in co-fermentation. This work will help optimize the fermentation functional microbiota to obtain better soy sauce flavor.

Metabolism and release of characteristic components and their enzymatic mechanisms in Pericarpium Citri Reticulatae co-fermentation.

A co-fermentation strategy was explored to rapidly improve the characteristic components and quality of Pericarpium Citri Reticulatae (PCR) using Monascus anka and Saccharomyces cerevisiae, and the enzymatic mechanism was investigated. The results showed that the free flavonoid content of fermented PCR was 48.12% higher than that of unfermented PCR after 12 days of co-fermentation, resulting in stronger antioxidant activity. d-Limonene, γ-terpinene, proline (Pro), arginine (Arg), and serine (Ser) contributed the most to the flavors of citrus, herb, and sweet citrus based on odor and taste activity value analysis. Metabolomics and multivariate statistics showed that 55 components were differentially metabolized during co-fermentation, and ten metabolic pathways were closely related to metabolism. Furthermore, five hydrolases participated in the release and conversion of the active ingredients. This study provides an effective processing method for PCR and is conducive to the development of new PCR functional health foods.

Antifungal and antibacterial activities of Cannabis sativa L. resins.

ETHNOPHARMACOLOGICAL RELEVANCE: Cannabis sativa L. (Cannabaceae) is a plant native to Eastern Asia spread throughout the world because of its medicinal properties. Despite being used for thousands of years as a palliative therapeutic agent for many pathologies, in many countries research on its effects and properties could only be carried out in recent years, after its legalization. AIMS OF THE STUDY: Increasing resistance to traditional antimicrobial agents demands finding new strategies to fight against microbial infections in medical therapy and agricultural activities. Upon legalization in many countries, Cannabis sativa is gaining attention as a new source of active components, and the evidence for new applications of these compounds is constantly increasing. METHODS: Extracts from five different varieties ofCannabis sativa were performed and their cannabinoids and terpenes profiles were determined by liquid and gas chromatography. Antimicrobial and antifungal activities against Gram (+) and Gram (-) bacteria, yeast and phytopathogen fungus were measured. To analyze a possible action mechanism, cell viability of bacteria and yeast was assessed by propidium iodide stain. RESULTS: Cannabis varieties were grouped into chemotype I and II as a consequence of their cannabidiol (CBD) or tetrahydrocannabinol (THC) content. The terpenes profile was different in quantity and quality among varieties, with (-)b-pinene, b-myrcene, p-cymene and b-caryophyllene being present in all plants. All cannabis varieties were effective to different degree against Gram (+) and Gram (-) bacteria as well as on spore germination and vegetative development of phytopathogenic fungi. These effects were not correlated to the content of major cannabinoids such as CBD or THC, but with the presence of a complex terpenes profile. The effectiveness of the extracts allowed to reduce the necessary doses of a widely used commercial antifungal to prevent the development of fungal spores. CONCLUSION: All the extracts of the analysed cannabis varieties showed antibacterial and antifungal activities. In addition, plants belonging to the same chemotype showed different antimicrobial activity, demonstrating that the classification of cannabis strains based solely on THC and CBD content is not sufficient to justify their biological activities and that other compounds present in the extracts are involved in their action against pathogens. Cannabis extracts act in synergy with chemical fungicides, allowing to reduce its doses.

Antipyretic and antinociceptive effects of methanolic extract of C. iria L. tuber.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus iria L. is a sedge belongs to Cyperaceae family. Tuber of this plant is traditionally used in fever. AIM OF THE STUDY: This study aimed to verify the effectiveness of this plant part against fever. Additionally, antinociceptive effect of the plant was evaluated. MATERIALS AND METHODS: Antipyretic effect was evaluated by yeast induced hyperthermia experiment. Antinociceptive effect was determined by acetic acid induced writhing test and hot plate test. Four different doses of plant extract were used in mice model. RESULTS: Extract at dose of 400 mg/kg.bw produced greater effect than paracetamol; reduction of elevated mice body temperature was observed by 2.6 °F and 4.2 °F after 4 h by paracetamol and 400 mg/kg.bw extract respectively. In acetic acid writhing test, extract at 400 mg/kg.bw and diclofenac were found to have equivalent effects producing percentage inhibition of writhing of 67.68% and 68.29% respectively. In hot plat test, significant reduction in latency was also observed after administration of plant extracts. Mean percent maximal effect was 83.55% and 67.26% for ketorolac and extract (400 mg/kg.bw) respectively. CONCLUSION: Our study endorsed the traditional use of C. iria tuber in fever with possible antinociceptive effects.

Optimization of Molasses and Soybean Meal Content to Enhance Tetramethylpyrazine Yield by Bacillus sp. TTMP20.

Microbial fermentation for the production of tetramethylpyrazine (TTMP) is considered to be the most promising method, and the development of a cheap fermentation substrate is of great importance for large-scale TTMP production. In this study, inexpensive by-products from the food industry, i.e., molasses and soybean meal (instead of glucose and tryptone), were used as substrates for TTMP fermentation. The pretreatment of soybean meal was explored in order to achieve a better fermentation effect. The contents of each component in the fermentation medium were optimized by central composite design (CCD). The optimum contents were as follows: 72.5 g/L of molasses, 37.4 g/L of diammonium hydrogen phosphate (DAP), 53.4 g/L of soybean meal, and 5 g/L of yeast powder. The software predicted a maximum TTMP yield of 1469.03 mg/L, and the actual TTMP yield was 1328.95 mg/L for the validation experiment in the optimum medium. Under the optimum conditions (72.5 g/L of molasses, 37.4 g/L of DAP, 53.4 g/L of soybean meal, and 5 g/L of yeast powder), the actual maximum TTMP yield (1328.95 mg/L) in this study was much higher than the TTMP yield (895.13 mg/L) under the conditions (150 g/L of molasses, 30 g/L of DAP, 30 g/L of tryptone, and 10 g/L of yeast powder) of our previous study published in Molecules. In this study, the TTMP yield improved by 48.46%, with decreased molasses (more than half), decreased yeast powder (half) and by-product soybean meal instead of tryptone compared to our previous study. In summary, the cheaper fermentation medium had a higher TTMP yield in this study, which improves the application potential of Bacillus sp. TTMP20.

A Yeast Cell Wall Derived Hybrid Hydrogel with Photothermal and Immune Combined Modality Therapy for Enhanced Anti-Melanoma Efficacy.

Introduction: The effect of traditional treatment for melanoma is quite limited, especially for its recurrence. As the major components of yeast cell wall, chitin and ß-glucan exhibit good immune activation effect and are promising candidates for adjuvant. Therefore, melanoma cell membrane (CM) and indocyanine green (ICG) was loaded in a chitin and ß-glucan hybrid hydrogel to achieve an enhanced anti-melanoma therapy. Methods: The novel hybrid hydrogel was prepared, and its physicochemical properties were examined. Its effect towards melanoma prevention and treatment was evaluated via a melanoma-bearing mice model. Results: The CM-ICG-hybrid hydrogel was successfully prepared with excellent injectability, self-healing, drug loading, rheological, in vitro and in vivo photothermal stability, and retention properties. It also exhibited good cellular and in vivo safety profiles. In the primary melanoma mice model, it quickly ablated the in-situ melanoma, effectively inhibited the tumor growth, increased the survival rate of melanoma-bearing mice, and increased the level of IFN-γ and TNF-α. In the distal secondary melanoma model, it efficiently prevented the reoccurrence of melanoma and activated the memory T cells. In both models, a synergistic effect of photothermal therapy and immune therapy was found. The hydrogel effectively recruited CD3+ CD4+ T cells and CD3+ CD8+ T cells, inhibited the proliferation of melanoma cells, and induced the apoptosis of melanoma cells. Conclusion: The hybrid hydrogel was successfully prepared, and it showed excellent efficacy towards melanoma prevention and treatment due to its efficient tumor ablation and immune activation capability.

Yeast-driven valorization of agro-industrial wastewater: an overview.

The intensive industrial and agricultural activities currently on-going worldwide to feed the growing human population have led to significant increase in the amount of wastewater produced. These effluents are high in phosphorus (P), nitrogen (N), chemical oxygen demand (COD), biochemical oxygen demand (BOD), and heavy metals. These compounds can provoke imbalance in the ecosystem with grievous consequences to both the environment and humans. Adequate treatment of these wastewaters is therefore of utmost importance to humanity. This can be achieved through valorization of these waste streams, which is based on biorefinery idea and concept of reduce, reuse, and recycle for sustainable circular economy. This concept uses innovative processes to produce value-added products from waste such as wastewater. Yeast-based wastewater treatment is currently on the rise given to the many characteristics of yeast cells. Yeasts are generally fast growing, and they are robust in terms of tolerance to stress and inhibitory compounds, in addition to their ability to metabolize a diverse range of substrates and create a diverse range of metabolites. Therefore, yeast cells possess the capacity to recover and transform agro-industrial wastewater nutrients into highly valuable metabolites. In addition to remediating the wastewater, numerous value-added products such as single cell oil (SCO), single cell proteins (SCPs), biofuels, organic acid, and aromatic compounds amongst others can be produced through fermentation of wastewater by yeast cells. This work thus brings to limelight the potential roles of yeast cells in reducing, reusing, and recycling of agro-industrial wastewaters while proffering solutions to some of the factors that limit yeast-mediated wastewater valorization.

Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81.

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

Short tandem repeats bind transcription factors to tune eukaryotic gene expression.

Short tandem repeats (STRs) are enriched in eukaryotic cis-regulatory elements and alter gene expression, yet how they regulate transcription remains unknown. We found that STRs modulate transcription factor (TF)-DNA affinities and apparent on-rates by about 70-fold by directly binding TF DNA-binding domains, with energetic impacts exceeding many consensus motif mutations. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density, with impacts well predicted by statistical mechanics. Confirming that STRs also affect TF binding in cells, neural networks trained only on in vivo occupancies predicted effects identical to those observed in vitro. Approximately 90% of TFs preferentially bound STRs that need not resemble known motifs, providing a cis-regulatory mechanism to target TFs to genomic sites.

Promising application of probiotic microorganisms as Pickering emulsions stabilizers.

The purpose of this work was to study the ability of nineteen food-grade microorganisms as Pickering emulsion (PE) stabilizers. Medium-chain triacylglycerol (MCT) oil-in-water (50:50) PEs were fabricated by 10 wt% or 15 wt% of thermally-inactivated yeast, cocci, Bacillus spp. and lactobacilli cells. The characteristics of microorganisms related to "Pickering stabilization" including morphology, surface charge, interfacial tension, and "contact angle" were firstly studied. After that, the cells-stabilized PEs were characterized from both kinetic and thermodynamic viewpoints, microstructure and rheological properties. The interfacial tension and "contact angle" values of various microorganisms ranged from 16.33 to 38.31 mN/m, and from 15° to 106°, respectively. The mean droplet size of PEs ranged from 11.51 to 57.69 µm. Generally, the physical stability of cell-stabilized PEs followed this order: lactobacilli > Bacillus spp. > cocci > yeast. These variations were attributed to the morphology and cell wall composition. Increasing the microorganism concentration significantly increased the physical stability of PEs from a maximum of 12 days at 10 wt% to 35 days at 15 wt% as a result of better interface coverage. Shear-thinning and dominant elastic behaviors were observed in PEs. Physical stability was affected by the free energy of detachment. Therefore, food-grade microorganisms are suggested for stabilizing PEs.

Role of Candida in the bioremediation of pollutants: a review.

The population and modernization of society have increased dramatically from past few decades. In order to meet societal expectations, there has been a massive industrialization and resource exploitation. Anthropogenic practices like disposal of hazardous waste, large carbon footprint release variety of xenobiotic substances into the environment, which endanger the health of the natural ecosystem. Therefore, discovering proper long-term treatment approaches is a global concern. Various physical and chemical approaches are employed to remove contaminants. However, these technologies possess limitations like high cost and low efficacy. Consequently, bioremediation is regarded as one of the most promising remedies to these problems. It creates the option of either totally removing pollutants or transforming them into nonhazardous compounds with the use of natural biological agents. Several microorganisms are being utilized for bioremediation among which yeasts possess benefits such as high biodegradability, ease of cultivation etc. The yeast of Candida genus has the capability to effectively eliminate heavy metal ions, as well as to degrade and emulsify hydrocarbons which makes it a promising candidate for this purpose. The review highlights many potential uses of Candida in various remediation strategies and discusses future directions for research in this field.

Performance and mechanism of co-culture of Monascus purpureus, Lacticaseibacillus casei, and Saccharomyces cerevisiae to enhance lovastatin production and lipid-lowering effects.

To facilitate lipid-lowering effects, a lovastatin-producing microbial co-culture system (LPMCS) was constituted with a novel strain Monascus purpureus R5 in combination with Lacticaseibacillus casei S5 and Saccharomyces cerevisiae J7, which increased lovastatin production by 54.21% compared with the single strain R5. Response Surface Methodology (RSM) optimization indicated lovastatin yield peaked at 7.43 mg/g with a fermentation time of 13.88 d, water content of 50.5%, and inoculum ratio of 10.27%. Meanwhile, lovastatin in LPMCS co-fermentation extracts (LFE) was qualitatively and quantitatively analyzed by Thin-Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). Cellular experiments demonstrated that LFE exhibited no obvious cytotoxicity to L-02 cells and exhibited excellent biosafety. Most notably, high-dose LFE (100 mg/L) exhibited the highest reduction of lipid accumulation, total cholesterol, and triglycerides simultaneously in oleic acid-induced L-02 cells, which decreased by 71.59%, 38.64%, and 58.85% than untreated cells, respectively. Overall, LPMCS provides a potential approach to upgrade the lipid-lowering activity of Monascus-fermented products with higher health-beneficial effects.

Yeast vacuolar enzymes as novel hatching inhibitors for aquatic organisms, Daphnia magna and Danio rerio eggs.

Concerns over the spread of non-native species in aquatic environments have led to the need for effective methods to prevent and control their spread while protecting native species. This study investigated the potential of yeast vacuolar enzymes as a natural hatching inhibitor for controlling aquatic organisms. Hatching experiments with Daphnia magna eggs demonstrated that exposure to yeast vacuole enzymes inhibited hatching in a concentration-dependent manner, suggesting their potential as an effective inhibitor of egg hatching in aquatic organisms. Interestingly, the protease used for comparative purposes did not inhibit hatching, but instead increased the mortality of hatched D. magna. Additionally, chorionic changes were observed in non-hatched D. magna eggs and zebrafish eggs exposed to yeast vacuole enzymes, suggesting that the enzyme can alter the chorion and interfere with hatching. These findings suggest that yeast vacuolar enzymes may be a promising and natural management tool for controlling the spread of harmful aquatic organisms, and further research is warranted to explore their potential for species-specific control.

Simultaneous DHA and organic selenium production by Schizochytrium sp.: a theoretical basis.

Docosahexaenoic acid (DHA) and selenium (Se) are nutrients that confer several health benefits to both humans and animals. Widespread use of DHA in milk powder and health products requires large-scale mass production via Schizochytrium sp., while Se intended for human consumption is produced as organic Se via yeast. However, producing these nutrients on an industrial scale is constrained by various factors. We found that supplementing Schizochytrium sp. with Na2SeO3 (0.5 mg/L) improves its biomass and DHA production and also provides organic Se. De novo assembled transcriptome and biochemical indicators showed that Na2SeO3 promotes forming acetyl coenzyme A and L-cysteine via the glycerol kinase and cysteine synthase pathways, promoting DHA synthesis through the polyketide synthase pathway. However, high doses of Na2SeO3 (5 mg/L) limited the biomass of Schizochytrium sp. and DHA content. This study provided a theoretical basis for the simultaneous production of organic Se and DHA via Schizochytrium sp.

Imidazo[1,2-c]quinazolines as a novel and potent scaffold of α-glucosidase inhibitors: design, synthesis, biological evaluations, and in silico studies.

α-Glucosidase inhibition is an approved treatment for type 2 diabetes mellitus (T2DM). In an attempt to develop novel anti-α-glucosidase agents, two series of substituted imidazo[1,2-c]quinazolines, namely 6a-c and 11a-o, were synthesized using a simple, straightforward synthetic routes. These compounds were thoroughly characterized by IR, 1H and 13C NMR spectroscopy, as well as mass spectrometry and elemental analysis. Subsequently, the inhibitory activities of these compounds were evaluated against Saccharomyces cerevisiae α-glucosidase. In present study, acarbose was utilized as a positive control. These imidazoquinazolines exhibited excellent to great inhibitory potencies with IC50 values ranging from 12.44 ± 0.38 µM to 308.33 ± 0.06 µM, which were several times more potent than standard drug with IC50 value of 750.0 ± 1.5 µM. Representatively, compound 11j showed remarkable anti-α-glucosidase potency with IC50 = 12.44 ± 0.38 µM, which was 60.3 times more potent than positive control acarbose. To explore the potential inhibition mechanism, further evaluations including kinetic analysis, circular dichroism, fluorescence spectroscopy, and thermodynamic profile were carried out for the most potent compound 11j. Moreover, molecular docking studies and in silico ADME prediction for all imidazoquinazolines 6a-c and 11a-o were performed to reveal their important binding interactions, as well as their physicochemical and drug-likeness properties, respectively.

Local flux coordination and global gene expression regulation in metabolic modeling.

Genome-scale metabolic networks (GSMs) are fundamental systems biology representations of a cell's entire set of stoichiometrically balanced reactions. However, such static GSMs do not incorporate the functional organization of metabolic genes and their dynamic regulation (e.g., operons and regulons). Specifically, there are numerous topologically coupled local reactions through which fluxes are coordinated; the global growth state often dynamically regulates many gene expression of metabolic reactions via global transcription factor regulators. Here, we develop a GSM reconstruction method, Decrem, by integrating locally coupled reactions and global transcriptional regulation of metabolism by cell state. Decrem produces predictions of flux and growth rates, which are highly correlated with those experimentally measured in both wild-type and mutants of three model microorganisms Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis under various conditions. More importantly, Decrem can also explain the observed growth rates by capturing the experimentally measured flux changes between wild-types and mutants. Overall, by identifying and incorporating locally organized and regulated functional modules into GSMs, Decrem achieves accurate predictions of phenotypes and has broad applications in bioengineering, synthetic biology, and microbial pathology.

Ustilago maydis PR-1-like protein has evolved two distinct domains for dual virulence activities.

The diversification of effector function, driven by a co-evolutionary arms race, enables pathogens to establish compatible interactions with hosts. Structurally conserved plant pathogenesis-related PR-1 and PR-1-like (PR-1L) proteins are involved in plant defense and fungal virulence, respectively. It is unclear how fungal PR-1L counters plant defense. Here, we show that Ustilago maydis UmPR-1La and yeast ScPRY1, with conserved phenolic resistance functions, are Ser/Thr-rich region mediated cell-surface localization proteins. However, UmPR-1La has gained specialized activity in sensing phenolics and eliciting hyphal-like formation to guide fungal growth in plants. Additionally, U. maydis hijacks maize cathepsin B-like 3 (CatB3) to release functional CAPE-like peptides by cleaving UmPR-1La's conserved CNYD motif, subverting plant CAPE-primed immunity and promoting fungal virulence. Surprisingly, CatB3 avoids cleavage of plant PR-1s, despite the presence of the same conserved CNYD motif. Our work highlights that UmPR-1La has acquired additional dual roles to suppress plant defense and sustain the infection process of fungal pathogens.