Fermentation

Use of low-salt fish sauce (Yulu) is limited due to its perishable property and rapid loss of unique flavor. In this study, a salt-tolerant strain, Bacillus subtilis B-2, with high protease production was used as a microbial starter for low-salt Yulu fermentation. A total of 133 volatile compounds were detected through HS-SPME-GC-MS. Most aldehydes, alcohols, ketones, furans, and hydrocarbons reached their maximum at 15 d, while most esters, aromatic compounds, acids, nitrogen compounds, and sulfur compounds peaked at 45 d. The 16S rRNA gene high-throughput sequencing showed that Bacillus remained in high abundance during fermentation, reaching 93.63% at 45 d. The characteristic volatile flavor was obviously improved while the microbial contamination was significantly reduced in low-salt Yulu fermented with B. subtilis, compared with those without a microbial starter. The correlation network map suggested that the significant decrease in Pseudomonas, Achromobacter, Stenotrophomonas, Cyanobium, Rhodococcus, Brucella, Tetragenococcus, and Chloroplast contributed most to the decreasing richness and evenness of the microbial community, while Bacillus was the only genus playing a key role in the inhibition of spoilage microorganisms and improvement of volatile flavor after B. subtilis addition. This study suggests the potential use of salt-tolerant B. subtilis as a special starter for industrial Yulu fermentation in the future. Full article

Olive leaves are important by-products for the recovery of phenolic compounds and extracts with high phenolic content using lactic acid bacteria during fermentation. Lactiplantibacillus plantarum (L. plantarum) strains as glucosidase-positive strains are starter cultures used to control the fermentation process. The main objective of the present work is to determine the most effective strain for the biodegradation of oleuropein to hydroxytyrosol using two L. plantarum strains for the fermentation of olive leaves. Box–Behnken experimental design was applied to determine the optimum ultrasound-assisted extraction (UAE) conditions to obtain hydroxytyrosol-rich extract using the brine of the fermented leaves. L. plantarum ATCC 14917 (hydroxytyrosol; 126.89 ± 1.59 mg/L) strain showed higher oleuropeinolytic activity than L. plantarum ATCC-BAA 793 (85.93 ± 0.70 mg/L) in olive leaf brine. When the UAE method was applied, it was seen that the hydroxytyrosol concentration of samples that were inoculated with L. plantarum ATCC 14917 (362.29 ± 2.31 mg/L) compared to L. plantarum ATCC-BAA 793 (248.79 ± 4.14) increased. The optimum UAE conditions were determined as 30% amplitude-5 min-30% ethanol for ATCC 14917 and 45% amplitude-9 min-10% ethanol for BAA 793 strain. This study showed that the brines of fermented olive leaves with oleuropeinolytic strains can be considered high added value products. Full article

Grape seed proanthocyanidin (GSP) contains polyphenolic bioflavonoids ubiquitously found in the lignified portions of grape seeds from the winery and distillery industries, as an antioxidant. To explore its potential as a rumen modifier in methanogenesis inhibition, a 2 × 5 factorial experiment was conducted to determine the effect of GSP at 0, 15, 30, 60 and 120 mg/g of substrate on the rumen fermentation and methanogenesis of two representative total mixed rations (HY, a diet for high-yield (>2 kg/d) lactating cows, and LY, a diet for low-yield (<25 kg/d) lactating cows). By using the MIXED procedure, after a 48 h in vitro rumen incubation, increasing the GSP addition linearly decreased the in vitro dry matter digestion (IVDMD) and slowed down the rates of ration fermentation (RmaxS, g/h) and kinetic gas production (RmaxG, mL/h), with the decreases being more pronounced in the LY than HY group (p < 0.05). The GSP addition decreased hydrogen recovery (2Hrec) and altered the fermentation gas composition. The molar CH₄ proportion was significantly reduced with both 60 and 120 mg GSP addition (p < 0.01). The total volatile fatty acid production was linearly decreased with the increasing GSP addition (p < 0.01). In addition, the GSP addition significantly decreased the ratio of methanogens to total bacteria (p < 0.05), and the reduction was notably greater in the HY than in the LY substrate (45.3% vs. 15.2% decrease), although the diversity of rumen methanogenic archaea was not affected in either the HY or the LY group. Bioinformatic analysis illustrated that the rumen archaeal community was predominated by a Methanobrevibacter genus (>72.5%), followed by Methanomassiliicoccus (>20.9%) and Methanosphaera (>1.0%). Methanobrevibacter could play an important role in methanogenesis in the presence of GSP, though it is usually considered to be the main hydrogenotrophic methanogen. In brief, the GSP addition presented high potential as a rumen modifier to mitigate methanogenesis by decreasing the ratio of methanogens to total bacteria. Methanobrevibacter could play an important role in methanogenesis in the presence of GSP. However, a relatively low administration level of GSP should be taken into consideration in order to obtain its inhibitory effect on CH₄ emission, with a minimal negative effect on rumen digestion and fermentation. Full article

Sludge management is considered a difficult and challenging task and is a priority of environmental policy. This study evaluates the transformation of the anaerobic sludge of agroindustrial wastes (cow manure, anaerobic sludge mixtures) directly to compost using Eisenia fetida earthworms (vermicomposting) in relation to sludge quality; moreover, it investigates the effects of different sludge compositions on vermicompost physicochemical properties. In particular, the biostabilization of anaerobic sludge (AS1) produced in excess from the wastewater treatment plant (WWTP) of Patras, Greece, and mixed anaerobic sludge (AS2) originating from the effluents of a laboratory anaerobic co-digestion system treating an agroindustrial waste mixture (olive mill wastewater, cheese whey, and liquid cow manure in a ratio of 55: 40: 5 (w/w), respectively) mixed with cow dung (CD) using Eisenia fetida earthworms was examined. Comparing the mixtures of CD-AS1 to CD-AS2, superior results were obtained with the use of AS2 since an increase in N-P-K was observed when either 10% (22%, 51.8%, and 2.4%, respectively) or 15% of AS2 (38.7%, 14.1%, and 8.1% respectively) was used. Although a reduction in earthworms’ growth was observed compared to 100% CD, during the vermicomposting of the CD-AS mixtures, 410 and 250 mg/earthworm was sustained in the mixtures of 85% CD-15% AS2 and 80% CD-20% AS2 after a period of 63 and 70 days of vermicomposting, respectively. Full article

Alfalfa cultivated in salt–alkali soil was used for fermentation, to which an inoculum of Lactobacillus plantarum YQM48 was added, to assess its effect on the feed quality and the microbial community structure of the fermented silage. A control was included without inoculum. The nutritional components, fermentation quality, pH, and microbial community of the silage were measured after 30 and 60 days of anaerobic fermentation. The results showed that after 30 and 60 days of fermentation in the presence of the inoculum, the content of water-soluble carbohydrates, crude protein content, and dry matter were all significantly higher than those of the control silage, the pH and butyric acid content were lower, and the content of lactic acid and acetic acid were higher (p < 0.05 for all). There was no significant difference in pH and butyric acid content between 30 and 60 days of fermentation in the presence of the inoculum (p > 0.05), while the lactic acid and acetic acid contents were significantly lower in the 60-day silage compared to the 30-day silage (p < 0.05). Fermentation reduced the abundance of Cyanobacteria and Proteobacteria (the abundant phyla in the fresh alfalfa), while the abundance of Firmicutes increased, reaching 92.3% after 60 days of fermentation with inoculum. The dominant genus in that sample was Lactobacillus (70.0%), followed by Enterococcus (12.9%), while fermentation of 60 days without inoculum resulted in only 29.2% Lactobacillus, together with 27.8% Cyanobacteria, and 12.2% Enterococcus. In summary, the addition of L. plantarum YQM48 can improve the nutritional components and fermentation quality of alfalfa silage cultivated on a salt-alkali soil. Full article

Hyaluronic acid (HA) is a natural linear polysaccharide extensively used in many fields, including the food, medicine, and cosmetics industries. Currently, species that produce HA synthetase (HAS) from microbial sources are relatively small and mainly pathogenic, such as Streptococcus pyogenes and Pasteurella multicide. Moreover, there is limited research on the safe microbial sources of HAS. Thus, we characterized SthasA, a HAS derived from the probiotic Streptococcus thermophilus, and used it for the de novo synthesis of HA in a chassis strain of Bacillus amyloliquefaciens. Metabolic engineering of the precursor supply modules suggested that hasB (encoding UDPG dehydrogenase), which was derived from Corynebacterium glutamicum ATCC 13032, effectively promoted the accumulation of HA products. Furthermore, by combining the expression of the global regulatory factor CcpA, HA yield from the recombinant strain reached 3.20 g/L. Finally, we obtained a yield of 5.57 g/L HA with a molecular weight of 1.7 × 10⁶ Da using various process optimization strategies in a 5 L bioreactor. This study enriches our understanding of obtaining HAS from non-pathogenic bacteria and provides a safe and effective process for producing HA, which has the potential to promote the industrial applications of HA further. Full article

Yarrowia lipolytica, a GRAS (generally recognized as safe) nonconventional yeast, has been used widely in industrial fermentation to produce chemicals, fuels, and functional sugars such as erythritol and mannitol. Although Y. lipolytica is a promising organism for bioconversion and has substantial potential in industrial production, its utilization is restricted by the high cost of cooling during the fermentation process; the optimum growth or fermentation temperature of Y. lipolytica is 28–30 °C, which is lower than that of some fermenting species. Therefore, it is necessary to breed a thermoresistant Y. lipolytica for use in a fermentation system. Here, we report a new thermoduric Y. lipolytica strain (a thermoresistant clone, HRC) that can grow at 35 °C—higher than the starting strain Y. lipolytica CGMCC7326 (maximum growth temperature at 33 °C)—by laboratory adaptive evolution. Based on the transcriptome analysis of the mutant strain HRC and the parental strain Y. lipolytica CGMCC7326 at different temperatures, 22 genes with increased expression at high temperatures were identified and 10 of them were overexpressed in Y. lipolytica CGMCC7326. HRC1, HRC2, and HRC3 (with YALI0B21582g, YALI0C13750g, and YALI0B10626g overexpressed, respectively) were assessed for growth at higher temperatures. This revealed that these three genes were related to thermotolerance. This study provides insights into the metabolic landscape of Y. lipolytica under heat stress, enabling future metabolic engineering endeavors to improve both thermoresistance and sugar alcohol production in the yeast Y. lipolytica. Full article

Four parameters affecting hydrothermal pretreatment (HTP) of municipal sludge prior to anaerobic digestion and fermentation were investigated. Partial factorial design including several key HTP parameters at two distinct levels, including temperature (170 and 190 °C), retention time (RT) (10 and 30 min), pH (4 and 10), and solid content (SC) (4% and 16%), were studied. Further, the impact of HTP parameters on mesophilic and thermophilic fermentation was explored and compared. Results revealed a significant effect of all HTP parameters on COD solubilization, VFA, and methane yield. There were correlations between HTP parameters and process responses such as VFA yield and methane yield. HTP was found to increase COD solubilization and VFA production between 15 and 20% during thermophilic fermentation in relation to mesophilic treatment. All parameters, including SC, temperature, pH, and RT, were important contributing factors affecting methane production during anaerobic digestion. The highest methane production yield of 269 mL CH₄/g TCOD added was observed at the highest SC (16%) and pH (10) and at the lower temperature (170 °C) and RT (10). HTP is expected to be combined with other intensification routes to treat waste with high solid contents improving the fermentation and anaerobic digestion processes. Full article

A distinctive isolate was discovered and visually recognized as a member of the genus Didymella during a routine examination of Coelomycetes isolated from diverse fruit juices. Based on sequencing of the internal transcribed spacer (ITS), the fungus was identified as Didymella keratinophila since it showed a 100% identity to the type strain. The strain thrived and produced keratinase and collagenase enzymes by hydrolyzing native chicken feathers in submerged fermentation (SmF). After 10 days of fermentation at 30 °C, pH 9 using sodium nitrate as a nitrogen supply produced the highest keratinase activity of 8780 ± 620 U/mL/min, while pH 6 and beef extract produced the maximum collagenase activity of 11,230 ± 1290 U/mL/min. The partially-purified keratinase enzyme worked best at pH 7.0 and 45 °C, exhibiting a specific activity of 44,903 ± 1555 U/mg protein. The activity of the partially-purified collagenase enzyme was excellent at pH 6.0 at 35 °C, generating 15,753 ± 110 U/mg enzyme-specific activity. Mn²⁺ and K⁺ were the most efficient inhibitors of keratinases and collagenase, respectively. Both EDTA and metal ions significantly decreased the activity of keratinase and collagenase. This report identified a workable supplier of collagenase and keratinase enzymes derived from chicken feathers, offering a reliable way to exploit and manage these wastes for obtaining high-value products. Full article

Lytic polysaccharide monooxygenase (LPMO) could oxidize and cleavage the glycosidic bonds of polysaccharides in lignocellulose, thereby promoting the hydrolysis of polysaccharide substrates by glycoside hydrolases and significantly improving the saccharification efficiency of lignocellulose. Brown-rot fungi are typical degraders of lignocellulose and contain multiple LPMO genes of the AA14 family and AA9 family, however, the AA14 LPMO from brown-rot fungi was rarely reported. Herein, the transcriptomic analysis of Serpula lacrymans incubated in the presence of pine exhibited that an AA14 LPMO (SlLPMO14A) was significantly upregulated and there were redox interactions between LPMOs and other enzymes (AA3, AA6, and hemicellulose degrading enzyme), indicating that SlLPMO14A may be involved in the degradation of polysaccharides. Enzymatic profiling of SlLPMO14A showed the optimal pH of 8.0 and temperature of 50 °C and it had higher reaction activity in the presence of 40% glycerol and acetonitrile. SlLPMO14A could significantly improve the saccharification of pine and xylan-coated cellulose substrate to release glucose and xylose by cellulase and xylanase by disturbing the surface structure of lignocellulose based on environmental scanning electron microscope and atomic force microscopy analysis. Full article

Astaxanthin is a xanthophyll carotenoid possessing impressive nutraceutical, antioxidant, and bioactive merits. Traditionally, astaxanthin is extracted from crustacean wastes via solvent extraction methods. However, the rigid structure of shells that comprise complex proteins and chitin challenges the extraction process. This investigation addressed an efficient microbial-assisted method to facilitate astaxanthin recovery from crab exoskeleton waste utilizing chitinolytic and proteolytic microorganisms. Herein, we evaluated the effect of pretreatment of the exoskeleton waste with a newly isolated probiotic strain, Bacillus amyloliquefaciens CPFD8, showing remarkable protease and chitinase activity and a proteolytic Saccharomyces cerevisiae 006-001 before solvent extraction, using acetone/hexane, on astaxanthin recovery. Furthermore, the antioxidant and anti-inflammatory activities of the recovered astaxanthin were inspected. Results revealed that both strains boosted the astaxanthin yield from the crab (Callinectes sapidus) exoskeleton compared with solvent extraction using acetone/hexane. Under optimum conditions, astaxanthin yield was 217 and 91 µg/g crab exoskeleton in samples treated with B. amyloliquefaciens CPFD8 and S. cerevisiae 006-001, respectively. Interestingly, pretreatment of crab exoskeleton waste with B. amyloliquefaciens CPFD8 yielded more than 6-fold astaxanthin compared with the solvent extraction method that yielded just 35 µg/g. This increase could be attributed to the proteolytic activity of B. amyloliquefaciens CPFD8 that rendered deproteinized shell chitin accessible to chitinase, facilitating the penetration of solvents and the recovery of astaxanthin. The recovered astaxanthin exhibited excellent antioxidant activity in scavenging DPPH or ABTS free radicals with IC₅₀ values of 50.93 and 17.56 µg/mL, respectively. In addition, the recovered astaxanthin showed a remarkable anti-inflammatory impact on LPS-induced murine macrophage RAW264.7 cells and significantly inhibited the production of nitric oxide, TNF-α, and IL-6 compared with the untreated control. These findings suggest the potential use of the developed microbial-assisted method utilizing chitinolytic and proteolytic B. amyloliquefaciens CPFD8 to maximize the recovery of bioactive astaxanthin from crab (C. sapidus) exoskeleton waste. Full article

This study aimed to assess the impact of essential oils (EOs) on in vitro gas formation and the degradability of dairy and beef cattle diets. This study also aimed to investigate the effects of different types of EOs on nutrient utilization and rumen microbial activity. The current study was conducted using a fully randomized design consisting of eight experimental treatments, including two control treatments without any additives, and treatments with cinnamon essential oil (CEO), flaxseed essential oil (FEO), and lemon seed essential oil (LEO) at a concentration of 60 mg/kg fresh mass. Two control treatments were used, one with alfalfa silage and dairy concentrate (DC, CON-DC) and the other with alfalfa silage and fattening concentrate (FC, CON-FC). Gas formation, dry matter (DM) digestibility, crude protein (CP) digestibility, effective degradability (ED), and soluble fractions of DM and organic matter (OM) were evaluated. CEO had a substantial effect on gas formation (p < 0.05). When EOs were added to the diets, they increased dry matter digestibility after 24 h of incubation as compared to control treatments. After 24 h of incubation, FCCEO and FCFEO had the highest CP digestibility among the diets. FCLEO considerably enhanced ED, as well as the soluble fraction of DM (a) at a passage rate of 2% per hour. Treatment with FCCEO resulted in a significant increase in soluble fractions compared to the control diets. At a passage rate of 2% h, DCCEO had the maximum ED value. When EOs were introduced to the diet, they dramatically decreased the insoluble portion of CP (b). Compared to the control treatments, gas production was significantly lower in the presence of LEO (FCLEO; p < 0.05). The addition of EOs to cattle diets may increase nutrient utilization and enhance rumen microbial activity. EOs extracted from lemon seeds (at a dose of 60 mg/kg of diet) lowered gas production in both dairy cattle and fattening diets. Full article

Fermentation is a widely used process in the biotechnology industry, in which sugar-based substrates are transformed into a new product through chemical reactions carried out by microorganisms. Fermentation yields depend heavily on critical process parameter (CPP) values which need to be finely tuned throughout the process; this is usually performed by a biotech production expert relying on empirical rules and personal experience. Although developing a mathematical model to analytically describe how yields depend on CPP values is too challenging because the process involves living organisms, we demonstrate the benefits that can be reaped by using a black-box machine learning (ML) approach based on recurrent neural networks (RNN) and long short-term memory (LSTM) neural networks to predict real time OD_600nm values from fermentation CPP time series. We tested both networks on an E. coli fermentation process (upstream) optimized to obtain inclusion bodies whose purification (downstream) in a later stage will yield a targeted neurotrophin recombinant protein. We achieved root mean squared error (RMSE) and relative error on final yield (REFY) performances which demonstrate that RNN and LSTM are indeed promising approaches for real-time, in-line process yield estimation, paving the way for machine learning-based fermentation process control algorithms. Full article

Rumen fermentation (RF) is a complex system of oxidation–reduction reactions governed by the rumen microbiota, and its end-products are the volatile fatty acids (VFAs) which constitute the main energy source for the host animal. We speculated that the process could be manipulated through electrofermentation (EF) by utilizing solid-state electrodes as electron sources. This study aimed to evaluate the effects of single-chamber EF applied (0.4 vs. 0.9 V) for 6, 12, or 24 h to rumen fluid on the resulting microbial biomass, dry matter digestibility (DMD), VFA production, NADH/NAD ratio, and composition of the rumen fluid’s bacterial communities. The application of a 0.9 V electric potential for 24 h produced the most significant changes in RF compared to the control treatment (0V), increasing microbial biomass (12%), DMD (58%), and VFA concentrations (~84%), and reducing NAD/NADH ratio (~50%) without a significant impact on bacterial diversity. These results suggest that the application of a 0.9 V electric potential allows the microbiota to better hydrolyze and digest the feed’s components and could offer an electron sink for the production of VFAs. Hence, EF can provide innovative solutions to a range of challenges that limit the RF process. Nonetheless, more studies are needed to understand how the solid electrodes and external electric energy function. Full article

Astaxanthin is a carotenoid pigment extensively used in various industries. Rhodotorula sp. CP72-2, isolated from Calotropis gigantea, showed potential astaxanthin production. In this study, strain CP72-2 was identified as a putative new species in the genus Rhodotorula based on the 26S rRNA gene sequence (98% identity). It was first used as the microbial source for producing astaxanthin. Strain CP72-2 was screened for its astaxanthin production and was identified and quantified by High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and UV-Vis spectrophotometer. After a screening of astaxanthin production, various carbon sources, pH, temperature, and incubation period were evaluated for their effect on the astaxanthin production of strain CP72-2. Among the several experimental factors, the most efficient conditions for astaxanthin production were glucose (50 g/L), pH 4.5, 25 °C, and three days of cultivation. The assembly genome of strain CP72-2 has a total length of 21,358,924 bp and a GC content of 64.90%. The putative candidate astaxanthin biosynthesis-associated genes (i.e., CrtE, CrtYB, CrtI, CrtS, CrtR, CrtW, CrtO, and CrtZ) were found. This research presents the first report on the production and optimization of astaxanthin from strain CP72-2 and its genome analysis, focusing on the biotechnological potential of the astaxanthin producer. Full article

In this study, the effects of mixed silage on the fermentation quality, chemical composition and bacterial communities of corn straw and soybean straw were assessed. Corn straws, soybean straws or a mixture of corn straws and soybean straws in a 1:1 mass ratio were uniformly mixed and processed using the same method to produce bagged silage weighing 50 kg per bag. At days 0, 14 and 60 of the fermentation process, the nutritional composition and fermentation quality indicators of each group were measured. A complete factorial design to assess the effect of the ensiling time, types of fermented straw (treatment group) and their interactions on the assessed parameters was performed. In addition, metabolomics sequencing and microbial community analysis were performed on the samples on the 60th day of fermentation. Compared with the corn straw group and the soybean straw group, the silage fermentation quality of the corn straw–soybean straw group is better, which is reflected in higher crude protein rate (p < 0.05), higher lactic acid content (p < 0.05) and lower ammonia nitrogen content (p < 0.05). The combined analysis of differential microbial taxa (species level) and differential metabolites revealed a positive correlation between certain bacterial species, such as manihotivorans, brevis and zeae, and significantly downregulated metabolites, such as pyrocatechol and N-benzoyltyramine. Similarly, positive correlations were observed between certain bacterial species, such as dublinensis, marcescens and agglomerans, and upregulated metabolites, such as D-xylonic acid and neochlorogenic acid. These findings suggest that the key microbial–metabolite pathways identified are responsible for the good fermentation quality observed during mixed fermentation of corn straw and soybean straw at a 1:1 mass ratio. Full article

The brown seaweed Alaria esculenta is the second most cultivated species in Europe, and it is therefore of interest to expand its application by developing food products. In this study, a lactic acid bacteria consortium (LAB consortium) consisting of three Lactiplantibacillus plantarum strains (relative abundance ~94%) and a minor amount of a Levilactobacillus brevis strain (relative abundance ~6%) was investigated for its ability to ferment carbohydrates available in brown seaweed. The consortium demonstrated the ability to ferment glucose, mannitol, galactose, mannose, and xylose, of which glucose and mannitol were the most favored substrates. No growth was observed on fucose, mannuronic and guluronic acid. The consortium used different pathways for carbohydrate utilization and produced lactic acid as the main metabolite. In glucose fermentation, only lactic acid was produced, but using mannitol as a carbohydrate source resulted in the co-production of lactic acid, ethanol, and succinate. Xylose fermentation resulted in acetate production. The consortium was also able to utilize laminari-oligosaccharides (DP2-4), obtained after enzymatic hydrolysis of laminarin, and produced lactic acid as a metabolite. The consortium could grow directly on A. esculenta, resulting in a pH decrease to 3.8 after 7 days of fermentation. Incubation of the same seaweed in corresponding conditions without inoculation resulted in spoilage of the seaweed by endogenous bacteria. Full article

Many efforts in norovirus vaccine development have focused on subunit or recombinant protein vaccines, such as subviral P particles formed by the protruding (P) domain of VP1. P particles are immunogenic and have a region with a human histo-blood group antigen binding site, an interaction critical for infecting the host. In the past, only intracellular NoV P proteins expressed in Escherichia coli and Pichia pastoris were reported, and the low yield and difficulty in purification limited their applications. In this study, the Taiwan-native NoV P domain was successfully expressed and secreted by P. pastoris. The secretion efficiency was greatly enhanced by integrating oligosaccharyl transferase (Ost1) into the α-factor signal peptide and coexpressing Hac1. The production of NoV P in fermentation cultures reached 345 mg/L, and the purity and recovery were 94.8% and 66.9%, respectively, after only ion-exchange chromatography. Transmission electron microscopy analysis showed that the small P particles were mostly ring-, square-, and triangle-shaped, with diameters of 10-15 nm. The biological activity of NoV P was confirmed by saliva-binding assay using human histo-blood group antigen. This study describes the secretion and characterization of the Taiwan-native norovirus P domain in P. pastoris. Particles formed from the P domain were similar in size, morphology, and binding ability to those expressed intracellularly. The strategy described in this study provides great potential in scale-up production and antiviral vaccine development. Full article

SARS-CoV-2 was identified as the pathogenic agent causing the COVID-19 pandemic. Among the proteins codified by this virus, the Spike protein is one of the most-external and -exposed. A fragment of the Spike protein, named the receptor binding domain (RBD), interacts with the ACE2 receptors of human cells, allowing the entrance of the viruses. RBD has been proposed as an interesting protein for the development of diagnosis tools, treatment, and prevention of the disease. In this work, a method for recombinant RBD production using Pichia pastoris as a cell factory in a stirred-tank bioreactor (SRTB) up to 7 L was developed. Using a basal saline medium with glycerol, methanol, and compressed air in a four-stage procedure, around 500 mg/L of the raw RBD produced by yeasts (yRBD) and 206 mg/L of purified (>95%) RBD were obtained. Thereby, the proposed method represents a feasible, simple, scalable, and inexpensive procedure for the obtention of RBD for diagnosis kits and vaccines’ formulation. Full article