Semi-defined Medium Research Articles

Bifidobacterium animalis subsp. lactis HN019 live probiotics and postbiotics: production strategies and bioactivity evaluation for potential therapeutic properties.

Introduction: B. animalis subsp. lactis HN019 is a commercially available well-characterized probiotic with documented effects on human health, such as the ability to enhance the immune function and to balance the intestinal microbiome. Therefore, optimizing the manufacturing process to improve sustainability, increasing biomass yields and viability, and avoiding animal -derived nutrients in the medium to meet vegan consumer's needs, is currently of interest. Besides the established use of live probiotic cells, alternative supplements indicated as postbiotics, like non-viable cells and/or probiotics derived bioactive molecules might be considered as potential next generation biotherapeutics. In fact, advantages of postbiotics include fewer technological limitations, such as easier production processes and scale-up, and even higher specificity. Methods: In this work, medium design together with different fermentation strategies such as batch, fed-batch and in situ product removal on lab-scale bioreactors were combined. Medium pretreatment by ultrafiltration and protease digestion was performed to reduce polysaccharidic contaminants and facilitate the purification of secreted exopolysaccharides (EPS). The latter were isolated from the fermentation broth and characterized through NMR, GC-MS and SEC-TDA analyses. The expression of TLR-4, NF-kb and IL-6 in LPS challenged differentiated CaCo-2 cells treated with EPS, live and heat-killed B. lactis cells/broth, was evaluated in vitro by western blotting and ELISA. Zonulin was also assessed by immunofluorescence assays. Results and Discussion: The titer of viable B. lactis HN019 was increased up to 2.9 ± 0.1 x 1010 on an animal-free semidefined medium by applying an ISPR fermentation strategy. Medium pre-treatment and a simple downstream procedure enriched the representativity of the EPS recovered (87%), the composition of which revealed the presence of mannuronic acid among other sugars typically present in polysaccharides produced by bifidobacteria. The isolated EPS, live cells and whole heat inactivated broth were compared for the first up to date for their immunomodulatory and anti-inflammatory properties and for their ability to promote intestinal barrier integrity. Interestingly, EPS and live cells samples demonstrated immune-stimulating properties by downregulating the expression of TLR-4 and NF-kb, and the ability to promote restoring the integrity of the intestinal barrier by up-regulating the expression of zonulin, one of the tight junctions forming proteins. Postbiotics in the form of heat killed broth only reduced NF-kb expression, whereas they did not seem effective in the other tested conditions.

First Report of White Mucus Disease Caused by Fuligo gyrosa on Agrocybe chaxingu in China.

Agrocybe chaxingu is an edible and medicinal mushroom widely cultivated in China (Liu et al. 2021). Agrocybe chaxingu is extremely well-liked for the unique flavor and nutritional value. In May 2021, a serious white mucus disease was observed in the farms of A. chaxingu in the Ganxian district of Ganzhou City, Jiangxi Province, China, with an approximate disease incidence of 20%. In the years of 2022 and 2023, the same white mucus disease on A. chaxingu was observed in the farms in Nanchang City, Jiujiang City and Guangchang County, Jiangxi Province, China. The disease generally occurs on the media, stipe or pileus of A. chaxingu under condition of high humidity. The plasmodial slime molds migrated from the surface of culture media (78% hardwood sawdust, 15% wheat bran, 5% tea seed shell, 1% lime, and 1% gypsum) to the base of fruiting bodies, stipes and finally to pilei, showing as moist, sticky, and white reticulated structures. The infected fruiting bodies of A. chaxingu were completely covered by reticulated plasmodia, displaying a white or pale-yellow color. This resulted in the growth cessation, wilting and eventual death of fruiting body. Microscopic observation found that the plasmodia of slime mold enveloped the hyphae of A. chaxingu, resulting in the fragmentation of the hyphae. The disease can spread quickly, resulting in a 30% reduction in production. Slime mold cultures were isolated by transferring diseased fruiting bodies of A. chaxingu onto oat-agar medium (2% agar and 1% oatmeal) at 25 ℃. The isolates can be obtained after being subcultured for two to three generations. Purified plasmodia were placed on the semi-defined medium (1% tryptone, 1% glucose, 0.15% yeast extract, chick embryo extract and a balanced salt solution) to confirm the absence of bacteria (Daniel et al. 1964) and thus obtained the pure culture. Specimen of the voucher has been deposited in the Institute of Agricultural Applied Microbiology, Jiangxi Academy of Agricultural Sciences as number IAAM-W0002. The vegetative plasmodia have a large and well-developed scalloped structure that were white or milky white in colour. The white plasmodium became opaque pale yellow when exposed to light before fruiting. The veins merged and thickened. Fruiting bodies can be formed on the lid or side of the Petri dish under light condition. The fruiting bodies formed papillae with irregular shape, and then the color changed from translucent yellow to greyish black. Spores were usually spherical or subglobose, free, greyish brown in mass, purplish brown, 7-12 μm in diameter under light microscopy. These morphological characteristics were found to be consistent with those of Fuligo gyrosa (Synonym: Physarum gyrosum) (Kim et al. 2009; Shi et al. 2005; Jahn 1902). The identity of the isolates was further confirmed by sequence analysis of the 18S ribosomal RNA gene with primer SMNUR101/NS4 (Rusk et al. 1995; White et al. 1990). Using BLASTn searches, the sequence of 18S rRNA gene (GenBank accession number OR186216) matched the sequence of F. gyrosa (GenBank accession number LC744593) with the identity of 99.91% and coverage of 97%. A phylogenetic tree based on the 18S rRNA gene also demonstrated that the slime mold clustered with F. gyrosa. Over ten isolates have been obtained from the diseased A. chaxingu samples in different factories and identified as F. gyrosa. To test the pathogenicity of F. gyrosa, five healthy young fruiting bodies (three to five days of primordium) of A. chaxingu cultivated in mushroom-growing room were gently inoculated by a 12 mm diameter oat-agar medium with plasmodia at 24 ± 2 ℃ and then were kept with relative humidity of 90%-95%. Five fruiting bodies inoculated with a 12 mm oat-agar medium served as controls. After 5 days, white mucus characteristics and three fifths of death symptoms were observed on the fruiting bodies inoculated with the plasmodia, while the controls remained asymptomatic. The slime mold on the inoculated fruiting bodies was morphologically identical to F. gyrosa that was observed on the initial diseased fruiting bodies. It was also observed the envelopment A. chaxingu hyphae by the plasmodia of slime mold and fragmentation of the hyphae, and the fragmentation was not observed in the controls. Reisolations were prepared from the inoculated fruiting bodies and confirmed to be F. gyrosa based on morphological characteristics and 18S rRNA sequence, thus fulfilling Koch's postulates. Fuligo gyrosa has been reported to cause severe disease in oriental melon in Korea (Kim et al. 2009). This is the first report of F. gyrosa causing white mucus disease in cultivated A. chaxingu. The findings will provide important information on prevention and control of the disease, and be helpful for the development of A. chaxingu industry.

Predictive Modeling of Riboflavin Production in Lactiplantibacillus plantarum MTCC 25432 Using Fuzzy Inference System.

Riboflavin (Vitamin B2) is an essential vitamin and a microbial metabolite produced by some lactic acid bacteria (LAB). This investigation aims to study the overproduction of riboflavin in selected Lactiplantibacillus plantarum strain by using the one factor at a time (OFAT) tool coupled with the Fuzzy Inference System (FIS) and its validation through fermentative production in semi-defined media. Out of three Lactiplantibacillus strains used in this study, the maximum riboflavin producing strain was selected based on its ability to grow and produce higher levels of riboflavin. In results, Lactiplantibacillus plantarum strain MTCC 25432 was able to produce 346 µg/L riboflavin in riboflavin deficient assay medium and was investigated further. By using the OFAT-fuzzy FIS system, casamino acid in the range of 5-20 g/L, GTP 0.01-0.04 g/L, sodium acetate 5-15 g/L, and glycine 5-15 g/L were used to predict their effect on riboflavin production. The conditions optimized with modeling showed a 24% increment in riboflavin production (429 µg/L) by Lactiplantibacillus plantarum MTCC 25432 vis-a-vis the unoptimized counterpart (346 µg/L). In conclusion, an FIS-based predictive model was effectively implemented to estimate the riboflavin within an acceptable limit of 3.4%. Riboflavin production enhancing effects observed with various levels of sodium acetate, casamino acid, and GTP could be useful to re-design matrices for riboflavin production.

Dextrans of Weissella cibaria DSM14295: Microbial production, structure and functionality

Lactic acid bacteria of the genus Weissella contribute to spontaneous fermentation in, e.g., sourdough or sauerkraut, but are not registered as starter cultures because of their pending safety assessment. Some strains are able to produce high amounts of exopolysaccharides. This study aims to demonstrate the techno-functionality of five dextrans from W. cibaria DSM14295, produced under varying cultivation conditions, with respect to structural and macromolecular properties. A maximum of 23.1 g/L dextran was achieved by applying the “cold shift” temperature regime. The dextrans differed in molecular mass (9–22∙108 Da, determined by HPSEC-RI/MALLS), intrinsic viscosity (52–73 mL/g), degree of branching (3.8–5.7 % at position O3, determined by methylation analysis) and their side chain length and architecture, determined by HPAEC-PAD after enzymatic hydrolysis. Stiffness of acid gels from milk spiked with these dextrans increased linearly with dextran concentration. Principal component analysis showed that dextrans produced in a semi-defined medium are primarily described by moisture sorption and branching properties, whereas dextrans produced in whey permeate were similar because of their functional and macromolecular properties. Overall, dextrans from W. cibaria DSM14295 have a high potential because of the high production yield and their functionality which can be tailored by the conditions during fermentation.

Evaluation of Xylose-Utilising Yeasts for Xylitol Production from Second-Generation Ethanol Vinasse and Effect of Agitation Intensity in Flask-Scale Xylitol Production

This study aimed to select a yeast strain that effectively utilises xylose to produce xylitol from the vinasse of ethanol broth obtained from the fermentation of sugarcane bagasse hydrolysate. Eleven strains of xylose-fermenting yeasts were evaluated for their abilities to utilise xylose and produce xylitol. Two strains that showed outstanding performance in the semi-defined xylose medium were selected for further testing with a vinasse medium. Candida guilliermondii TISTR 5068 showed a superior xylitol production of 7.03 ± 0.08 g/L with the xylitol yield of 0.70 g/gxylose when cultured in bagasse-based ethanol vinasse. The strain was further tested for its xylitol production performance when cultured at four different agitation intensities. Excessive agitation resulted in a rapid xylitol production rate but caused xylitol consumption once the xylose was depleted. Moderate agitation resulted in the highest xylitol yield of 0.79 g/gxylose. The results of this study have provided important information for the development of the xylitol production process using waste streams from cellulosic ethanol production.

Biotechnological Conversions of Mizithra Second Cheese Whey by Wild-Type Non-Conventional Yeast Strains: Production of Yeast Cell Biomass, Single-Cell Oil and Polysaccharides

The cultivation of oleaginous yeasts on various agro-industrial residues and the subsequent production of microbial lipids (single-cell oils), which can be used as starting materials for the synthesis of both “second generation” biodiesel and various types of oleochemicals, is considered as one of the most important approaches of the Industrial Biotechnology, in terms of circular economy and green and sustainable development. In this study, seven wild-type non-conventional yeast strains were evaluated for their growth on a semi-defined medium with cheese whey lactose as a sole carbon source. Five of these strains were further batch-cultivated into the complex substrate that contained second cheese whey, which is the main by-product of Mizithra whey cheese manufacture, after centrifugation and filtration without any extra mineral salts, carbon, or nitrogen source addition. All these five strains grown in second cheese whey produced mainly yeast biomass and to lesser extent microbial lipids and other interesting metabolites, such as polysaccharides. The strain Cryptococcus curvatus ATCC 20509 produced the highest total dry weight (TDW) amount (22.4 g/L), that contained 3.4 g/L of intra-cellular lipids, followed by C. curvatus NRRL Y-1511, which produced 20.6 g/L of TDW and 3.2 g/L lipids. A novel, non-previously systematically studied strain, namely Papiliotrema laurentii NRRL Y-2536, produced significant quantities of TDW (22.0 g/L) and, interestingly, secreted quantities of exopolysaccharides. Fed-batch shake-flask cultivation of C. curvatus ATCC 20509 in pretreated second cheese whey, pulse-supplemented with condensed cheese whey-derived lactose, led to the significant TDW quantity of 38.1 g/L that contained c. 57% w/w of total lipids (lipids at a concentration 21.7 g/L were produced). Cellular lipids of all microorganisms, mainly stored as triacylglycerols, contained in variable quantities the fatty acids Δ9C18:1, C16:0, Δ9,12C18:2 and C18:0, constituting perfect candidates for the synthesis of “second generation” biodiesel.

Genome Sequence of Eubacterium limosum B2 and Evolution for Growth on a Mineral Medium with Methanol and CO2 as Sole Carbon Sources

Eubacterium limosum is an acetogen that can produce butyrate along with acetate as the main fermentation end-product from methanol, a promising C1 feedstock. Although physiological characterization of E. limosum B2 during methylotrophy was previously performed, the strain was cultured in a semi-defined medium, limiting the scope for further metabolic insights. Here, we sequenced the complete genome of the native strain and performed adaptive laboratory evolution to sustain growth on methanol mineral medium. The evolved population significantly improved its maximal growth rate by 3.45-fold. Furthermore, three clones from the evolved population were isolated on methanol mineral medium without cysteine by the addition of sodium thiosulfate. To identify mutations related to growth improvement, the whole genomes of wild-type E. limosum B2, the 10th, 25th, 50th, and 75th generations, and the three clones were sequenced. We explored the total proteomes of the native and the best evolved clone (n°2) and noticed significant differences in proteins involved in gluconeogenesis, anaplerotic reactions, and sulphate metabolism. Furthermore, a homologous recombination was found in subunit S of the type I restriction-modification system between both strains, changing the structure of the subunit, its sequence recognition and the methylome of the evolved clone. Taken together, the genomic, proteomic and methylomic data suggest a possible epigenetic mechanism of metabolic regulation.

Limosilactobacillus fermentum from buffalo milk is suitable for potential biotechnological process development and inhibits Helicobacter pylori in a gastric epithelial cell model

Probiotics are living microorganisms that give beneficial health effects while consumed, and each strain possesses diverse and unique properties and also different technological characteristics that affect its ability to be produced at large scale. Limosilactobacillus fermentum is a widely studied member of probiotics, however, few data are available on the development of fermentation and downstream processes for the production of viable biomasses for potential industrial applications.In the present study a novel L. fermentum strain was isolated from buffalo milk and used as test example for biotechnological process development. The strain was able to produce up to 109 CFU/mL on a (glucose based) semi-defined medium deprived of animal-derived raw materials up to the pilot scale (150 L), demonstrating improved results compared to commonly used, although industrially not suitable, media rich of casein and beef extract. The study of strain behavior in batch experiments indicated that the highest concentration of viable cells was reached after only 8 h of growth, greatly shortening the process. Moreover, initial concentrations of glucose in the medium above 30 g/L, if not supported by higher nitrogen concentrations, reduced the yield of biomass and increased production of heterolactic fermentation by-products. Biomass concentration via microfiltration on hollow fibers, and subsequent spray-drying allowed to recover about 5.7 × 1010CFU/gpowder of viable cells, indicating strain resistance to harsh processing conditions.Overall, these data demonstrate the possibility to obtain and maintain adequate levels of viable L. fermentum cells by using a simple approach that is potentially suitable for industrial development. Moreover, since often exopolysaccharides produced by lactobacilli contribute to the strain's functionality, a partial characterization of the EPS produced by the newly identified L. fermentum strain was carried out.Finally, the effect of L. fermentum versus H. pylori in a gastric epithelial cell model was evaluated demonstrating its ability to stimulate the response of the immune system and displace the infective agent.

High Cell Density Cultivation of Escherichia coli DH5α in Shake Flasks with a New Formulated Medium

High cell density cultivation necessitates cell division and biomass formation, the mechanisms of which remain poorly understood, especially from the cellular energetics perspective. Specifically, the sensing of energy abundance and the channelling of nutritional energy into biomass formation and cell maintenance remains enigmatic at the sensory, effector and decision levels. Thus, optimization of cell growth remains an iterative trial and error process where the principal parameters are growth medium composition and incubation temperature. In this study, a new semidefined formulated medium was shown to be useful for high cell density cultivation of Escherichia coli DH5α (ATCC 53868). Comprising K2HPO4, 12.54; KH2PO4, 2.31; D-Glucose, 4.0; NH4Cl, 1.0; Yeast extract, 12.0; NaCl, 5.0; MgSO4, 0.24; the medium possessed a high capacity phosphate buffer able to moderate pH fluctuations during cell growth known to be detrimental to biomass formation. With glucose and NH4Cl providing the nutrients for initial growth, followed by a lag phase of 3 hours, a maximal optical density of 12.0 was obtained after 27 hours of cultivation at 37 oC and 230 rpm. Yeast extract provides a secondary source of carbon and nitrogen. Maximal optical density obtained in formulated medium was higher than the 10.1, 4.2, and 3.4 obtained in Tryptic Soy Broth, M9 with 1 g/L of yeast extract, and LB Lennox, respectively. Cultivation of E. coli DH5α in formulated medium with 6 g/L of glucose resulted in a longer lag phase of 8 hours and a longer time (68 hours) to attainment of maximal optical density, which marked the upper limit of glucose concentration beyond which biomass formation would be reduced. Specifically, glucose concentration above 6 g/L markedly reduced biomass formation possibly due to the environmental stress arising from low pH in the culture broth. Glucose concentration below 4 g/L, on the other hand, reduced biomass formation through a smaller pool of nutrients serving as biomass building blocks. Deviation from 1:1 molar ratio between glucose and NH4Cl was not detrimental to biomass formation and growth rates. Collectively, a semi-defined formulated medium could increase optical density of E. coli DH5α beyond that of LB Lennox and Tryptic Soy Broth, and may find use in cultivation of cells for applied microbiology research.

Xylitol and ethanol co-production from sugarcane bagasse and straw hemicellulosic hydrolysate supplemented with molasses

Co-production systems are a strategy used to diversify production chains and to enable more profitable processes. In addition, the valorization of the sugar-and-alcohol industry through by-product conversion into value-added products is environmentally friendly and enhances their competitiveness to other products available in the market. Thus, the aim of the current study is to evaluate xylitol and ethanol co-production by Candida tropicalis deriving from sugarcane by-products, such as bagasse and straw, supplemented with molasses. Sugarcane bagasse and straw hemicellulosic hydrolysate was obtained through dilute-acid hydrolysis with H2SO4; next, it was concentrated and detoxified with activated charcoal. Different commercial sucrose concentrations (5, 25 and 50 gL−1) were added to the bagasse and straw hemicellulosic hydrolysate in semi-defined medium. In addition, hydrolysate supplementation with sugarcane molasses was evaluated based on sucrose content (10 and 50 gL−1) adjustment. Batch fermentations were carried out in 125-mL Erlenmeyer flasks filled with 50 mL of medium, at 30 °C, 200 rpm, pH 5.5, for 48 h. Molasses supplementation adjusted to 50 gL−1 sucrose in sugarcane bagasse and straw hemicellulosic hydrolysate recorded the maximum xylitol (30.61 gL−1) and ethanol (47.97 gL−1) concentrations observed in the current study. Besides, volumetric xylitol (0.64 gL−1h−1) and ethanol (1.00 gL−1h−1) productivity was observed under the very same condition. Results have shown the likelihood of co-producing xylitol and ethanol through C. tropicalis by using sugarcane by-products to compose the fermentation medium as strategy to intensify and integrate these bioprocesses in sugarcane biorefineries.

Towards Sustainable Bioinoculants: A Fermentation Strategy for High Cell Density Cultivation of Paraburkholderia sp. SOS3, a Plant Growth-Promoting Bacterium Isolated in Queensland, Australia

Paraburkholderia sp. SOS3 is a plant growth-promoting bacterium (PGPB) that displays pleiotropic effects and has the potential to be applied at a large scale across several agronomically important crops. The use of SOS3 is a suitable option to reduce the use of chemical fertilisers. While the benefits of SOS3 have been demonstrated in vitro, its potential applications at large scale are limited due to low biomass yield in current batch culture systems. Here, we developed a strategy for high-cell density cultivation of SOS3 in instrumented bioreactors, moving from low-biomass yield in a complex medium to high-biomass yield in a semi-defined medium. We achieved a 40-fold increase in biomass production, achieving cell densities of up to 11 g/L (OD600 = 40). This result was achieved when SOS3 was cultivated using a fed-batch strategy. Biomass productivity, initially 0.02 g/L/h in batch cultures, was improved 12-fold, reaching 0.24 g/L/h during fed-batch cultures. The biomass yield was also improved 10-fold from 0.07 to 0.71 gbiomass/gsolids. Analysis of the fermentation profile of SOS3 indicated minimal production of by-products and accumulation of polyhydroxybutyrate (PHB) during the exponential growth phase associated with nitrogen limitation in the medium. By implementing proteomics analysis in fed-batch cultures, we identified the expression of four metabolic pathways associated with growth-promoting effects, which may be used as a qualitative parameter to guarantee the efficacy of SOS3 when used as a bioinoculant. Ultimately, we confirmed that the high-cell density cultures maintained their plant growth-promoting capacity when tested in sorghum and maize under glasshouse conditions.

Calcium (Ca2+)-regulated exopolysaccharide biosynthesis in probiotic Lactobacillus plantarum K25 as analyzed by an omics approach

Exopolysaccharide (EPS)-producing lactic acid bacteria have been widely used in dairy products, but how calcium, the main metal ion component in milk, regulates the EPS biosynthesis in lactic acid bacteria is not clear. In this study, the effect of Ca2+ on the biosynthesis of EPS in the probiotic Lactobacillus plantarum K25 was studied. The results showed that addition of CaCl2 at 20 mg/L in a semi-defined medium did not affect the growth of strain K25, but it increased the EPS yield and changed the microstructure of the polymer. The presence of Ca2+ also changed the monosaccharide composition of the EPS with decreased high molecular weight components and more content of rhamnose, though the functional groups of the polymer were not altered as revealed by Fourier transform infrared spectral analysis. These were further confirmed by analysis of the mRNA expression of cps genes, 9 of which were upregulated by Ca2+, including cps4F and rfbD associated with EPS biosynthesis with rhamnose. Proteomics analysis showed that Ca2+ upregulated most of the proteins related to carbon transport and metabolism, fatty acid synthesis, amino acid synthesis, ion transport, UMP synthesis. Specially, the increased expression of MelB, PtlIIBC, EIIABC, PtlIIC, PtlIID, Bgl, GH1, MalFGK, DhaK, and FBPase provided substrates for the EPS synthesis. Meanwhile, metabolomics analysis revealed significant change of the small molecular metabolites in tricarboxylic acid cycle, glucose metabolism and propionic acid metabolism. Among them the content of active small molecules such as polygalitol, lyxose, and 5-phosphate ribose increased, facilitating the EPS biosynthesis. Furthermore, Ca2+ activated HipB signaling pathway to inhibit the expression of manipulator repressor such as ArsR, LytR/AlgR, IscR, and RafR, and activated the expression of GntR to regulate the EPS synthesis genes. This study provides a basis for understanding the overall change of metabolic pathways related to the EPS biosynthesis in L. plantarum K25 in response to Ca2+, facilitating exploitation of its EPS-producing potential for application in probiotic dairy products.

26 Effect of different cryoprotectant concentrations on vitrification of invitro-matured bovine oocytes in paper containers

A great challenge for successful oocyte vitrification is the development of a low-cytotoxic cryoprotectant solution in a safe device allowing ultra-rapid cooling. This study compared different concentrations of cryoprotectants for bovine IVM-oocyte vitrification in a safe paper container device on oocyte survival and cleavage rates. Abattoir ovaries were obtained and cumulus–oocyte complexes (COCs) were recovered by aspirating follicles of 3 to 6mm in diameter. A total of 470 COCs with homogeneous cytoplasm oocytes, surrounded by several layers of cumulus cells were selected, in 5 replicates. Groups of ∼50 COCs were matured in 500µL of semi-defined IVM medium for 22h at 38.8°C in a humidified atmosphere with 5% CO2. After IVM, COCs were allocated to 1 of 3 groups of 20 to 30 COCs, differing only in final concentration of cryoprotectants. A nonvitrified control group (CG) was also tested, totalling 4 groups. Before vitrification, each group was transferred to 500µL of TCM-199 HEPES with 20% fetal bovine serum (FBS) (Base medium, BM) for 5min at 34°C, and COCs were partially denuded by gentle pipetting. Vitrification followed a 3-step protocol at room temperature and groups of 4 to 5 COCs were transferred to BM solution drops containing (1) 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO) for 30 s; (2) 10% EG + 10% DMSO + 0.25M sucrose for 30 s; and (3) vitrification solution (VS), according to each group: high (HG), 20% EG + 20% DMSO + 0.5M sucrose; medium (MG), 15% EG + 15% DMSO + 0.5M sucrose; or low (LG), 10% EG + 10% DMSO + 0.5M sucrose for 30s. Afterwards, COCs were loaded in <1µL of solution and placed in a homemade paper container device, and immediately plunged in liquid nitrogen. Warming was performed placing the paper container in 3mL of 1M sucrose in BM for 2min. After warming, a 3-step protocol was conducted and COCs were transferred to (1) 500µL of 0.5M sucrose in BM for 2 min; (2) 500µL of 0.25M sucrose for 2 min; (3) 500µL of BM for 2min. Then, COCs from each group were transferred to 250µL of semi-defined IVF medium. Motile sperm were recovered by Percoll washing from one bull and added to IVF medium (Day 0) at final concentration of 106 sperm mL−1 for 18h. At Day 1, all presumptive zygotes were cultured in 25µL of SOF medium with 5% FBS under mineral oil at 38.8°C with 5% CO2 and 5% O2. Normal data were subjected to ANOVA and post hoc Tukey test. Cleavage rate was recorded at Day 2 after IVF. Oocyte survival rate was similar (P>0.05) among vitrified groups (HG, 80%; MG, 86%; LG,87%). Cleavage rate differed (P<0.05) in all vitrified groups compared with control (CG, 82%; HG, 10%; MG, 16%; LG, 16%). Although no difference (P>0.05) was observed among vitrified groups, MG and LG showed a slightly increased oocyte survival and cleavage rates compared with HG. In conclusion, the use of either medium or low concentrations of cryoprotectants may be a less toxic alternative for vitrification of IVM bovine oocytes on paper device. This research was funded by CAPES/COFECUB (#88881.142966/2017-01).

Evaluation of carbon:nitrogen ratio in semi-defined culture medium to tacrolimus biosynthesis by Streptomyces tsukubaensis and the effect on bacterial growth

Tacrolimus is an immunosuppressant produced by several Streptomyces species, widely used in transplantolgy and dermatology. In this report, attempts have been made to enhance tacrolimus productivity by altering the primary carbon source on culture media. In order to better understand the effects of glucose and maltose on tacrolimus biosynthesis, different carbon contents of these sugars were evaluated in the culture media, thus altering the carbon:nitrogen ratio. The highest specific production achieved on this report was 10.46 mg/g at a carbon:nitrogen ratio of 8.7:1.0 and maltose as carbon source. Using glucose as primary carbon source, the highest specific production achieved was 9.06 mg/g at a carbon:nitrogen ratio of 8.5:1.0. The range of carbon:nitrogen ratios evaluated for each sugar presented distinct behaviors. Glucose containing-media exhibited an initial linear downward trend, by the other hand, maltose containing-media exhibited an increasing tendency. Both glucose and maltose presented the highest specific production at 3 % of carbon source on media. Media containing more than 3 % of carbon content exerted carbon catabolite repression.The range of carbon:nitogen ratios evaluated presented an increasing tendency on tacrolimus biosynthesis up to 3 % of glucose or maltose in the media, both carbon sources exerted the phenomenon of carbon catabolite repression with more than 3 % of carbon source on the media.

Statistical optimisation of the exopolysaccharide production by Lactobacillus fermentum Lf2 and analysis of its chemical composition

Lactobacillus fermentum Lf2 produces high amounts of exopolysaccharides (EPS) (~1 g/L) with demonstrated functional and technological roles when applied as a food ingredient in dairy matrices, properties that made these EPS interesting in comparison with other similar molecules from lactic acid bacteria (LAB). Those characteristics encouraged us to optimise the production. The EPS extract is composed of a high molecular mass β‐glucan and a medium molecular mass heteroglycan. In the present work, the optimal conditions that doubled the EPS yield using a semidefined medium (SDM, 0.63% yeast nitrogen base, 0.53% bacto casitone, 0.53% ammonium citrate, 6.25% sucrose, pH 6.5) were found by means of response surface methodology (RSM). The chemical characterisation indicated that under optimised conditions the synthesis of the heteroglycan was favoured compared with that of the β‐glucan.

Porcine blastocyst viability and developmental potential is maintained for 48 h of liquid storage at 25 °C without CO2 gassing

Short- and medium-term storage of pig embryos has become relevant for commercial application of non-surgical deep uterine embryo transfer (NsDU-ET) in the light of the strict legal and administrative requirements posed by the International Association for Air Transport (IATA) to allow shipment of liquid nitrogen (LN2) containers and the technical drawbacks when using vitrified embryos. Therefore, this study developed an efficient method for the liquid storage of in vivo-derived porcine blastocysts for a moderate duration (48 h) without controlled CO2 gassing. We evaluated two storage temperatures (25 °C and 37 °C) and three HEPES-supplemented media: the chemically defined media TL-PVA and NCSU-PVA and the semi-defined medium NCSU-BSA. We observed no differences in survival, hatching rate or final developmental stage between the two temperatures, but storage at 25 °C was more efficient to preserve zona pellucida (ZP) integrity. Blastocysts were successfully stored for 24 h in a chemically defined medium. Yet, only 48 h storage in NCSU-BSA medium supported blastocyst development. Although all storage conditions resulted in an embryonic developmental delay, blastocysts stored in NCSU-BSA at either tested temperature could hatch and attain the same final developmental stage as control blastocysts when cultured under standard conditions after storage. Moreover, blastocysts stored at 25 °C for 48 h in NCSU-BSA medium could produce pregnancies after surgical transfer. In conclusion, porcine blastocysts maintain their viability and developmental potential after storage in the semi-defined medium NCSU-BSA for at least 48 h at 25 °C.

Isolation and characterization of a high molecular mass β-glucan from Lactobacillus fermentum Lf2 and evaluation of its immunomodulatory activity

When grown in a semi-defined medium, L. fermentum Lf2 synthesizes significant quantities (∼2 g/L) of two exopolysaccharides (EPS). The two EPS were separated by preparative size exclusion chromatography to give a high molecular mass β-glucan (1.23 × 106 Da) and a medium molecular mass heteroglycan (8.8 × 104 Da). The structure of the high molecular mass β-glucan was determined using a combination of NMR spectroscopy, monomer and linkage analysis. The EPS has the following structure:▪The immunomodulatory activity of the high molecular mass EPS was studied in peripheral blood mononuclear cells (PBMC). Exposure of PBMC to an aqueous solution of the EPS for 24 h led to increased cell proliferation, changes in expression of the cytokines CD14 and TLR2, and to an increase in production of TNF-α compared to controls. In contrast, when cells that had been treated with EPS for 24 h and from which the EPS had been removed, were subsequently exposed to the bacterial antigen LPS very low levels of TNF-α production were observed. This result indicates that the EPS imparts immunotolerance in PBMC. An ability to modulate the release of the proinflammatory mediators, such as TNF-α, is an important goal in the development of therapies for the treatment of diseases, such as Crohn's disease and ulcerative colitis, associated with excessive release of inflammatory mediators.

Culture of Preimplantation Rabbit Embryos.

It is surprising that so little attention is currently given to in vitro culture of preimplantation rabbit embryos, even though the rabbit is the only laboratory animal in which there is very considerable embryo growth before implantation, resulting in a 300-fold increase in protein content of embryonic cells during the preimplantation period and the formation of more than a 100,000 cells in the blastocyst. This growth pattern explains why blastocyst formation in vitro has an absolute requirement for amino acids, and vitamins, particularly inositol, are esssential for blastocyst growth. A semi-defined medium supplemented with 1.5% BSA (variously known as BSM II or modified F10) was developed at Cornell University at the end of the 1960s and allowed the systematic investigation of the requirements for development of 1-cell rabbit embryos to blastocysts. However, the requirements for in vitro blastocyst growth comparable to in vivo growth still remain an unsolved problem. Citrate, often found as a contaminant in serum albumin, may have an essential role in rabbit blastocyst growth, which would fit in with its role in the development of serum-free media for culture of various types of mammalian cells.A comprehensive account of the methodology is given to enable a researcher with experience culturing embryos of a different species to work on the rabbit embryo. This account covers medium preparation, hormonal stimulation of superovulation, natural breeding/artificial insemination, and collection of embryos of different stages from 1-cell to blastocyst either after euthanasia or under anesthesia. Peculiarities of the rabbit embryo such as the presence of the mucoprotein coat and its effects on behavior of cultured and transferred embryos are described. Suggestions are made for future avenues of research.

Selection of lactic acid bacteria for exopolysaccharide production

An important source of natural alternative to commercial additives that are commonly extracted from plants and animals is the exopolysaccharide (EPS) produced by lactic acid bacteria (LAB). A screening for EPS production by Lactobacillus delbrueckii, Lactobacillus rhamnosus NBRC 3425 and Weisella paramesenteroides was conducted to identify which among these three LAB would produce the highest yield of EPS. The test organisms were grown in a Semi-defined Medium (SDM) of Sanchez et al (2006) with some modifications. EPS production was confirmed by the formation of precipitate after mixing the broth medium with 95% absolute ethanol. Results of total sugar analysis by phenol-sulfuric acid assay revealed that estimated EPS yield of L. rhamnosus NBRC 3425 was significantly higher at p<0.05 than those of W. paramesenteroides and L. delbrueckii ssp. lactis with values of 0.1355g/L, 0.0652g/L and 0.0544g/L, respectively even though their viable count did not differ significantly from each other. Correspondingly, the pH of L. rhamnosus NBRC 3425 media was also significantly higher (pH 4.03) than L. delbrueckii (pH 3.60) and W. paramesenteroides (pH 3.83).

Construction and co-cultivation of two mutant strains harboring key precursor genes to produce prodigiosin

The biosynthesis of prodigiosin (PG) from Serratia marcescens involves the coupling of a bipyrrole, 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC), with a monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP), and formation of a linear tripyrrole (PG). We constructed mutant strains in which either the MBC biosynthesis by S.marcescens BMJ816 or the MAP biosynthesis by S.marcescens AMJ817. S.marcescens BMJ816 and AMJ817 confirmed that they lose the ability to synthesize PG when they are cultivated alone. An experiment was also conducted in which cultures of the two mutant strains were grown to the early exponential phase in a semi-defined medium, and one suspension culture was inoculated with the other. This approach yielded 103mg/L PG. The findings suggest that the addition of precursors may enhance PG production by microorganisms.