Optimization of culture conditions of SARS-CoV-2 four Omicron variants in Vero cells / 中国生物制品学杂志

@#Objective To optimize the culture conditions of four vaccine candidates of severe acute respiratory symptom coronavirus 2(SARS-CoV-2) Omicron variants BA.1,BA.1.1,BA.2 and BA.5 in Vero cells.Methods The harvest time(24,48,72 and 96 h) and MOI(0.01,0.001,0.0001 and 0.000 01) of four Omicron variants cultured in Vero cells were optimized by using cytopathic effect(CPE),viral nucleic acid copy number and viral titer as evaluation indexes.Results The optimum harvest time of the four Omicron variants BA.1,BA.1.1,BA.2 and BA.5 in Vero cells was 72 h,and the optimum MOI was 0.001～0.000 01,0.001～0.000 01,0.01～0.000 01 and 0.01～0.000 01,respectively.Conclusion The culture conditions of four Omicron variants in Vero cells were optimized,which laid a foundation of the development of SARS-CoV-2 Omicron variant inactivated vaccine based on Vero cells.

Characterization of bacterial endophytes from Myanmar medicinal plants for antimicrobial activity against human and plant pathogens

This research aimed to investigate the antagonistic activity of the bacterial endophytes from Myanmar medicinal plants. Thirty-one bacterial isolates were isolated from Myanmar medicinal plants: Tinospora cordifolia (Wild.) Miers., Catharanthus roseus G. Don., Tectona hamiltoniana Wall. and Boscia variabilis Collett & Hemsl. (Capparaceae). Dual culture and agar well diffusion methods were used for antimicrobial assay. One endophyte from Catharanthus roseus and two bacterial isolates from Boscia variabilis Collett & Hemsl. (Capparaceae) had not only the antibacterial activity towards the human pathogenic bacteria but also the antifungal activity against the plant pathogenic fungi. From 16S rRNA sequencing, one strain from Catharanthus roseus G. Don. was Bacillus amyloliquefaciens DSM7 and two antagonistic strains from Boscia variabilis Collett & Hemsl. (Capparaceae) were Bacillus subtilis subsp. subtilis str. 168 and Bacillus amyloliquefaciens DSM7, respectively. The best medium for the maximum production of the bioactive compounds was Bacillus medium supplemented with the 4% of starch and 0.3% of peptone for B. amyloliquefaciens DSM7 and B. subtilis subsp. subtilis str. 168 showed the maximum antimicrobial compounds production when it was incubated in the medium amended with 3% of starch and 2% of peptone. The optimum conditions for the the maximum production of the antimicrobial compound were the medium pH of 6 at 35 ºC after two days of incubation for B. amyloliquefaciens DSM 7 and B. subtitlis subsp. subtilis str. 168 secreted the maximum concentration of the bioactive compounds at pH 7.5 and 35˚C on second day incubation period. In conclusion, the isolated endophytic bacteria showed the strong antimicrobial activity towards the pathogenic microbes and they could be used in medicine and agriculture as well.

Advances in Bioculture Technology of Bupleuri Radix and Biosynthesis Pathways of Saikosaponins / 中国实验方剂学杂志

Synthetic biology is an emerging discipline that analyzes the biosynthesis pathways of active constituents in traditional Chinese medicine and explores genes involved in biosynthesis. Bupleuri Radix is one of the most commonly used Chinese medicinal materials with remarkable medicinal value, its index component is saikosaponins, which has significant anti-inflammatory, anti-viral and anti-tumor activities. However, the current wild resources of Bupleuri Radix have been destroyed, and there were some problems in the process of artificial cultivation. The application of biological culture technology and synthetic biology can expand the sources of saikosaponins and protect resources of Bupleuri Radix. The culture conditions of different plants can be followed without a fixed pattern, and the biosynthetic pathways of different medicinal active ingredients are also inconsistent. At present, there is no review report on the culture technology of Bupleuri Radix and the research on the biosynthesis pathway of saikosaponins. This paper introduces the research progress of biological culture techniques, such as callus culture, adventitious root culture, hairy root culture and suspension cell culture used in synthetic biology, and the biosynthesis pathway of saikosaponins and its key enzyme functional genes. It is suggested to optimize the biological culture technology of Bupleuri Radix by referring to the tissue culture technology of other traditional root medicinal materials, so as to provide a reference for the in-depth study on the biosynthesis pathway and metabolic regulation of saikosaponins.

Bioflocculant production from Streptomyces platensis and its potential for river and waste water treatment

ABSTRACT A bacterium isolated from Sterkfontein dam was confirmed to produce bioflocculant with excellent flocculation activity. The 16S rDNA nucleotide sequence analyses revealed the bacteria to have 99% similarity to Streptomyces platensis strain HBUM174787 and the sequence was deposited in the Genbank as Streptomyces platensis with accession number FJ 486385.1. Culture conditions for optimal production of the bioflocculant included glucose as a sole carbon source, resulting in flocculating activity of 90%. Other optimal conditions included: peptone as nitrogen source; presence of Mg2+ as cations and inoculum size of 1.0% (v/v) at neutral pH of 7. Optimum dose of the purified bioflocculant for the clarification of 4 g/L kaolin clay suspension at neutral pH was 0.2 mg/mL. Energy Dispersive X-ray analysis confirmed elemental composition of the purified bioflocculant in mass proportion (%w/w): carbon (21.41), oxygen (35.59), sulphur (26.16), nitrogen (0.62) and potassium (7.48). Fourier Transform Infrared Spectroscopy (FTIR) indicated the presence of hydroxyl, carboxyl, methoxyl and amino group in the bioflocculant. The bioflocculant produced by S. platensis removed chemical oxygen demand (COD) in river water and meat processing wastewater at efficiencies of 63.1 and 46.6% respectively and reduced their turbidity by 84.3 and 75.6% respectively. The high flocculating rate and removal efficiencies displayed by S. platensis suggests its industrial application in wastewater treatment.

Effect of different culture conditions on biological characteristics of fibroblasts derived from human hypertrophic scar / 中华整形外科杂志

Objective@#To investigate the effect of different culture conditions on cell proliferation, apoptosis, collagen synthesis and TGF-β1 expression of fibroblasts derived from human hypertrophic scar(HSF).@*Methods@#Fibroblasts were isolated from human hypertrophic scar and cultured in vitro. Cells of passage 4 to 6 were cultured with 10%O2+ 100% culture medium for 48 h, and then divided into four groups, 10%O2+ 100% culture medium group (control group), 7%O2+ 70% culture medium group (experimental group 1), 4%O2+ 40% culture medium group (experimental group 2) and 1%O2+ 10% culture medium group (experimental group 3) according to the different culture conditions. There are 4 samples in each group. MTT assay, Flow cytometer and the Annexin V-FITC/PI double-staining were performed to detect the cell proliferation, S phase cell ratio and cell apoptosis. Hydroxyproline assay kit was adopted to detect the collagen synthesis of the fibroblasts. Protein expression levels of transforming growth factor β1 (TGF -β1)were determined with Western-blotting. SPSS 13.0 software was used to analyze the data. Analysis of variance ( oneway ANOVA) and SNK test were used to evaluate significant differences among these groups.@*Results@#①Compared with control group, the cell proliferation rate was higher in the experimental group 1(P<0.05), while it significantly decreased in experimental group 3 (P<0.05). There was a statistically significant difference among groups (F=163.32, P<0.01). ②The collagen synthesis was much higher in experimental group 1 compared with control group (0.56±0.01 vs 0.42±0.01, P<0.05), and was lower in experimental group 3 (0.48±0.01 , P<0.05). There was a statistically significant difference among groups (F=357.69, P<0.01). ③The S phase cell ratio in the four groups were(19.40±0.37)%, (22.83±0.28)%, (20.94±0.38)% and(14.54±0.21)% respectively. Compared with control group, the S phase cell ratio was significantly higher in experimental group 1 and experimental group 2 (P<0.05), while it was significantly lower in experimental group 3(P<0.05). There was a statistically significant difference among groups (F=357.69, P<0.01). ④ The expression of TGF-β1 increased significantly in experimental group 1 and 2(P<0.05)compared with control group. However, experimental group 3 showed less TGF-β1 expression. There was a statistically significant difference among groups (F=357.69, P<0.01). ⑤The ratio of living cells in the four groups was (96.21±0.29)%, (96.61±0. 24)%, (96.61±0.17)% and(95.36±0.15)% respectively. There was no significant difference between control group and experimental group 1 and 2, while it was significantly decreased in experimental group 3 (P<0.05). There was a statistically significant difference among groups(F=12.38, P<0.01).@*Conclusions@#Mild hypoxia and low concentration of culture medium promote cell proliferation, collagen synthesis, TGF-β1 expression and cell viability of fibroblasts derived from hypertrophic scar. However, severe hypoxia and low concentration of culture medium decrease cell biological characteristics and promote cell apoptosis.

Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

A novel pullulanase from a fungus Hypocrea jecorina QM9414: production and biochemical characterization.

Pullulanase production from a fungus Hypocrea jecorina QM9414 that produces native extracellular hydrolases having industrial applications was carried out in a shaking flask culture containing 0.5% amylopectin at a pH of 6.50 at 30°C. The enzyme was purified 11-fold by ammonium sulfate fractionation, anion-exchange and gel-filtration chromatographies with a yield of 10.12% and a specific activity of 1.36 ± 0.14 U/mg protein. The molecular mass of pullulanase was estimated to be 130.56 kDa by PAGE and SDS-PAGE, indicating that the native enzyme was a monomer. The optimum pH and temperature for purified enzyme was 6.5 and between 35°-65°C, respectively. The Km values for amylopectin, starch and pullulan as substrates were 10.7, 15.5 and 38.4 mg/mL, respectively. The Vmax values were found to be 3.32, 3.32 and 3.82 ΔA/min for amylopectin, starch and pullulan, respectively. The enzyme was stable at 40-70°C for 30 min, but lost about 33% of its activity at 80°C and about 43% of activity at 90°C and 100°C for the same incubation period. Pullulanase activity was stimulated by CoCl2, NiCl2, KI, NaCl, MgCl2, and LiSO4. The enzyme was slightly inhibited by urea, CaCl2 and b-mercaptoethanol. The enyzmatic characteristics, substrate specificity and the products of hydrolysis indicated that the enzyme was similar to those of type II pullulanases.

Optimization of Culture Conditions for Enhanced Antimicrobial Activity of Rhodococcus erythropolis VLK-12 Isolated from South Coast of Andhra Pradesh, India.

Aims: To Isolate and characterize the antimicrobial actinomycetes from the marine habitats of south coast of Andhra Pradesh, India. Place and Duration of the Study: Marine habitats of south coast of Andhra Pradesh, India, between June 2011 and July 2012. Methodology: The soil samples were collected, pre-treated and plated on yeast extractmalt extract dextrose agar medium. Identification of the strain was carried out by employing the polyphasic taxonomical studies including the 16S rRNA sequence based analysis. Phylogenetic tree was constructed using the Molecular Evolutionary Genetic Analysis (MEGA) version 5. The influence of culture conditions and the effect of environmental factors on the biomass and antimicrobial activy\ity of the strain was the focus of this study. Results: A total of 20 actinobacteria were isolated from the marine habitats of south coast of Andhra Pradesh, India, and screened for antimicrobial activity against test bacteria and fungi. The potent bioactive metabolite producing strain was designated as VLK-12. Further polyphasic studies revealed that the Isolate VLK-12 belongs to the genera Rhodococcus. Phylogenetic analysis of 16S rRNA sequencing studies revealed that the strain is closely related to Rhodococcus erythropolis. The crude ethyl acetate extract obtained by culturing the strain on YMD inhibited Gram positive and Gram negative bacteria along with fungi. Conclusion: Rhodococcus erythropolis isolated from the marine habitats of south coast of Andhra Pradesh exhibited antimicrobial activity against pathogens.

Optimization of flask culture medium and conditions for hyaluronic acid production by a streptococcus equisimilis mutant nc2168

A mutant designated NC2168, which was selected from wild-type Streptococcus equisimilis CVCC55116by ultraviolet ray combined with60Co-γ ray treatment and does not produce streptolysin, was employed to produce hyaluronic acid (HA). In order to increase the output of HA in a flask, the culture medium and conditions for NC2168 were optimized in this study. The influence of culture medium ingredients including carbon sources, nitrogen sources and metal ions on HA production was evaluated using factional factorial design. The mathematical model, which represented the effect of each medium component and their interaction on the yield of HA, was established by the quadratic rotary combination design and response surface method. The model estimated that, a maximal yield of HA could be obtained when the concentrations of yeast extract, peptone, glucose, and MgSO4 were set at 3 g/100 mL, 2 g/100 mL, 0.5 g/100 mL and 0.15 g/100 mL, respectively. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a remarkable increase in the output of HA and the maximum of the predicted HA production was 174.76 mg/L. The model developed was accurate and reliable for predicting the production of HA by NC2168.Cultivation conditions were optimized by an orthogonal experimental design and the optimal conditions were as follows: temperature 33ºC, pH 7.8, agitation speed 200 rpm, medium volume 20 mL.

Optimal subculture methods for passaging and growing human epidermal keratinocytes.

Background: Human epidermal keratinocytes (HEKs) cover the outer layer of the skin and play a key role in wound repair. Although the methods for isolation and cultivation of primary HEKs from epidermis have been used successfully in both laboratory and clinical settings, the ability to subculture (passage) these cells has yet to be established and is the primary factor hindering their usage. Objectives: We conducted this study to identify optimal subculture conditions for HEKs. Methods: We first harvested the primary HEKs from prepuce tissue specimens, and then compared three different reagent compositions (0.25% trypsin, 0.25% trypsin plus 0.01% EDTA, and 0.25% dispase digestion solution) for various periods of time at 4oC with the conventional 0.25% trypsin or 0.25% trypsin plus 0.01% EDTA digestion at room temperature. Results: Our data indicated that the cold digestion conditions yielded higher cell numbers and more viable cells than the conventional methods. Furthermore, the subcultured HEKs also adhered and grew better after four hours of a 0.25% trypsin cold digestion or after six hours of a 0.25% dispase cold digestion. These procedures produced higher numbers of HEK passages than that commonly seen experienced with conventional methods. Conclusion: The data from the current study demonstrated that the optimal subculture condition for passaging and growing HEKs in vitro is four hours digestion with 0.25% trypsin.

Cell suspension culture of Dendrobium huoshanenes and its optimal conditions / 中草药

Objective: Single-cell of Dendrobium huoshanenes was cultured by using cell suspension culture for solving the problem of resource shortage of medicinal plant D. huoshanenes. Methods: The single-cell of D. huoshanenes was obtained by different preparing ways, effects of various factors on cell growth were studied and the optimal growing conditions of cell suspension culture were investigated by orthogonal design. Results: Through the orthogonal test, the results indicated that the optimum conditions for suspension culture of D. huoshanenes cell were as follow: is that the concentration of sucrose was 35 g/L, NH4NO3 was 1.3 g/L, pH value was 5.6, the hormone IAA was 0.4 mg/L , 6-BA was 1.0 mg/L, the best culture time was 14-18 d. Under optimal culture conditions, the concnentration of single cells in this test was 9.42 X 105 cell/mL. Conclusion: After four successive subculture, cellular morphology of D. huoshanenes shows circular shape or elliptic shape and the chip of cell is decreased. The higher concentration of D. huoshanenes cell could be obtained by using the optimal growing conditions of cell suspension culture.

Role of brown-rot fungi in the bioremoval of azo dyes under different conditions

The present study is vital to the understanding of bioremediation of structurally different azo dyes by some unusual Brown-rot fungi. Bioremoval of each dye (20 mg l-1) was tested in two different culture media under static and shaking conditions by taking inocula from different fungi. Fungal strains showed varying dyes removal abilities, though considerable high in case of Acid Red (AR) 151(di-azo) as compared to Orange (Or) II (mono-azo). With an exception of Aspergillus tereus SA3, all the fungal isolates showed higher removal of dyes in SDB. Under static condition, the maximum decolorizing fungal strains were; Aspergillus flavus SA2 (67 percent) and Alternaria spp. SA4 (57 percent) in AR 151, while Penicillium spp. (34 and 33 percent) in Orange II, in SDB and STE, respectively. Bioremoval of dyes was considerably increased when experiments were shifted from static to shaking mode. It was specifically increased ( percent) in; AR 151 (255) with Penicillium spp., Or II with A. flavus SA2 (112) and Alternaria spp. (111). The primary mechanism of dyes removal proved to be fungal biosorption. However, reduction of dyes (onto fungal) with formation of their products (α. naphthol, sulphalinic acid and aniline) furthermore revealed that dyes (specifically azo) were actually biodegraded.

Optimization of Solid-State Fermentation for Improved Conidia Production of Beauveria bassiana as a Mycoinsecticide

The production of conidia of entomopathogenic Beauveria bassiana by solid-state fermentation was studied for the development of a biocontrol agent against aphid Myzus persicae. The optimal conditions for conidia production on polished white rice were 40% moisture content, 25degrees C culture temperature, 2-day-old seeding culture grown in 3% corn meal, 2% rice bran, 2% corn steep powder medium, initial conidia concentration of 107 conidia/g in the wet rice, 10% inoculum size, and use of a polyethylene bag as a container. The polyethylene bag containing inoculated rice was hand-shaken every 12 hr during fermentation. Using optimal conditions, the maximum conidia production obtained was 4.05 g conidia/100 g dry rice after 14 days of cultivation, a rate 2.83 times higher than conidia yield of pre-optimization.

Production of Blastospore of Entomopathogenic Beauveria bassiana in a Submerged Batch Culture

The principal objective of this study was to determine the optimal liquid culture conditions in shake flasks for maximal sporulation of Beauveria bassiana. The optimal initial pH for the spore production of B. bassiana using Potato Dextrose Broth was 5.2. The screening in shake flasks of carbon and nitrogen sources resulted in the identification of an optimal medium based on 3% sucrose and 1% casamino acid, with a C : N ratio of 22 : 4. Using this medium, a production level of 5.65 x 107 spores per ml was obtained after 5 days of culture. Using 3% corn meal, 2% corn steep powder, and 2% rice bran, the maximum spore concentration of 8.54 x 108/ml was achieved 8 days after inoculation at 25degrees C in a rotary shaking incubator operated at 200 rpm. This represents a yield gain of approximately 2.89 times that of pre-optimization.

Physicochemical Requirement for the Vegetative Growth of Schizophyllum commune Collected from Different Ecological Origins

Schizophyllum commune is an edible and medicinal mushroom widely distributed in the world. The optimal growth conditions for the mycelia of 10 strains of the fungus were investigated. The temperature suitable for the mycelial growth and density was obtained at 30~35degrees C. Among the tested conditions, the minimum mycelial growth was found at 15degrees C. In case of pH, the most favorable growth was found at pH 5. The results indicated that this mushroom well adapted to high temperature and low pH for its mycelial growth. Considering growth phenotype of mycelia, Hamada, Hennerberg, PDA and YM were the most suitable and Lilly, Glucose triptone, Glucose peptone and Hoppkins were the most unfavorable among tested media for the mycelial growth of S. commune. Out of tested carbon sources, dextrin and fructose were the most suitable and lactose, mannose and sorbitol were the unsuitable for the fungus. Compact mycelial density was obtained from most of the carbon sources. Among used nitrogen sources, calcium nitrate, potassium nitrate and alanine were the most appropriate and the most incompatible were ammonium phosphate, histidine, urea and arginine for mycelial growth of S. commune on the culture media. Calcium nitrate, histidine and potassium nitrate showed moderately thin or thin, and rest of nitrogen sources showed compact or moderately compact mycelial density.

Cultural Conditions for Production of Glutathione by Mutant Saccharomyces J-X25 / 中国生物工程杂志

The production conditions of glutathione with shaking flask fermentation by the mutant Saccharomyces J-X25, a methionine-defected strain were studied, and the optimum culture conditions are as follows: initial pH6. 0, temperature 30℃, 100ml in 500ml flask, the inoculum size 10% and agitation rate 220r/min. The emphasis was on the stimulating effect on the cells by dioxogen and the sodium lactate as surfactant. Both of which were added at the logarithmic phase of fermentation, and the GSH production was up to 0. 253g/L , 52% higher than the control without the additions. Compared with the production of GSH initial strain that by the mutant in optimum conditions was increased by 79%.

Three Dimensional Reconstitution of Oral Mucosal Keratinocytes and Its Biologic Characteristics

The purposes of this study were to develop an in vitro co-culture model of epithelial tissue with dermal equivalent, cultured at an air-liquid interface, and to evaluate the effects of extracellular matrix and concentration of calcium and fetal bovine serum in medium to find optimized culture condition. Oral keratinizing epithelial cells in monolayer culture were grown in Mitomycin-treated 3T3 feeder. Primary cultured oral epithelial cells were reconstituted onto the dermal equivalents consisting of 3T3 fibroblast and type I collagen, and co-culture was grown at the air-liquid interface. The histomorphological development of reconstituted oral epithelium in vitro for 21 days revealed 10~12 layered statified epithelium, closely similar to the parakeratinized gingival epithelium. Neither laminin nor type IV collagen was able to induce keratinocyte differentiation. But a mixture of laminin and type IV collagen induced well-polarized keratinizing tissue with anchoring structure of basal cells. When the reconstituted oral epithelium was incubated in 1.0% and 0.5% serum-containing medium, the granular cell layers with orthokeratinization developed. The reconstituted epidermis generated in serum-free keratinocyte growth medium (KGM)-containing pituitary extract showed features of incomplete differentiation. The present study shows that the dermal equivalents containing fibroblasts will support epidermal morphogenesis and differentiation. And these results suggest that extracellular matrix and calcium concentration are important factors during the reconstitution of keratinizing epithelium in vitro.

Investigation on Characteristics and the Yield of the Secondary Metabolites of a Myxobacterium Isolated from Beach Soil / 微生物学通报

A myxobacterial strain, So ce cpu-1, was isolated from a soil sample near the Huanghai beach. So ce cpu-1 had broad antimicrobial activity, active component has maxima] absorption at 210 nm. The effects of different culture conditions on the yield of the secondary metabolites were investigated. The results showed that, when cultivating the strain in the M, medium (containing 10% w/v D312 neutral absorber resin ), the air up to 70% of the whole flask volume, adding 5?L the secondary metabolites as the revulsant, at 30℃, 200r/min, for 6 days, the yield of the secondary metabolites achieved the maximum.

CULTURE CONDITIONS FOR CREATININASE FORMATION BY PSEUDOMONAS SP. K9510 / 微生物学通报

From air bacteria capable of decomposing creatinine, three single independent strains K9510、K9511 and K9512 have been isolated. The highest creatinine amidohydrolase (EC 3. 5. 2. 10; creatininase) producing strain K9510 was screened out. The strain K9510 was identified as Pseudomonas sp. The results of culture condition for creatininase formation by strain K9510 were obtained as follows: creatinine and creatine were found to be the effective inducers for enzyme formation; the solution of mixed metallic salts could stimulate cell growth and enzyme formation. The suitable medium for creatininase formation was consisted of 0. 9% creatinine、0. 15% yeast extract、 0. 09% malt extract、0. 05% NH4Cl and some amount of the solution of mixed metallic salts at pH5 5. When the bacterium was grown in 250mL conic flask containing 50mL of the medium mentioned above on the rotary shaker(250r/min) at 35℃ for 33 h, about 50 u creatininase was obtained.