Controlled rate slow freezing with lyoprotective agent to retain the integrity of lipid nanovesicles during lyophilization

We designed a novel lyophilization method using controlled rate slow freezing (CSF) with lyoprotective agent (LPA) to achieve intact lipid nanovesicles after lyophilization. During the freezing step, LPA prevented water supercooling, and the freezing rate was controlled by CSF. Regulating the freezing rate by various liquid media was a crucial determinant of membrane disruption, and isopropanol (freezing rate of 0.933 °C/min) was the optimal medium for the CSF system. Lyophilized lipid nanovesicle using both CSF and LPA retained 92.9% of the core material and had uniform size distributions (Z-average diameter = 133.4 nm, polydispersity index = 0.144), similar to intact vesicles (120.7 nm and 0.159, respectively), after rehydration. Only lyophilized lipid nanovesicle using both CSF and LPA showed no changes in membrane fluidity and polarity. This lyophilization method can be applied to improve storage stability of lipid nanocarriers encapsulating drugs while retaining their original activity.

Development of a method for obtaining healthy mini-tubers of potatoes of the Yubilyar variety using a humic preparation from peat

The influence of various nutrient medium compositions on the growth of potato plants during in vitro cultivation and the possibility of using biological fertilizer from peat in different concentrations to improve the adaptation of plants to growing in ground for the development of a method for obtaining healthy mini-tubers of potatoes of the Yubilyar variety were studied. The influence of various nutrient medium compositions on the height, biomass, intensity of rhizogenesis, the number of internodes of improved potato micro-plants, as well as humic fertilizer from Humostim peat on the survival rate and height of potato plants when adapting it to ground conditions is analyzed. Taking into account the data obtained, as well as the cost of the studied variants of the medium, the optimal medium for growing potato micro-plants in laboratory conditions in vitro is a nutrient medium with a content of mineral components 1/3 from the norm. The use of humic preparation from peat Humostim in a concentration of 0.001% caused an acceleration of plant growth and an increase in their height, and this concentration is recommended for use.

Isolation, Identification and Characterization of Bioflocculant-Producing Bacteria from Activated Sludge of Vulindlela Wastewater Treatment Plant

The low microbial flocculant yields and efficiencies limit their industrial applications. There is a need to identify bacteria with high bioflocculant production. The aim of this study was to isolate and identify a bioflocculant-producing bacterium from activated sludge wastewater and characterise its bioflocculant activity. The identification of the isolated bacterium was performed by 16S rRNA gene sequencing analysis. The optimal medium composition (carbon and nitrogen sources, cations and inoculum size) and culture conditions (temperature, pH, shaking speed and time) were evaluated by the one-factor-at-a-time method. The morphology, functional groups, crystallinity and pyrolysis profile of the bioflocculant were analysed using scanning electron microscope (SEM), Fourier transform infrared (FTIR) and thermogravimetric (TGA) analysis. The bacterium was identified as Proteus mirabilis AB 932526.1. Its optimal medium and culture conditions were: sucrose (20 g/L), yeast extract (1.2 g/L), MnCl2 (1 g/L), pH 6, 30 °C, inoculation volume (3%), shaking speed (120 rpm) for 72 h of cultivation. SEM micrograph revealed the bioflocculant to be amorphous. FTIR analysis indicated the presence of hydroxyl, carboxyl and amino groups. The bioflocculant was completely pyrolyzed at temperatures above 800 °C. The bacterium has potential to produce bioflocculant of industrial importance.

Optimization of an Economical Medium Composition For The Coculture of Clostridium Butyricum And Bacillus Coagulans

Clostridium butyricum is a spore-forming probiotic existing in the intestines of humans and animals which can promote the enhancement of beneficial bacteria and maintain intestinal microecological balance. However, it is difficult to improve the production level of C. butyricum by conventional fermentation process. In this study, a co-fermentation process of C. butyricum DL-1 and Bacillus coagulans ZC2-1 was established to improve the viable counts and spore yield of C. butyricum, and the formula of coculture medium was optimized by flask fermentation. The results showed that the optimum medium composition is bran 10 g/L, corn steep powder 15 g/L, peptone 15 g/L, K2HPO4 1 g/L and MnSO4 0.5 g/L. Cultured stationarily in the optimal medium for 36 h, the number of viable bacteria of C. butyricum DL-1 reached 1.5×108 CFU/mL and the spore forming rate was 92.6%. The results revealed an economical and effective medium composition for the coculture of C. butyricum and B. coagulans. The co-fermentation process established in this study provides a new fermentation mode for the industrial production of other absolute anerobic bacteria.

Optimization of Fermentation Conditions for Carrageenase Production by Cellulophaga Species: A Comparative Study

Carrageenases appear in various species of marine bacteria and are widely used for the degradation of carrageenans, the commercially significant sulphated polysaccharides. The carrageenase production ability of six different Cellulophaga species was identified, with ι-carrageenase being the most abundant carrageenolytic enzyme. C. algicola was the most potent strain, followed by C. fucicola and C. geojensis, whereas C. pacifica was the least effective carrageenase producer among the studied strains. The enzyme production was maximized using the one-factor-at-a-time optimization method. The optimal incubation temperature was identified as 25 °C and the incubation time was set as 48 h for all tested species. The optimal medium composition for Cellulophaga strains was determined as 30 g/L sea salt, 1.4 g/L furcellaran, and 3 g/L yeast extract. An ultrafiltered enzyme extracted from C. algicola had the highest activity at around 40 °C. The optimal pH for enzymatic degradation was determined as 7.8, and the enzyme was fairly stable at temperatures up to 40 °C.

An Improved Protocol for Asymbiotic Seed Germination and Seedling Development of Paphiopedilum tigrinum

Paphiopedilum tigrinum is an endangered orchid with high ornamental value. However, seed germination and seedling regeneration in P. tigrinum is very difficult in vitro. Little is known about why P. tigrinum seedlings are difficult to propagate or how to improve the seed germination and seedling rates of this species. In this study, we investigated the developmental process of P. tigrinum from asymbiotic seed germination to seedling rooting by comparing it with P. appletoniantum, a much easier species for germination and seedling formation. We found that asymbiotic seed germination in P. tigrinum is limited by severe browning of the protocorm at the seed germination stage, and protocorm rooting at the differentiation stage was also proved to be difficult. The optimal medium for seed germination of P. tigrinum was a modified Harvais (mHa) medium supplemented with 0.5 mg·L−1 kinetin (Kin), 0.1 g·L−1 activated charcoal (AC) and 100 mL·L−1 coconut water (CW). At the protocorm differentiation stage, seedlings with 1–2 leaves were obtained on a 1/4 MS medium supplemented with 1.0 mg·L−1 6-benzylaminopurin (BA), 0.3 g·L−1 AC and 50–100 mL·L−1 CW after culturing for 120 day. At the seedling subculture stage, a 1/2 MS medium supplemented with 0.5–1.5 g·L−1 AC and 100 mL·L−1 CW was better for leaf and root growth of P. tigrinum. At the rooting stage, a 1/2 MS medium supplemented with 1.0 g·L−1 AC, 0.5 g·L−1 dolomite flour, 15 g·L−1 potato homogenate and 30 g·L−1 banana homogenate was most suitable for the growth and rooting of seedlings. This study has established an effective protocol for seed germination and seedling regeneration of P. tigrinum.

An Advanced Lyophilization Toward Intact Lipid Nanovesicles: Liquid-mediated Freezing With Cryoprotectant to Retain the Integrity of Lipid Nanovesicles

A novel lyophilization method using liquid-mediated freezing (LMF) with cryoprotectant (CPA) was designed to achieve intact lipid nanovesicles after lyophilization. During the freezing step, CPA prevented water supercooling, and the freezing rate was controlled by LMF. Regulating the freezing rate by various liquid media was a crucial determinant of membrane disruption, and isopropanol (freezing rate of 0.933°C/min) was the optimal medium for the LMF system. Lyophilized lipid nanovesicle using both LMF and CPA retained 92.9% of the core material and had uniform size distributions (Z-average diameter = 135.5 nm, polydispersity index = 0.074), similar to intact vesicles (112.3 nm and 0.184, respectively), after rehydration. Only lyophilized lipid nanovesicle using both LMF and CPA showed no changes in membrane fluidity and polarity. This lyophilization method can be applied to improve storage stability of lipid nanocarriers encapsulating drugs such as a mRNA vaccine, while retaining its original activity.

Production enhancement of the glycopeptide antibiotic A40926 produced by an engineered N. gerenzanensis lcu1

Objective To enhance the production of A40926 by implementing a strategy of the combination of genetically engineered strain construction and medium optimization. Results The engineered strain of Nonomuraea gerenzanensis presented an increment of 30.6 percent in A40926 production compared with that of the parent strain. Subsequently, an assembling medium, which was defined as M9 medium and mainly comprised glucose, maltodextrin, soybean meal, peptone, L-valine, and other inorganic salts, was determined as the optimal medium among the tested nine media. The optimum concentration of medium components was glucose 10 g/l, maltodextrin 37.9 g/l, soybean meal 34.5 g/l, peptone 30.0 g/l, and L-valine 4.3 g/l, respectively. The optimized medium was verified experimentally, and A40926 yield increased significantly from 257 mg/l to 332 mg/l, as compared to the non-optimized medium. The strategy brought a significant increase of A40926 yield by 65.2 percent. Conclusions The engineered mutant with the genetic attributes of the co-expression of the dbv3 and dbv20 genes and the deletion of the dbv23 gene could obviously enhance the production of A40926. In addition, the optimization of medium was an effective and essential tool for the improvement of the secondary metabolites in Actinomyces.

Establishment of callus induction system, histological evaluation and taxifolin production of Larch

Taxifolin in larch is an important natural active ingredient, which prevents and treats liver diseases and cardiovascular diseases, and has many health benefits and application values ​​such as anti-tumor, anti-virus, and anti-aging. However, the traditional extraction method is not conducive to the protection of the ecological environment, and also limits the industrial production of taxifolin from larch. Therefore, the purpose of this research is to establish a larch callus culture system and obtain taxifolin from callus. The shoots of the stem-tips of Larix olgensis were used as explants to be cultured. The light conditions, optimal medium and hormone ratio for callus induction were screened to evaluate the callus induction characteristics of larch and its influence on the accumulation of taxifolin. The results showed that: callus formation started on 28–51 days, and the best conditions for inducing callus were explored: 12 h/d light culture MS + 2,4-D 2 mg/L + 6-BA 1 mg/ L + KT 0.1 mg/L; the callus induction rate is 50%-80%; the highest condition for producing taxifolin 6-BA 1 mg/L + NAA 0.1 mg/L, reaching 0.414%. In conclusion, this study represents a suitable induction condition for larch callus culture and taxifolin accumulation.