Scientific results:
We have created a full-cycle research and production complex based on the development of environmentally-friendly hi-tech biotechnology for the manufacture of high-quality product that can be used in pharmacology and food industry with the use of cultivated cells and organs of higher plants and microalgae. The complex, due to the combination of its parameters, is a unique platform for import substitution for the industrial production of biopreparatons with specified properties regardless of the conditions of the environment. The characteristics of the existing technological equipment allow to scale the laboratory designs to create an environmentally friendly and economically viable production scheme with capabilities from developing producer strains to their cultivation on an industrial scale with the possibility of subsequent assessment of the quality and the functional activity of the resulting product.
In particular, we produced new steadily growing in vitro callus and suspension cultures of medicinal plants of the genera Fabaceae, Alhagi persarum and Sutherlandia frutescens, for whose extracts we demonstrated antibacterial action with respect to Staphylococcus aureus. For a culture of A. persarum cells we have developed a technology for cultivation in a laboratory 20-liter bubble column bioreactor. For the earlier produced culture of Ajuga turkestanica cells grown in flasks and bioreactors, we have demonstrated the presence of turkesterone, ecdysterone, and other ecdysteroids, as well as phenylethanoids.
A methodology has been developed for the apparatus-based cultivation of suspension cell cultures of the medicinal plants Polyscias filicifolia and P. fruticosa, and we demonstrated the presence of five triterpene glycosides with oleanolic acid as the aglycone in the cell biomass. In Panax japonicus and Polyscias fruticosа cell cultures grown in flasks and bubble column bioreactors synthesize a rich mixture of triterpene glycosides with a considerable concentration of ginsenoside R0 (13.8 mg per gram dry weight).
The Laboratory has developed and optimized a scheme for scaling and cultivating suspension cultures of Dioscorea deltoidea, Taxus wallichiana, P. japonicus and P. Filicifolia cells in the fill and draw mode in bubble column bioreactors up to industrial volumes in a 20 l–75 l–630 l lineup with a yield of 9.00-12.00 grams of dry weight per liter of the medium and stable accumulation of target biologically active substances. The elicitation of a T. wallichiana culture by methyl jasmonate led to the appearance of taxoids of the С13-ОН group, to which paclitaxel belongs.
We have produced and are currently maintaining adventitious roots of 11 species of plants – potential producers of biologically active substances.
It has been shown that strains of Dunaliella salina IPPAS D-294 amd Scenedesmus rubescens IPPAS D-292 microalgae show high growth characteristics, while the IPPAS D-292 strain, apart from that, also shows high productivity. Both strains are able to store large volumes of starch and are able to accumulate carotenoids. The IPPAS D-292 stain can additionally switch to lipid accumulation at the later stages of their growth.
We have optimized the conditions and selected the cultivation regimes for microalgae of the Chlorella sorokiniana IPPAS C-1 strain in laboratory bioreactors at different values of illumination, CO2 concentration and temperature. The highest growth indicators (2.78 grams of dry weight per liter) were attained at 30–32 ºС and 1.5–3% CO2 over 9 days of cultivation. The overall harvest was 194.6 gDW. In the case of the optimization of semicontinuous and combined cultivation in bioreactors, the overall harvest amounted to 163.62 gDW over 16 days of cultivation. We have also successfully cultivated the C. vulgaris IPPAS C-2 and P. kessleri IPPAS C-9 strains in the periodical mode in the phytobioreactor PG-5.
We have developed and optimized a process for scaling cultivation of the selected strains producing microalgae in bioreactors in a lineup of bioreacttors ranging from 0,25 l to 100 l developed at the Institute of Plant Physiology of the Russian Academy of Sciences. Using these facilities, a set of experiments was conducted to cultivate Chlorella sorokiniana IPPAS С-1, C. vulgaris IPPAS C-2 and Parachlorella kessleri IPPAS C-9 in various conditions. In total, over a ten-year cycle of periodical cultivation with a transition from 250 ml to 70 l, an increase of the biomass by a factor of 168 was observed.
We have developed methods of processing and storing produced microalgae raw materials for various purposes. Storing the paste, which was produced as a result of biomass concentration, at -20 °С was found to be most efficient for long-term storage. Storing the algae suspension under reduced lighting is more economically feasible while preserving the maximum viability of cells.
The results produced within the mega-grant project can be viewed as import substitution technologies ready for implementation in industrial enterprises for the production of functional food products, feeds and feed supplements, ingredients for cosmetics and pharmaceuticals.
Implemented results of research:
On the basis of the results of the conducted work we have compiled preliminary recommendations for the production and use of pharmaceutical substances in the form of water suspensions of Dioscorea deltoidea, Tribulus terrestris and Panax spp. Biomassp produced using biotechnological methods for preventing and correcting lipid and carbohydrate exchange in humans. Our researchers have produced Projects of pilot industrial regulations for producing cell biomass of the selected strains using a deep method in bioreactors. The data collected during the implementation of the project will substantiate the subsequent implementation of results of basic research in the pharmaceutical industry for the development of innovative pharmeceuticals for preventing and treatment of pathologist related to lipid and carbohydrate exchange disorders (atherosclerosis and diabetes), on the basis of Dioscorea deltoidea, Tribulus terrestris and Panax japonicus cell culture.
The results of the conducted R&D are used by the «Chemistry and Biological Association «VITA company» and «BioPharmos Group».
Education and career development:
Two education programs have been developed for the training of highly qualified researchers and engineering personnel in the following topics: «Biobanking of cell cultures, cultures of organs of higher plants and microalgae» (in two parts), «Cell biology and foundations of industrial phytobiotechnology» (in two parts). The programs were tested at a branch of of the Department of Plant Physiology of the Faculty of Biology of the Moscow State University.
Collaborations:
- Department of Plant Physiology and Department of Bioengineering of the Faculty of Biology of Moscow State University, V.M. Gorbatov Federal Research Center for Food Systems of the Russian Academy of Sciences, Saint Petersburg State Chemical and Pharmaceutical University of the Ministry of Health of Russia, «BIOPHARMOS» Ltd («BioPharmos Group» company), «Chemistry and Biological Association «VITA company» (Russia).
The results of this collaboration are:
1 An analysis of the macro- and microelement composition of the biomass produced in bioreactors. The research relied on mass spectrometry to study Panax ginseng and Polyscias filicifolia biomass using inductively coupled plasma mass spectrometry (ICP-MS). It has been determined that the presence of both cell cultures of all vitally important macro- and microelements in biomass of the suspension culture of both species. The collected data allow to characterize Panax japonicus and Polyscias fruticosa cell biomass as an environmentally friendly raw material and recommend its use in functional food products and fodder.
2 A demonstration of the high antioxidant activity of samples of cell culture biomass of 7 species, Polyscias fruticosa P. filicifoliа, P. vietnamensis P. japonicus, Ajuga turkestanica, Dioscorea deltoidei and Tribulus terrestris, cultivated in the research and production complex of the Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences in bioreactors. The highest values of antioxidant activity (AOA) for samples of cell cultures were registered in cultures of Panax vietnamensis, Polyscias filicifolia and Tribulus terrestris. The АОА values for these cultures are comparable to or exceed those in cell cultures already used in the commercial production of cosmetics. A high АОА allows to speak of high prospects of these cultures for their use in the food and cosmetics industries.
3 A biological test system based on D. magna crustaceans for the fast screening of the toxicity of cell cultures of higher plants produced using the biotechnological method.
4 In collaboration with Moscow State University we are continuing work to create innovative polymer materials based on chitosan and biomass of suspension cultures of plant cells. We have synthesized a hybrid polymer polycationic material based on chirosan with a molecular mass of 600 kDa and biomass of Ajuga turkestanica and Polyscias fruticosa cells. It has been demonstrated that the new material with the addition of biomass of A. turkestanica cells have a higher surface area in comparison with chitosan and almost completely adsorbs cells of model microalgae in one hour.
5 In collaboration with the Saint Petersburg State Chemical and Pharmaceutical University of the Ministry of Health of Russia we have conducted work to determine acute oral toxicity and to assess the antihypoxic action of water suspensions of Dioscorea deltoidea, Tribulus terrestris and Panax japonicus biomass samples produced in bioreactors. According to the results of our research, the samples were assigned «class 5 toxicity or not classified». Also for the researched substances (water-based suspensions) we have demonstrated antihypoxic action, which is most pronounced in Tribulus terrestris and Dioscorea deltoidea.
6 A research of the hypoglycemic properties of phytopreparations based on ginseng, Dioscorea and Tribulus. Our research demonstrates the efficiency of the use phytopreparations Panax, Dioscorea and Tribulus in type 2 diabetes in rats, based on decreasing indicators of blood glucose, urine glucose, daily diuresis and total serum cholesterol. The decrease in the indicators of carbohydrate and lipid exchange are comparable to the effect of the reference preparation (metformin). Water extracts of cell cultures of Dioscorea, Tribulus and Panax also demonstrated hypocholesterolemic action in animals with obesity. Following the results of the conducted research, the most efficient phytopreparation showing hypocholesterolemic and hypoglycemic action was an extract of Dioscorea deltoidea cell cultures.
- Sunchon National University (South Korea): we have developed and are currently testing a system for the long-term storage of a genetic database of wild and rare medicinal plants.
- Chungbuk National University (South Korea): production of a culture of adventitious roots for a number of species of medicinal plant, including Digitalis lanata, Polyscias filicifolia, Polyscias scutellaria, Polyscias balfouriana, Maackia amurensis. We are currently conducting a comprehensive assessment of the produced cultures as potential producers of biologically active substances characteristic of these species.
