Project name: Biologically active additives and biostimulants based on microalgae for use in the agricultural and food industries

Scientific supervisor of the project: Doctor of Biological Sciences, Professor, Academician of the National Academy of Sciences of the Republic of Kazakhstan Zayadan B.K. The

purpose of the project is to develop a technology for obtaining biologically active additives and biostimulators for use in the agricultural and food industries based on production-valuable strains of microalgae.

KNOW-HOW

Microalgae, being a source of many valuable compounds, are of great interest in the agricultural, medical, cosmetic and food industries as new and safe sources of biologically active drugs. They are characterized by immunomodulatory, antioxidant, antimetastatic action, capable of inhibiting the development of malignant tumors in humans and animals. Biologically active additives based on these organisms are natural, affordable, environmentally safe preventive biological products that do not cause adverse reactions, do not have contraindications to use.

Relevance and novelty

Microalgae contain up to 50-70% easily digestible protein (compared to 50% in meat and 15-17% in wheat), up to 30% lipids, more than 40% glycerin, up to 8-14% pigments, including carotene and a fairly high concentration of vitamins B1, B2, B3, B6, B12, E, K, D compared to other plants or animals. Food microalgae are a good source of proteins, essential amino acids, phytosterols, carbohydrates, vitamins, characterized by a unique combination of biologically active compounds, polyunsaturated fatty acids with a high content of gamma-linolenic acid and many other healthy compounds. They, having unique biochemical and physiological properties, are sources of pigments such as chlorophyll, alpha- and beta-carotene, lycopene, lutein, zeaxanthin and astaxanthin, phycocyanin, which, having antioxidant and antitumor properties, are used in the medical, food, nutraceutical and cosmetic industries.

Microalgae are well known for their diverse biological actions, such as antibacterial, antifungal, cytotoxic, immunosuppressive, antiallergic properties and antiviral activity, which also predetermine their great potential in medicine and pharmacology, when obtaining biologically active additives and biostimulants to strengthen immunity.

Practical significance

A large number of sunny days and the favorable geographical location of our Republic of Kazakhstan, the relative ease of cultivation of microalgae to obtain its biomass create prerequisites for the development of production facilities for the production of biologically active additives (dietary supplements) and biostimulants based on them. It is shown that the use of microalgae in animal husbandry as a source of protein, vitamins and other physiologically active substances increases the resistance of animals to various diseases, primarily associated with vitamin deficiency, accelerates metabolic processes, and thus contributes to an increase in the volume and quality of marketable products. In addition, it should be noted about the possibilities of creating new jobs at enterprises producing biological products, bio-feeds and dietary supplements.

Expected results

The technology of mass cultivation of productive and valuable strains of microalgae will be developed to obtain biologically active substances from producers - active strains of microalgae.

The technology of obtaining new biologically active additives and biostimulants based on production - valuable strains of microalgae will be developed and their biological properties will be studied.

The formulation and commercial forms of new biologically active additives and biostimulants based on the biomass of production- valuable microalgae strains will be developed.

Implementation object

Biologically active additives and biostimulants based on microalgae biomass containing essential amino acids, vitamins B, C, D, provitamin A, polyunsaturated fatty acids, carbohydrates, polysaccharides and having high bioavailability and great potential in the food, therapeutic industry and animal husbandry.

Prospects for implementation

The technology of obtaining dietary supplements based on microalgae has great real prospects for its introduction into production. The prospects of this project are determined not only by economic profitability, but also by the possibility of obtaining new, environmentally friendly, unique biological products based on microalgae. Therefore, the issues of industrial production of microalgae attract more and more attention of researchers and industrial firms. Thus, having high productivity, these facilities are at the same time producers of new valuable products that are widely used in the food industry (as biologically active additives) and in the agricultural sector, in particular in animal husbandry, poultry farming, fishing (biologically active feed additives). At the same time, it should be borne in mind that obtaining biologically active additives based on microalgae can be economically advantageous, since it does not require expensive equipment and nutrient media for their cultivation.

Consumers

Agro-industrial sector (poultry farms, pig breeding, livestock farms, aquaculture), food industry, pharmacology and medicine.

Competitiveness

The competitiveness of this project is determined by the above-mentioned unique properties of microalgae and the advantages of their cultivation technology (cheap nutrient medium, sun, not expensive equipment). The proposed project is original, while there are no analogues in the Republic. Microalgae biotechnology is well developed in the USA, Japan, India, Russia, China, the Czech Republic and other countries of the world. There are no enterprises in Kazakhstan for the production of biological products based on microalgae and other phototrophic microorganisms. The project has a good commercial perspective and a great potential for commercialization.

Name of the project. New antibacterial drugs for the treatment of diseases of sturgeon fish.

The purpose of the project. Investigation of the therapeutic potential of parental and engineered chimeric endolysins with increased lytic activity against Gram-negative bacteria P. fluorescens, P. putida, A. hydrophila, A. salmonicida and A. sobria. These bacteria are the main pathogens in sturgeon aquaculture, demonstrating an increased level of antibiotic resistance and causing significant morbidity and mortality in sturgeon fish. The goal provides for the following tasks: - isolation and physiological, biochemical, molecular identification of bacterial pathogens P. putida, P. fluorescens, A. hydrophila, A. salmonicida and A. sobria from infected sturgeon fish; - construction of chimeric endolysins with increased lytic activity against bacterial pathogens causing diseases of sturgeon fish in aquaculture; - characterization of antibacterial activity in vitro and in vivo of parental and engineered new chimeric endolysins.

KNOW-HOW. The cultivation of sturgeon in aquaculture is one of the important actions aimed at solving the problem of population reduction and restoring the almost interrupted life cycle of sturgeon species. However, the rapid development of aquaculture is accompanied by outbreaks of diseases caused by bacterial infection, which lead to high mortality and catastrophic economic losses in sturgeon aquaculture. The most severe bacterial diseases in sturgeon in aquaculture are infections caused by Pseudomonas and Aeromonas. Currently, due to the widespread and often uncontrolled use of antibiotics, the number of antibiotic-resistant bacteria has increased dramatically and is the main cause of morbidity and mortality. This phenomenon can not only lead to the failure of antimicrobial therapy, but also cause concerns about the safety of fish products. For this reason, new strategies to combat these drug-resistant pathogens are urgently needed.

Currently, endolysin therapy is considered a very promising alternative to the treatment of complex infections. Over the past ten years, the field of endolysin research has accelerated significantly. Some endolysins developed by various companies, including chimeric endolysins, primarily against gram-positive and gram-negative pathogens of humans and animals, are currently at the stage of preclinical and clinical trials. However, the potential of endolysins as antibacterial agents has not yet been studied in the field of aquaculture, although cultured fish, like other animals and humans, are constantly under threat of microbial attacks.

The presented project is aimed at developing new effective chimeric endolysins with increased lytic activity against gram-negative and antibiotic-resistant bacteria, which are the main cause of sturgeon diseases in aquaculture.

Relevance and novelty. The production of sturgeon in aquaculture has increased significantly in recent years due to the high demand for caviar on the world market. However, due to the widespread and often uncontrolled use of antibiotics, the number of antibiotic-resistant bacteria has increased dramatically and is the main cause of morbidity and mortality. Bacterial species, most often Aeromonas and Pseudomonas, have been registered as the main pathogens (Figure 1).

Aeromonosis (A) and pseudomonosis of sturgeon fish (B); Infected sturgeon fish from LLP "Educational and scientific complex of experimental industrial production of aquaculture" (Uralsk, West Kazakhstan region).

Figure 1 - Hemorrhagic septicemia of sturgeon fish

For this reason, new strategies to combat these drug-resistant pathogens are urgently needed. Endolysins are possible alternatives to antibiotics. Endolysins are classified according to enzymatic activity into the following three groups (Figure 2A): (i) Glycosidases, which include glucosaminidases (EC 3.2.1.52), muramidases (EC 3.2.1.17) and lytic transglycosylases (EC 4.2.2.n1), the action of these enzymes is aimed at cleavage of β-(1,4)-glycoside bonds of the sugar backbone; (ii) amidases (EC 3.5.1.28), which hydrolyze the amide bond between the sugar main chain and the peptide branches; and (iii) endopeptidases (EC 3.4.-.-), which hydrolyze the bond between two amino acids. The structure of endolysins can be either globular or modular (Figure 2).

A)    1 - Glucosaminidase; 2 - Muramidase (lysozyme) and lytic transglycosylase; 3 - L-alanine-amidase; 4-endopeptidase; GlcNAc - N-acetyl glucosamine; MurNAc - N-acetylmuramic acid. B) A diagram of the structure of gram-positive and gram-negative endolysins.

Most endolysins of gram-positive bacteria phages have a modular structure consisting of N-terminal catalytically active domains (CAD) and a C-terminal cell wall binding domain (CBD) separated by a short linker.

Thus, endolysins are able to effectively and quickly cause the death of a bacterium, regardless of its physiological state or antibiotic resistance.

Practical significance. The practical significance of the research project is to develop new effective chimeric endolysins with increased lytic activity against gram-negative and antibiotic-resistant bacteria, which are the main cause of diseases in sturgeon aquaculture.

Expected results: - Bacterial pathogens (A. hydrophila, A. salmonicida, A. caviae, A. sobria, P. putida and P. fluorescens) of sturgeon will be isolated and identified and their sensitivity to antibiotics in vitro will be tested;

- chimeric endolysins will be designed, as well as the genes of three parent and 7 chimeric endolysins will be cloned and their expression in E. coli will be optimized;

- the antibacterial activity of endolysins will be determined, and methods of treatment of infected sturgeon fish will be developed.

The object of implementation. The object of implementation is antibacterial drugs used to treat diseases of sturgeon caused by bacterial pathogens.

Prospects for implementation. Currently, endolysin therapy is considered a very promising alternative to the treatment of complex infections. Endolysins are phage-encoded enzymes that have peptidoglycan hydrolase activity and are therefore capable of destroying the bacterial cell wall, allowing the phage to leave the host cell after replication. The experimental data obtained under in vitro conditions demonstrate the impressive effectiveness of endolysins in the lysis of bacterial cells, including multidrug-resistant bacteria (Figure 3).

1 - control, 2 - endolysin (25 mcg), 3 - endolysin (50 mcg)

Figure 3 - Results of antibacterial activity of endolysin in vitro

Unlike antibiotics and bacteriophages, bacterial strains do not develop resistance to endolysins, which contributes to the widespread use of antibacterial drugs.

Consumers. The main consumers are fisheries enterprises, including industrial aquaculture complexes and fish hatcheries, as well as fish farmers.

Competitiveness (Advantages of technology) and commercialization of the project. The results of the research project are commercialized because they have practical significance in the field of ichthyopathology and are aimed at the treatment and prevention of diseases of fish grown in regulated systems. In general, the use of anti-bacterial drugs based on lysing proteins - endolysins is not limited to the use only in the treatment of fish diseases, there is also the possibility of use as a prevention of bacterial diseases and farm animals, which makes the proposed drugs universal, thereby significantly increasing competitiveness.

The aim of the project is to create a production facility for the production of microbial preparations for the restoration of disturbed and polluted soils, as well as for agriculture.

KNOW-HOW is the production of biological products and the organization of a small innovative enterprise for the production of biological products for oil-producing and agro-industrial enterprises of Kazakhstan.

Relevance and novelty - Pollution of the natural environment by oil and petroleum products today is one of the complex and multifaceted problems of ecology and environmental protection. No other pollutant can be compared by the breadth of distribution, the number of sources of pollution, the levels of chemical loads on all components of landscapes.

In Kazakhstan, oil fields occupy huge areas. When oil and petroleum products enter the soil, significant, and often irreversible changes in its chemical, physical, and microbiological properties occur, sometimes leading to the restructuring of not only the entire soil profile, but also the soil cover over large areas.

The most promising method of purification is the complex use of bioremediation, including agro- and microbiological techniques. In this case, the deficiency of microorganisms can be eliminated by introducing preparations from destructive microorganisms.

Currently, a promising direction is the creation of multicomponent plant-microbial systems that reproduce optimal natural agrophytocenoses and ensure high sustainability of agriculture. Microbial preparations developed by both foreign and Kazakhstani scientists based on microorganisms of various taxonomic groups are already used in agricultural production.

Preparations developed in other countries are not applicable in the conditions of Kazakhstan, it is necessary to take into account the soil and climatic conditions in the cleaning sites. The production of biological products based on microorganisms adapted to the soil and climatic conditions of various regions of the Republic of Kazakhstan is environmentally friendly, has no carcinogenic waste and harmful emissions into the atmosphere. The estimated volume of production per year will be determined taking into account the size of the market and the possibility of export.

Practical significance - Biological products based on destructor strains are adapted to the soil and climatic conditions of the oil-producing regions of the republic. Designed to clean soil and water from oil and petroleum products pollution in order to improve the environmental situation of the environment and reclamation of oil-contaminated lands. When using the drug, the processes of biodegradation of hydrocarbons caused effective purification of the soil from oil hydrocarbons and improvement of its ecological condition. The introduction of the drug into the oil-contaminated soil increased the efficiency of the processes of biodegradation of petroleum hydrocarbons, the loss was more than 85%.

In Kazakhstan, there is enough territory with a level of contamination of the surface layers of soils from 30 to 40 percent, deeper layers are polluted, for example, on the coastal plain oil penetrates to a depth of 45-50 cm. Under these conditions, bioremediation methods are of particular importance.

Working out the use of experimental samples of biological products in order to obtain the required level of high-quality crop yields, preserve and increase soil fertility, and protect the environment from pollution is an important stage in their development. For Kazakhstan, the study of the effectiveness of the use of biological preparations in the cultivation of cereals (winter and spring wheat, barley, oats) and fodder crops in conditions and the establishment of the most optimal combinations of the use of various types of fertilizers and biological products is relevant.

The object of implementation is Bioorta LLP (2010), Ozenmunaigas JSC (2013), Embamunaigas JSC (2018).

Prospects for implementation - The preparation for cleaning is applicable in practice for the technology of recovery of oil-contaminated soils, neutralization of smeared soils, liquid drilling waste and solid combustible oil-containing waste. Microbial preparations for the agro-industrial complex will provide import substitution in the agricultural sector related to ensuring food security of the region and the country. Organization of obtaining experimental batches of various biological products based on microorganisms for cleaning polluted soils with pollutants and for agro-industrial enterprises.

Consumers - the oil-producing and oil-refining industry of the Republic of Kazakhstan, agro-industrial enterprises, nature protection services, the private sector.

Competitiveness - The transition to a "green" economy, associated with the creation of environmentally friendly technologies and products such as microbial preparations for cleaning from pollution and improving soil fertility, are designed to help and benefit nature by improving the ecological condition of disturbed lands. Innovative, modern and safe biological product will be in demand for oil and gas sector enterprises as one of the leading sectors of the economy of the Republic of Kazakhstan.

Soils before cleaning                                                 Soils after cleaning

The scientific supervisor of the project: PhD, professor A.M. Kustubaeva

1. Name of the project (according to the media plan). "Psychophysiological markers of depression"

2. The goal of the project: determination of EEG biomarkers of depressive states for their objective diagnostics in order to reduce the burden of the socially significant disease. 

3. KNOW-HOW. The uniqueness of this research work lies in the determination of new EEG biomarkers that reliably determine the depressive state during cognitive loads. The commercialization of the research outcomes consists in the development of a computerized complex psychophysiological diagnostic tool, which allows its implementation for the survey of the broad masses of the country's population.

4. Relevance and novelty.

Today depression remains one of the most common diseases leading to disability of a wide range of the population. According to WHO estimations, depression and anxiety disorders cost the global economy approximately a trillion dollars annually. Patients with depression, accompanied by impaired cognitive functions, choose tactic of absenteeism (absence from work) or presentism (imitation of activity at work) and reduces the working capacity of the population (Greensberg, 2019). Behavioral disorders often induce criminal acts and therefore are a direct threat to society. An untreated disease leads to a high risk of suicide and decreases quality of life. Thus, in Kazakhstan, suicide mortality is 26.9 per 100,000 population. The lack of objective methods for diagnosing depressive states, along with stigma, complicates the situation. All this has determined the urgent need to develop new objective methods for diagnosing depressive states.

Obtained results revealed the most informative indicators of spontaneous brain activity during rest and features of event related potentials during the performance of cognitive tasks for executive control and decision making. These results were discussed and published at annual international conferences and showed the significance of the final results for implementation in practice. The psychometric methods developed in the current study were promptly introduced free of charge into the psychotherapeutic practice by our psychotherapists during the pandemic due to the increase in the number of depression cases (in cooperation with UNISEF, UNESCO). On the base of results of completed study, a methodological manual with a full description of the integrated approach carried out for this project was published as well as scientific articles in international journals with a non-zero impact factor. EEG biomarkers are ready for the technical development of equipment for EEG diagnostics of depression.

5. Practical relevance.`

The results of this study confirmed that precisely well-developed cognitive and emotional tasks could reveal deviations in patterns of brain signals associated with the depressive state.The selected cognitive tasks for our studies on the Attentional Network Test (ANT) and on decision making were the relevant loads for the identification of differences in brain activity of healthy and depressive subjects. The scientific and theoretical substantiation of the complex research methodology has confirmed its validity and good potential for using the obtained results for practical purposes. In general, our results showed that the deterioration of executive control in subjects with depression, which is a central regulatory link in cognitive processes, is associated with decreased brain activity in response to specific stimuli. Implementation of the adapted version of cognitive task developed according to the well-known M. Posner’s theory of executive control made it possible to determine such kind of impaired cognitive task performance in depressive subjects as a lower level of functioning of neural networks of cognitive control. The second cognitive decision-making task, aimed at the induction of emotional states and their influence on the complex cognitive decision-making process allowed to identify a deficit in brain activation for perception and feedback.

6. Expected outcomes.

The main scientific outcomes of the study can be implemented in the form of computer program or a device of domestic production for the diagnosis of depressive states and subsequent commercialization of the results in the future. The introduction into practice will allow timely diagnosis and treatment of the disease at an early stage, avoid severe consequences and suicide.

For the first time, a computer program will be developed on the basis of scientifically proved experimental complex which comprises of psychometric, cognitive tasks and psychophysiological methods of diagnostics of depressive states, already adapted and validated in the local population.

EEG biomarkers indicating the deficiency in brain activity and a decrease in brain lability in depression will be laid down as the main criteria in the diagnosis of depression. This device can be widely used in clinical practice to prevent depression and suicide.

7. The object of implementation.

A computer program or device for diagnosing an emotional states.

8. Prospects for implementation.

The results can be implemented in psychotherapeutic practice, security services, human resources services, medical services, research and educational institutions.

9. Consumers.

The data can subsequently be widely used in national and international practice: the intended consumers of the results obtained will be psychological and psychiatric services, security services, human resources services, medical services, research and educational institutions.

10. Competitiveness (Advantages of technology) and commercialization of the project.

The proposed product has a high competitiveness. At the moment there is no analogue of the proposed technology in the domestic market.