METHOD OF QUANTITATIVE ANALYSIS FOR THE ISOLATION OF SECONDARY METABOLITES OF Fusarium oxysporum FUNGUS

ABSTRACT

In recent years, the deterioration of the ecological environment on Earth has led to an increase in the number of fungi that cause various diseases in plants, animals and humans. The research that we studied showed that fungi belonging to the constum Fusarium are microorganisms that contain metabolites, which are destructive pathogens of plants and at the same time, show beneficial biological activity. The purpose of this research work was to obtain a high biomass in a certain time interval when choosing the optimal nutrient medium for the formation of the fungus Fusarium oxysporum and to analyze the amount of substances contained in this obtained biomass by the method of gravimetric analysis. In this research study, Potato Dextrose Broth (PDB): i.e. Potato extract + glucose (dextrose) was selected as a nutrient medium for the fungus Fusarium xysporum. At the same time, potato extract: 200 g of potato juice, glucose: 20 gr, 1 liter of water. (Sterilized for 15 minutes in an autoclave at 121°C.) The optimum conditions for the growth of fungus is: Temperature: 25-30°C, pH: 5.5 – 6.5. This environment is a favorable environment for mycelial growth and spore formation.  As a result of the study, it was decided that the feed composition and the environment for which it feeds is the most optimal and has been proven to give the highest biomass yield.

АННОТАЦИЯ

В последние годы ухудшение экологической обстановки на Земле привело к увеличению количества грибков, вызывающих различные заболевания у растений, животных и человека. Исследования показали, что грибы, относящиеся к констуму Fusarium, являются микроорганизмами, содержащими метаболиты, которые являются разрушительными возбудителями болезней растений и в то же время проявляют полезную биологическую активность. Целью данной исследовательской работы являлось получение высокой биомассы за определенный промежуток времени при выборе оптимальной питательной среды для формирования гриба Fusarium oxysporum и анализ количества веществ, содержащихся в этой полученной биомассе, методом гравиметрического анализа. В этом исследовании картофельный бульон декстрозы (PDB), т.е. картофельный экстракт + глюкоза (декстроза), был выбран в качестве питательной среды для гриба Fusarium xysporum. При этом картофельный экстракт: 200 г картофельного сока, глюкоза: 20 гр, 1 л воды. (Стерилизовано в течение 15 минут в автоклаве при 121°C.) Оптимальными условиями для роста грибка являются: Температура: 25-30°C, pH: 5,5 – 6,5. Эта среда является благоприятной средой для роста мицелия и образования спор.  В результате исследования было принято решение о том, что состав корма и среда, для которой он питается, являются наиболее оптимальными и, как было доказано, дают наибольший выход биомассы.

Keywords: Fusarium oxysporum secondary metabolite, microorganism, cultivated fluid, extract.

Ключевые слова: Fusarium oxysporum, вторичный метаболит, микроорганизм, культуральная жидкость, экст.

Introduction

In this study, we reviewed several scientific studies on the process of preparation of biomass from fungi of the Fusarium family and on secondary metabolites of the same fungus, and presented as an analysis several of the data studied. Of the secondary metabolites, terpenoids are well known for their diverse structures and for their large bioactive property which has a great potential in pharmaceuticals. However, natural products with lower yields are often overlooked in conventional and continuous chemical analysis. The current feature-based molecular network (FBMN) skeletons are designed to group chemical compounds that are similar to those that can quantify unknown compounds. Fusocyppen A is a chemical compound consisting of a unique 5/6/7/3/5 ring system of esterterpene synthesized by Fusarium oxysporum fusocypene synthase (FoFS) [1]. Terpenoids represent the largest segment of naturally occurring bioactive compounds, and are distinctly distinguished by their diverse structure and high range. The fungus of Fusarium oxysporum has unique and distinctive structures that exhibit its ability to synthesize di- and sesterterpenes into bifunctional terpene synthase (BFTS) [2,3]. At the same time, the introduction of a few genes in the tailor-gene group leads to the production of functional groups and a variety of novel substances in the terpenoid skeleton [4]. For example, ofiobolin F is changed to ofiobolin A. By the enzyme p450 have isolated testosterone·penoids with high anti-cancer activity. [5,6]. Similarly, the participation of the enzyme P450 gives rise to several families of norditerpenoids with a 5/5 bicyclic ring skeleton in the breakdown of VndE [7]. Advances in chromatographic and spectroscopy techniques are forcing researchers to innovate, i.e., accelerating the extraction of biologically active secondary metabolites from natural products and helping to identify substances from crude extracts by mass spectroscopy. [8,9,10]. Molecular network (FBMN) based on the in-network isomer property of compounds [11]. A method that included MS1 could further differentiate isomers within the network and retention time to classical MN [12]. We know that bioactive secondary metabolites, which are extracted from fungi with different potencies, are highly used in human health, industry, and agriculture [13,14]. Endophyte fungi form a variety of compounds that are structurally specific. These are biologically active natural products, such as alkaloids, benzoquinones, steroids, favonoids and terpenoids [15]. In recent times, it has been found that not endophytic microorganisms, but plants themselves, produce products with very high temperatures [16,17,18,19]. Polygala sinaica, a medicinal plant native to South Sinai, Egypt, belongs to the Polygalaceae family, but no chemistry studies have been conducted on the biology of this plant or its endophytic community. There is no paper in the literature about the plant Polygala sinaica or its endophytic community, based on the history of chemistry. They live alongside the lily of Fusarium oxysporum. Information on the continuous use of polygalaceae is very scarce. However, several traditional uses of Polygala spp. have been studied recently, including Polygala altomontana, Polygala caudata, Polygala favescens, Polygala glomerata, Polygala japonica, Polygala molluginifolia, Polygala sibirica, and Polygala tenuifolia. Their phytochemical studies revealed that they contain triterpene saponins, triterpenes, terpenoids, favonoids, coumarins, oligosaccharide esters, styrene benzoic compounds, benzophenones and polysaccharides. Medical studies on polygala sinaica have found that its neuroprotective, ischemic, antidepressant, analgesic, antitumor, and enzyme inhibitor properties have significant anti-inflammatory effects [20].

Materials and methods

Formation of fungal biomass. To determine the extraction efficiency by quantitative analysis for Fusarium oxysporum fungus. The fungus Fusarium oxysporum, which causes fatal damage to plants in the Kashkadarya region, was isolated from the soil, mainly infected with the spores of the same fungus, and it was multiplied in the culture liquid of its optimal nutrient medium, fusarium stamps were obtained, rainy and the amount of secondary metabolites in this fungus was determined and this process was carried out in the laboratory of the Department of Microbiology of Karshi State University in 7 days in 120 liters of cultivated nutrient liquid at a temperature of 25-30 degrees Celsius cultivated. The composition of the enriched liquid nutrient medium for Fusarium oxysporum is as follows:(relative to one liter). Potato Dextrose Broth (PDB): Contained potato extract + glucose (dextrose) It is a favorable environment for the growth of mycelium and the formation of spores. Potato extract: 200 g of potato juice, glucose: 20 gr, 1 liter of water. (Sterilized for 15 minutes in an autoclave at 121°C.) The optimum conditions for the growth of the fungus are: Temperature: 25-30°C, pH: 5.5 – 6.5 and produces a full biomass in a week.

Figure 1. Extraction of Fusarium oxysporum fungus and separation process by column chromatography

Technology of working with vacuum rotor equipment. In order to initially isolate the secondary metabolites, the fungus of Fusarium oxysporum was isolated from the liquid feed medium by centrifuge instrument, its wet mass was 25g per 1l (grown in a total of 120l of feed medium) and hexane extraction was performed in order to wash away the oils from the obtained biomass. The washed compounds in the hexane were isolated using a separator separator and the fungal biomass extraction process was repeated at least 6-10 times. The na'muna was collected in a special container and when the extract was dried with vacuum rotor equipment, it yielded an extraction sum of about 12 gr. In the next step, the remaining biomass was chloroform extracted and the substance was isolated again using a separator voron. The process of chloroform extraction of the fungal biomass was repeated at least 8-10 times. Na'muna was collected in a special container and when the extract was dried with vacuum rotor equipment, an extraction sum of 140.4 g was obtained. Then, when the remaining biomass was thoroughly dried in a place where the light of the tail was not touched, the dry biomass was 300 g and this biomass was extracted alcohol. Because methyl alcohol is expensive and toxic, ethyl alcohol has also been used for extracting purposes. The extraction process of the lily biomass was repeated at least 10-15 times. The na'muna was collected in a special container and when the extract was dried with vacuum rotor equipment, an extraction sum of 129.6 g was obtained. When the remaining biomass was extracted with water and dried the extract with vacuum rotor equipment, an extraction sum of 44.5g was formed. The total weight of the extraction sum was 216.5 grams (Fig. 1). The highest activity of the Fusarium oxysporum fungus synthesis of terpenoids from secondary metabolites is desociated during chloroform or ethyl acetate extract. Adequate biomass must be in place, including when temperature, humidity, and oxygen levels are sufficient, the fungus synthesizes its secondary metabolites at a high rate. These metabolites help the protective mechanisms of the fungus as well as enhance its biochemical activity. It was found that the process could be optimized using quantitative analyses. For example, ways to change suitable conditions (temperature, type of solvent or method) or save energy and time during the production process to increase extraction efficiency. These calculations are used to evaluate the effectiveness of the process and the amount of substance separated.

Results and discussion

To obtain maximum litter biomass using the above methods, Potato Dextrose Broth (PDB): Potato Dextrose Broth (PDB) containing potato extract + glucose (dextrose) as well as the temperature of this nutrient medium 25-30°C, pH 5.5 – 6.5 cultivation process was achieved and considered as optimal conditions. Next-stage litter from the litter biomass was extracted using four different solvents and pumped using a vacuum rotor instrument, during which extraction mass of varying values was obtained. (Table 1).

Table 1.

Results of a quantitative analysis of sum of Fusarium oxysporum fungus extracted in various solvents

From the results in this table, it can be seen that more biomass can be obtained from the cultivated fungus for one week, and that the extraction sum amount is higher in chloroform and ethyl alcohol extraction using this biomass.

Conclusion.

To sum up, it can be said that, according to the obtained results, the extractive efficiency was shown in the grown biomass of Fusarium fungus in a week of the highest indicators. Furthermore, even when the secondary metabolites were quantitatively analyzed, it was proved that the amount of secondary metabolites in Fusarium oxyporum fungal extract grown for 7 days was significantly higher. The highest activity of the Fusarium oxysporum fungus synthesis of terpenoids from secondary metabolites is desociated during chloroform or ethyl acetate extract.

References:

1. Kangjie Lv Yuyang Duan Xiaoying Li Xinye Wang Cuiping Xing Keying Lan1 Bin Zhu ·Guoliang Zhu Yuyang Qiu Songwei Li Tom Hsiang Lixin Zhang1 Lan Jiang Xueting Liu Identifying sesterterpenoids via feature-based molecular networking and small-scale fermentation.
2. Toyomasu T, Tsukahara M, Kaneko A, Niida R, Mitsuhashi W, Dairi T, Kato N, Sassa T (2007) Fusicoccins are biosynthesized by anunusual chimera diterpene synthase in fungi. Proc Natl Acad Sci USA 104(9):3084–3088. https://doi.org/10.1073/pnas.06084 26104
3. Mitsuhashi T, Abe I (2018) Chimeric terpene synthases possessing both terpene cyclization and prenyltransfer activities. Chem Bio-Chem19(11):1106–1114. https://doi.org/1002/cbic.2011800120
4. Keller NP (2019) Fungal secondary metabolism: regulation, function and drug discovery. Nat Rev Microbiol 17(3):167–180. https://doi.org/10.1038/s41579-018-0121-1
5. Chiba R, Minami A, Gomi K, Oikawa H (2013) Identification of ophiobolin F synthase by a genome mining approach: a sesterter-pene synthase from Aspergillus clavatus. Org Lett 15(3):594–597. https://doi.org/10.1021/ol303408a
6. Narita K, Chiba R, Minami A, Kodama M, Fujii I, Gomi K, OikawaH (2016) Multiple oxidative modifcations in the ophiobolin biosynthesis: P450 oxidationsfound in genome mining. Org Lett18(9):1980–1983. https://doi.org/10.1021/acs.orglett.6b00552
7. Jiang L, Lv K, Zhu G, Lin Z, Zhang X, Xing C, Yang H, Zhang W, Wang Z, Liu C, Qu X, Hsiang T, Zhang L, Liu X (2022a) Norditerpenoids biosynthesized by variediene synthase-associated P450 machinery along with modifcations by the host cell Aspergillus oryzae. Synth Syst Biotechnol 7(4):1142–1147. https://doi.org/10.1016/j.synbio.2022.08.002
8. Pye CR, Bertin MJ, Lokey RS, Gerwick WH, Linington RG (2017) Retrospective analysis of natural products provides insights for future discovery trends. Proc Natl Acad Sci U S A114(22):5601–5606. https://doi.org/10.1073/pnas.1614680114
9. Zhang C, Hong K (2020) Production of terpenoids by synthetic biology approaches. Front Bioeng Biotechnol 8:347. https://doi.org/10.3389/fbioe.2020.00347
10. Gong D-Y, Chen X-Y, Guo S-X, Wang B-C, Li B (2021) Recent advances and new insights in biosynthesis of dendrobine and sesquiterpenes. Appl Microbiol Biotechnol 105(18):6597–6606. https://doi.org/10.1007/s00253-021-11534-1
11. Nothias L-F, Petras D, Schmid R, Dührkop K, Rainer J, Sarvepalli A, Protsyuk I, Ernst M, Tsugawa H, Fleischauer M (2020) Feature-based molecular networking in the GNPS analysis environment. Nat Methods 17(9):905–908. https://doi.org/10.1038/s41592-020-0933-6
12. Afoullouss S, Balsam A, Allcock AL, Thomas OP (2022) Optimization of LC-MS2 data acquisition parameters for molecular networking applied to marine natural products. Metabolites 12(3):245. https://doi.org/10.3390/metabo12030245
13. El-Baz AF, El-Enshasy HA, Shetaia YM, Mahrous H, Othman NZ, Yousef AE (2018) Semi-industrial scale production of a new yeast with probiotic traits, Cryptococcus sp. YMHS, Isolated from the Red Sea. Probiotics Antimicrobial Proteins 10(1):77–88. https://doi.org/10.1007/s12602-017-9291-9
14. Adel A, El-Baz A, Shetaia Y, Sorour NM (2021) Biosynthesis of polyunsaturated fatty acids by two newly cold-adapted Egyptian marine yeast. 3 Biotech 11(11):461
15. Hashim AM, Alatawi A, Altaf FM, Qari SH, Elhady ME, Osman GH, Abouseadaa HH (2021) Phylogenetic relationships and DNA barcoding of nine endangered medicinal plant species endemic to Saint Katherine protectorate. Saudi Journal of Biological Sciences 28(3):1919–1930. https://doi.org/10.1016/j.sjbs.2020.12.043
16. El-Sayed ASA, Khalaf SA, Azaz HA, Hussein HA, El-Moslamy SH, Sitohy B, El-Baz AF (2021) Production, bioprocess optimization and anticancer activity of Camptothecin from Aspergillus terreus and Aspergillus favus, endophytes of Ficus elastica. Process Biochem 107:59–73. https://doi.org/10.1016/j.procbio.2021.05.007
17. El-Sayed ASA, Zayed RA, El-Baz AF, Ismaeil WM (2022) Bioprocesses optimization and anticancer activity of camptothecin from Aspergillus favus, an endophyte of in vitro cultured Astragalus fruticosus. Mol Biol Rep 49(6):4349–4364
18. Abdel-Fatah SS, Al-Sherbiny GM, Khalaf M, Baz AFE, Al-Sayed ASA, El-Batal AI (2022) Boosting the anticancer activity of aspergillus International Microbiology favus "endophyte of jojoba" taxol via conjugation with gold nanoparticles mediated by γ-irradiation. Appl Biochem Biotechnol 194(8):3558–3581. https://doi.org/10.1007/s12010-022-03906-8
19. Abdelhamid SA, Abo Elsoud MM, El-Baz AF, Nofal AM, El-Banna HY (2024) Optimisation of indole acetic acid production by Neopestalotiopsis aotearoa endophyte isolated from Thymus vulgaris and its impact on seed germination of Ocimum basilicum. BMC Biotechnol 24(1):46. https://doi.org/10.1186/s12896-024-00872-3
20. Lacaille-Dubois MA, Delaude C, Mitaine-Ofer AC (2020) A review on the phytopharmacological studies of the genus Polygala. J Ethnopharmacol 249:112417. https://doi.org/10.1016/j.jep.2019.112417