Optimization of Antibacterial Production of Endophytic Fungi with Various Sources of C, N, and pH using The Response Surface Methodology

Endophytic fungi are a source of antioxidant compounds in nature. The low yield of extracts and active compounds produced is one of the limiting factors for using endophytic fungi as a source of natural antioxidant compounds. Modification of growth media is an alternative solution to overcome this problem. This study aimed to determine the effect of media conditions, belonging to carbon and nitrogen sources and different initial pH of fermentation, on the antioxidant through DPPH radical scavenging activity of extract of endophytic fungi Cb.D1 isolated from cinnamon plant leaves. The culture was propagated using Czapek Dox Broth basal liquid medium with agitation speed 120 rpm at room temperature for 14 days. The carbon sources used were glucose, sucrose, and soluble starch. The nitrogen sources were natrium nitrate, ammonium nitrate, and yeast extract. The initial pH conditions used were 5, 7, and 9, and ethyl acetate as the extraction solvent. The results obtained that the variation of nitrogen and carbon sources and also initial pH conditions can increase the yield of extract of endophytic fungi Cb.D1. Glucose, yeast extract, and initial pH at 9 were the best growth media to gain it. The substitution of nitrogen sources and initial pH can increase the DPPH free radical scavenging activity of endophytic fungal extract compared to substitution for carbon sources. The highest activity from the Cb.D1 endophytic fungal extract was obtained from media that contain sucrose and natrium nitrate as a carbon and nitrogen sources and an initial pH of 5. The Variation of growth media of endophytic fungi Cb.D1 affected their extract in DPPH free radical scavenging activity.

Endophytic fungi associated with Nigerian plants have recently generated significant interest in drug discovery programmes due to their immense potential to contribute to the discovery of new bioactive compounds. This study was carried out to investigate the secondary metabolites of endophytic fungi isolated from leaves of Newbouldia laevis, Ocimum gratissimum, and Carica papaya The plants were collected from Agulu, Anambra State, South-East Nigeria. Endophytic fungal isolation, fungal fermentation; and extraction of secondary metabolites were carried out using standard methods. The crude extracts were screened for antimicrobial activities using the agar well diffusion method, and were also subjected to high performance liquid chromatography (HPLC) analysis to identify their constituents. A total of five endophytic fungi was isolated, two from N. laevis (NL-L1 and NL-L2), one from O. gratissimum (SL-L1), and two from C. papaya (PPL-LAC and PPL-LE2). In the antimicrobial assay, the extracts of NL-L2, SL-L1, and PPL-LE2 displayed mild antibacterial activity against both Gram negative and Gram positive test bacteria. PPL-LAC extract showed mild activity only against S. aureus, while no antimicrobial activity was recorded for NL-L1 extract. All the endophytic fungal extracts showed no activity against the test fungi C. albicans and A. fumigatus HPLC analysis of the fungal extracts revealed the presence of ethyl 4-hydroxyphenyl acetate and ferulic acid in NL-L1; ruspolinone in NL-L2; protocatechuic acid, scytalone, and cladosporin in SL-L1; indole-3-acetic acid and indole-3-carbaldehyde in PPL-LE2; and indole-3-acetic acid in PPL-LAC. The findings of this study revealed the potentials possessed by these plants as source of endophytes that express biological active compounds. These endophytes hold key of possibilities to the discovery of novel molecules for pharmaceutical, agricultural and industrial applications.

Ethnomedicinal plants are thought to have better prospects of harboring endophytes that produce natural products with pharmacological activities. This study aimed to investigate the antiplasmodial and anticancer properties of secondary metabolites of endophytic fungi from three medicinal plants. The endophytic fungi included Lasiodiplodia theobromae isolated from Cola acuminata, Curvularia lunata Bv4 isolated from Bambusa vulgaris, and Curvularia lunata Eg7 isolated from Elaeis guineensis. The identification of the fungi was based on the internal transcribed spacer (ITS-rDNA) sequence. The fungi were subjected to solid-state fermentation and the secondary metabolites were extracted with ethyl acetate. In vitro antiplasmodial screening of extracts was performed using the SYBR green I-based fluorescence assay on the chloroquine-resistant Plasmodium falciparum strain DD2. The cytotoxicity of the extracts on human red blood cells and Jurkat (leukemia) cells was assessed using the tetrazolium-based colorimetric MTT assay. Gas chromatography-mass spectrometry (GC–MS) analysis was used to identify the constituents of the fungal extracts. The extract of L. theobromae showed the best antiplasmodial activity against chloroquine-resistant P. falciparum (IC50 = 5.4 µg/mL) and was not harmful to erythrocytes (CC50 > 100 µg/mL). All three fungal extracts showed a weak cytotoxic effect against Jukart cell lines (CC50 > 100 µg/mL). GC–MS analysis of the three endophytic fungal extracts revealed the presence of forty major bioactive compounds, including: oxalic acid, isobutyl nonyl ester, 2,4-di-tert-butylphenol, and hexadecanoic acid, among others. The endophytic fungi from the medicinal plants in this study were promising sources of bioactive compounds that could be further evaluated as novel drugs for the treatment of malaria caused by P. falciparum-resistant strains.

Using inexpensive sources is a crucial point in the production of valuable compounds such as lactic acid. In the present study, the production of lactic acid by an efficient isolate of S. thermophilus was optimized using whey as a carbon source and yeast extract as a nitrogen source. MRS culture medium with 6.5 % NaCl was used for the isolation of streptococci from industrial yogurt samples and the selected isolate was identified using the 16S rRNA gene sequencing. Evaluation of lactic acid production was done by the Randox method. Lactic acid production by the selected isolate was optimized by response surface methodology (RSM) in MRS broth regarding to different concentrations of whey and yeast extract. The isolate that produced the highest amount (8.9 g.L-1) of lactic acid within 52 h growth in MRS broth was identified as a strain of S. thermophilus by molecular identification. Optimization of concentrations of carbon and nitrogen sources resulted in the induction of lactic acid production by Streptococcus thermophilus up to 24.18 g.L-1 in the presence of 3.5 % of whey and 5 % of yeast extract as external carbon and nitrogen sources in the MRS medium, which was similar to the value predicted by RSM. Yeast extract was found to be more effective on lactic acid production than whey. Optimization of lactic acid production by low-cost substrates whey and yeast extract resulted in high induction of lactic acid production by S. thermophilus compared to some other studies that used other cost-effective substrates and/or bacteria isolated from traditional dairy products.

This work aims at investigating the influence of the initial concentrations of carbon (glucose) and organic nitrogen (yeast extract) sources on Streptomyces rimosus ATCC10970 secondary metabolism in the stirred tank bioreactors. Additionally, glucose utilisation, biomass formation, pH, redox potential and dissolved oxygen levels, and the morphological development of S. rimosus pseudomycelium were studied. Eighteen secondary metabolites were detected by mass spectrometry and identified with the use of the authentic standard, or putatively with the use of literature and database of secondary metabolites. Varied initial yeast extract concentration acted much stronger on the formation of secondary metabolites than glucose did. For example, oxytetracycline was not biosynthesised at high yeast extract concentration while the formation of three other metabolites was enhanced under these conditions. In the case of glucose its increasing initial concentration led to higher secondary metabolite levels with the exception of an unnamed angucycline. High initial yeast extract concentration also drastically changed S. rimosus pseudomycelial morphology from the pelleted to the dispersed one. Ultimately, the cultivation media with the varied initial levels of carbon and nitrogen sources were proved to have the marked effect on S. rimosus secondary metabolism and to be the simplest way to either induce or block the formation of the selected secondary metabolites.

The production of many secondary metabolites such as carotenoids is influenced by the type of carbon and nitrogen sources and C:N ratio applied in culture medium. The present study discusses the role of C:N ratio and screening of carbon and nitrogen sources using mixture analysis design in carotenoids production by Blakeslea trispora. The C:N ratios of 20, 40, and 60 with six nitrogen sources were evaluated. Results indicated that limitation of nitrogen source (C:N of 60) could improve carotenoids production. Six nitrogen and carbon sources were then screened using mixture analysis design. The most effective nitrogen and carbon sources were soybean powder and glucose, respectively. The productivity of carotenoids (983.8 ± 31.5mg/L) based on consumed nitrogen and carbon source was 189.10mg/g soybean powder and 19.66mg/g glucose. Mixture analysis design indicated single carbon and nitrogen source were more effective than a mixture of sources for carotenoids production.

Diabetes mellitus is a serious health problem with an increasing number of sufferers. In this context, an important method for controlling blood sugar levels is the derivation of α-amylase enzyme inhibitors from natural materials such as endophytic fungi. Therefore, this research aimed to analyze the potential of endophytic fungi from Tithonia diversifolia leaves as α-amylase enzyme inhibitors, secondary metabolites, as well as the character and identity of potential endophytic fungi. The stages included isolation and identification of endophytic fungi, extraction of secondary metabolites, α-amylase inhibition tests, analysis of bioactive compounds using Thin Layer Chromatography (TLC), and identification of potential endophytic fungi. The results showed that DT3J1 (67.83 µg/mL), DM3J1 (69.36 µg/mL), DT2J2 (80.42 µg/mL), and DT4J1 (88.64 µg/mL) of the eight endophytic fungi isolates reported high potential as inhibitors of the α-amylase enzyme, with IC50 values ​classified as strong. Potential fungal extracts contain bioactive compounds such as alkaloids, flavonoids, phenols, and terpenoids. Meanwhile, molecular identification showed that potential endophytic fungi consisted of Curvularia pseudointermedia, Diaporthe passifloricola, Nodulisporium verrucosum, and Muyocopron laterale species. This research also provided scientific evidence on the potential of the endophytic fungus Tithonia diversifolia as a candidate for antidiabetic drugs.

BackgroundProstate cancer is a significant global health concern, particularly among ageing male populations, with a disproportionately higher burden in sub-Saharan Africa. Conventional treatments, though effective, are costly and cause devastating side effects which limit their clinical benefits. Hence, this study evaluated the in vitro antiprostate cancer properties and secondary metabolites of dichloromethane and ethyl acetate lead extracts of Vitex doniana to explore safer and efficacious natural alternatives based on ethnomedicinal claims.MethodsPhytochemical profiling was conducted using gas chromatography-mass spectrometry (GC-MS) analysis to identify secondary metabolites in the extracts. The cytotoxic effects of the extracts were determined through the MTT assay using Vero CCL-81 cells and DU-145 cells. The expression profile of the selected genes (ar, bcl2, caspase-3, cdk1, and p53) in DU-145 cells treated with the study extracts was investigated using RT-qPCR.ResultsGC-MS analysis revealed 10 secondary metabolites in the dichloromethane extract and 27 secondary metabolites in the ethyl acetate extract of V. doniana leaves, with the majority being sesquiterpenes, diterpenoids, and phytosterols. The dichloromethane and ethyl acetate leaf extracts of V. doniana exhibited low cytotoxicity against normal mammalian epithelial cells (Vero CCL-81), with CC50 values of 1,238.85 μg/mL and 964.81 μg/mL, respectively. Besides, the ethyl acetate leaf extract of the studied plant demonstrated potent anti-prostate cancer activity against DU-145 cells, with an IC50 of 35.68 μg/mL and a high selectivity index (SI) of 27.04. Likewise, the dichloromethane leaf extract of this plant displayed cytotoxic effects (IC50: 287.01 μg/mL) and a selectivity index of 4.32. The reference drug (Doxorubicin) showed a higher toxicity against Vero CCL-81(IC50: 0.41 μg/mL) and DU-145 (IC50: 0.28 μg/mL) cells and a lower selectivity index of 1.46. The DU-145 cells treated with the studied plant extracts exhibited notable upregulation of ar and bcl2, and normalization of caspase 3, cdk1 and p53 expression.ConclusionThe studied plant extracts possess in vitro anti-prostate cancer properties and could be promising candidates for further preclinical studies aimed at developing novel botanical-based therapies for the management of prostate cancer.

Endophytes possess several phytohormones and bioactive metabolites of medicinal importance and thus, continue to generate research interest as candidates in drug discovery programmes. This study was carried out to investigate the secondary metabolites of an endophytic fungus isolated from leaves of Psidium guajava. Endophytic fungal isolation, fungal fermentation; and extraction of secondary metabolites in ethyl acetate were carried out using standard methods. The crude extract was subjected to Vacuum Liquid Chromatography (VLC) using binary combinations of Hexane:Ethyl acetate and Dichloromethane:Methanol to obtain fourteen sub-fractions designated PG55-1 to PG55-14. The fungal crude extracts and VLC sub-fractions were screened for antimicrobial activity and were also subjected to high-performance liquid chromatography-diode-array detection (HPLC-DAD) analysis for the identification of bioactive compounds. An endophytic fungus, PG55 was isolated from the leaf of Psidium guajava. The fungal secondary metabolites showed antibacterial properties, with minimum inhibitory concentrations ranging from 0.0625 – 1 mg/ml. No antifungal activity was observed. HPLC-DAD analysis of the extract suggested the presence p-hydroxybenzoic acid, pentenedioic acid and palitantin in one of the fractions of PG55. Some of these compounds are known antimicrobial agents and may be responsible for the antimicrobial activities recorded for the fungal extracts. The results of this study, suggests the many potentials possessed by Nigerian plants as hosts of endophytes that could be reservoirs for excellent sources of pharmacologically active compounds.

Endophytes are endosymbiotic microorganisms that act as reservoir of novel bioactive secondary metabolites with antimicrobial, cytotoxic and anti-cancer activities. In the present study, the extracts of 26 different endophytic fungal strains were screened for their antimicrobial and anti-inflammatory activities. The results showed a wide variety of antimicrobial activities against 12 target microorganisms including three Gram (+) bacteria, three Gram (-) bacteria, 3 yeasts, 2 dermatophytic fungi and one keratinophilic fungus. Four fungal extracts ( Aspergillus versicolor , A. awamori , A. niger and Penicillium funiculosum ) displayed a broader antibacterial spectrum and inhibited the growth of all Gram (+) and Gram (-) bacterial species. The extracts of 8 endophytic fungi inhibited the growth of the two tested dermatophytic strains ( Trichophyton mentagrophytes and T. rubrum ). Only eight fungal extracts have an inhibition activity against the keratinophilic fungal strain ( Chrysosporium tropicum ). The anti-inflammatory assay showed that the extracts of Emericella nidulans , Pleospora tarda and Penicillium funiculosum had good activities in inhibition of protein denaturation reached to 83%, 82.5% and 81.4%, respectively. Also, Emericella nidulans and Pleospora tarda recorded the maximum inhibition effect on bovine serum albumin denaturation reached to 95% and 90.7%, respectively. On the other side, Emericella nidulans showed the maximum inhibition activity (69.5%) out of all tested endophytic strains against humun red blood cells membrane stabilization assay. In conclusion some secondary metabolites of endophytic fungi have a promising potential as antimicrobial and anti-inflammatory compounds.

There are differences in the extracellular enzymes produced from species of wood-rotting fungi and their activity due to variation in nutritional conditions such as carbon sources and nitrogen sources, as well as environmental conditions such as incubation temperature and pH. In this study, to determine the methods to promote the secretion of especially cellulase with high activity among the extracellular enzymes of wood-damaging fungi, the optimal nutrient sources and incubation conditions for the production of cellulase with high activity were investigated using response surface methodology based on a broth culture from Acanthophysium sp. KMF001, a novel strain of wood-damaging fungus. The nutrient sources that were optimal for the cellulase production with high activity from Acanthophysium sp. KMF001 were cellulose as a carbon source and tryptone:yeast extract (7:3) as a nitrogen source. The optimal incubation conditions were a temperature of 30 °C and a pH of 6. The optimal concentrations of carbon and nitrogen sources were cellulose at 31.1 g∙L-1 and tryptone:yeast extract (7:3) at 15 g∙L-1, with pH of 5.9.

A specific symbiotic Bacillus sp. isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce large number of bioactive compounds. The present study was conducted to determine the effect of carbon and nitrogen sources for the production of antimicrobial substances by Bacillus sp. The yield of the crude antimicrobial substances and antimicrobial activity against the test micro-organism also differed significantly when carbon and nitrogen sources in the fermentation media were changed. The antifungal activity was significantly high in yeast extract plus fructose (46.5 ± 2.12 mm) followed by yeast extract plus maltose, beef extract plus fructose and meat infusion plus glucose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention time indicating that they produced different compounds. When the carbon source was not included in the fermentation media, the antimicrobial production was substantially reduced. The results indicate that carbon source in the fermentation media plays a vital role in the production of antifungal substances. It is concluded that yeast extract and fructose as nitrogen and carbon sources produced maximum activity, which can effectively control the blue mould caused by Penicillium expansum in apples and pears.

The global threat to treatment of infections associated with multidrug-resistant microorganisms as well as side effects associated with combination therapy calls for the urgent development of new medicines to address these challenges. Endophytic fungi are efficient bio synthesizers and offer a wide array of these reliable bioactive compounds. The present study is aimed at evaluating the effect of combined extracts of endophytic fungi isolated from the leaves of Musa paradisiaca against multidrug-resistant Staphylococcus aureus (MRSA) strains. Screening for multidrug resistance was carried out using the Kirby-Bauer disc diffusion assay followed by a preliminary assay of endophytic fungal extracts bioactivity using agar diffusion assay against selected MRSA. A synergistic study using checkerboard assay technique was performed, then extracts were subjected to quantitative evaluation of secondary metabolites using Gas Chromatography with Flame Ionization Detector. Axenic cultures of five endophytic fungi from M. paradisiaca (EMp1-Emp5) were isolated from the sampled leaves. All the endophytic fungal extracts inhibited the growth of all the multi-drug resistant Staphylococcus aureus at 0.5 mg/mL. Varying inhibition zones that were concentration-dependent were observed. The combined effects of EMp3 and EMp5 extracts at 9:1; 8:2; 4:6; 3:7; and 2:8 combination ratios produced synergistic effects against S. aureus (S18) with very low Fractional inhibitory Concentrations of 0.03:0.01, 0.01:0.01, 0.006:0.02, 0.01:0.04 and 0.01:0.1 mg/mL respectively. Several bioactive secondary metabolites such as anthocyanin, (-) epicatechin, naringenin, resveratrol, catechin, rutin, phenol were produced by the fungal endophytes. Our findings further affirm fungal endophytes associated with M. paradisiaca leaves as producers of natural antibacterial agents with synergistic potentials.

The present study focused on production of mycelial chitosan from fungal mycelium by submerged fermentation with ecologically more balanced process. Different fungal strains were screened and Absidia butleri NCIM 977 was found to produce the highest mycelial chitosan. The one-factor-at-a-time method was adopted to investigate the effect of batch time, environmental factors (i.e. initial pH and temperature) and medium components (i.e. carbon and nitrogen) on the yield of mycelial chitosan. Among these variables, the optimal condition to increase in yield of mycelial chitosan was found to be batch time (72 h), pH (5.5), temperature (30°C), carbon source (glucose) and nitrogen source (tryptone and yeast extract). Subsequently, a three-level Box– Behnken factorial design was employed combining with response surface methodology (RSM) to maximise yield of mycelial chitosan by determining optimal concentrations and investigating the interactive effects of the most significant media components (i.e. carbon and nitrogen sources). The optimum value of parameters obtained through RSM was glucose (1.58%), tryptone (1.61%) and yeast extract (1.11%). There was an increase in mycelial chitosan yield after media optimization by one-factor-at-a-time and statistical analysis from 683 mg/L to 1 g/L. Mycelial chitosan was characterized for total glucosamine content (80.68%), degree of deacetylation (DD) (79.89%), molecular weight (8.07 × 104 Da) and, viscosity (73.22 ml/g). The results of this study demonstrated that fungi are promising alternative sources of chitosan with high DD and high purity.

Medium optimization for palmarumycin C13 production in liquid culture of endophytic fungus Berkleasmium sp. Dzf12 using response surface methodology