Optimization of the Production Parameters of a Novel Exopolysaccharide Chrysosporine from Endophytic Chrysosporium sp. KTL2, and Evaluation of its Antioxidative and Prebiotic Potentialities

Abstract

Chrysosporine, an exopolysaccharide, was produced by Chrysosporium articulatum KTL2, an endophyte of Ocimum sanctum L. It comprises arabinose, glucose, galactose, and fucose in a molar ratio of approximately 1:5:2:2 with a Mw ~ 2.95 × 10⁵ Da. One Variable at A Time, and Response Surface Quadratic Methodology analysed the maximum production of Chrysosporine at optimised laboratory conditions- 14 days of fermentation at 28 °C in fresh potato dextrose broth in a 250 mL conical flask with 50 mL medium provided with (g L⁻¹) glucose, 12; tryptone concentration, 1.75; NaCl, 0.05; with medium pH 6.5. Further addition of epigenetic modulator- Histone Methyltransferase Specific Probe BRD4770 promotes a 1.65-fold increase in Chrysosporine production. It is a potent radical scavenger and significantly uplifts some selected enzymatic antioxidant markers in peritoneal macrophage cells. Chrysosporine-fed rats have elevated counts of beneficial microflora of Lactobacillus sp. and less occurrence of putrefying Clostridium perfringens. Present outcomes suggest that Chrysosporine has a pharmaceutical interest as an exogenous antioxidant with prebiotic effectivities.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-024-01419-y.

Introduction

Bioactive compounds from natural sources, be they plants or microbes, are always of high pharmaceutical value and are widely accepted over synthetic ones. In recent times, scientists have been inclined towards microbial metabolites rather than phyto-molecules for industrial and medicinal applicability due to their easy production, optimisation, and harvesting procedures along with cosmopolitan utility, economic feasibility, and constancy from geographical and seasonal variation [27, 39]. The microbial metabolome represents a wide array of secondary metabolites, but the most prevalent and biologically efficient ones are the intra or extracellular polymers, commonly called EPS or IPS (Intracellular polysaccharides). Especially novel polymers from microbes that reside inside the host cells or tissues, called endophytes, have caught attention in the recent past [50].

Endophytic microbes, particularly fungi, are the symbiotic associates of green plants and are major entities contributing to the survival of their respective hosts in biotic or abiotic stressed situations. They are proven to be co-evolved with their hosts and share critically valued genes through horizontal and vertical routes, thus representing hotspots for generating high-end metabolites with unique industrial utility [56]. That’s why scientists have been attracted to the multidomain functionality of these polymers from endophytes i.e., antioxidation, anti-tumor, anti-microbial action, immunomodulation, and so on [20]. Out of different structural varieties—fructans, polygalactans, gellan, alginate, kefiran; β-glucans, are proved to be the most lucrative ones from the immunobiological, oncological, and prebiotic perspective [11]. Nonetheless, the utilisation of exopolysaccharide-producing novel fungal endophytes is limited, necessitating specific attention.

Other than exopolysaccharides, cell surface and capsular polysaccharides are also prevalent in nature, but EPSs are favoured for their elementary extraction, purification, non-toxic nature, and smooth administration into the targeted organisms. The commercial application of microbial secondary metabolites is dependent on structural characterisation as well as process escalation. Exopolysaccharide synthesis by endophytic fungi can be influenced by a range of fermentation conditions, such as composition and initial pH of the medium, as well as time and temperature of incubation.

In the twenty-first century, the problems of malignancy, immune deficiency, allergic responses, gut-related disorders, and hepatic anomalies are becoming frequent due to sedentary lifestyles, stress, unbalanced diets, and rapid environmental changes. There is a lack of proper medicines as well as treatment pathways to tackle these diseases. Research and development on novel EPS with health-promoting functionalities have recently boosted popularity in food, beverage, and pharmaceuticals due to the overall rise in health-related consciousness (Joulak et al., 2020).

In this context, endophytic fungi Chrysosporium articulatum KTL2 isolated from Ocimum sanctum L. (Krishna Tulsi), a common Indian ethnomedicinal plant, has been evaluated for the production of exopolysaccharide. β-glucan rich novel heteropolysaccharide, named Chrysosporine, is optimised through three different techniques, i.e., OVAT, RSM, and addition of histone methyltransferase specific probe- epigenetic modulator. Chrysosporine is immensely bioactive and exhibits in vitro antioxidative action, and prebiotic efficacy.

Materials and Methods

Identification of Chrysosporine-Synthesising Endophytic Fungi

Chrysosporium articulatum KTL2 was isolated as a sterile endophytic fungus from leaf tissues of Ocimum sanctum L., collected from Vidyasagar University Campus. The explants were cut into small pieces, surface sterilised and plated onto an isolation medium (water agar). Isolate lacks any characteristic sexual or asexual bodies and produces a profusely branched white-coloured tuft of mycelia. The identity of KTL2 was revealed based on the conserved ribosomal DNA sequences (ITS segment). Universal primers ITS-1 and ITS-4 were utilised for amplification following the standard protocols [58]. The sequencing of the amplified gene products was performed. The sequenced product was run through nucleotide BLAST. The different sequences of closely related associates and distant relatives of the isolate KTL2 were selected, and a phylogenetic tree was drawn [55]. The obtained nucleotide sequence of KTL2 was submitted to GenBank with an Accession Number of OQ372916.

Synthesis of Chrysosporine and Optimization of Fermentation Parameters

Chrysosporium sp. KTL2 was incubated in 250 mL conical flasks having 50 mL of the basal medium- Potato Dextrose Broth (PDB) with incubation at 26 °C and at an agitation of 120 rpm for an incubation period of 14 days. One Variable At a Time (OVAT) method was followed to fix the mandatory growth requirements. The organism was inoculated in varying growth temperatures, times, medium pH, carbon (i.e., fructose, glucose, galactose, maltose, rhamnose, raffinose, starch), and nitrogen (ammonium nitrate, ammonium sulphate, ammonium chloride, glycine, peptone, urea, tryptone, yeast extract) sources, salt/metal ion conditions (CaCl₂, MnCl₂, MgCl₂, FeCl₃, KCl, NaCl, HgCl₂ and phosphates- 0.05 g L⁻¹ K₂HPO₄, KH₂PO₄ and NaH₂PO₄). Different concentrations of each component were also assessed. During the whole process, medium conditions were kept constant, as reported in its earlier experimental step [34]. The necessity of oxygen was also checked by inoculating KTL2 at different inoculum volumes- 25 mL, 50 mL, 75 mL, and 100 mL in a 250 mL conical flask. The need for O₂ in the Erlenmeyer flask was implicitly predicted based on different interacting components- the surface area of inoculum, the volume of headspace/ air space, volume of inoculum, overall volume, and also the depth of inoculum [59]. The results of OVAT experiments were further analysed through Response Surface Quadratic Methodology- Box Behnken Design. Four impactful factors were selected and each one was evaluated at three levels i.e., + 1, 0, -1. An empirical model was established by the BBD approach that involved multiple regression techniques and that provided a polynomial equation- Y = β₀ + Σβ₁x₁ + Σβ_ijx_ixj + Σβ_iix_i². A different degree of Chrysosporine production was obtained using this model only. Here, the predicted response for Chrysosporine production is designated as Y. The independent factors are x_i, and x_j, whereas the intercept is β₀ and the linear coefficient is β_i. β_ii and β_ij are the quadratic and interaction coefficients, respectively. After this combined approach of OVAT-RSM, a third level of optimisation was made using epigenetic modulator histone methyl transferase-specific probe BRD4770. It was added at three varying concentrations- 10, 50, and 100 nano-molar to check at which concentration the maximum Chrysosporine production is achieved [40].

Estimation and Characterisation of Chrysosporine

The fermentation broth of KTL2 was sieved through filter paper, and the filtered product was again centrifuged (Reimi, India) at 10,000 g. The supernatant was taken, and the pellet was placed in a hot air oven to dry. The dried pellet was weighed. The supernatant was evaporated, and absolute chilled ethanol was added to the concentrated supernatant in a 5:1 ratio and stored at 4 °C overnight. Finally, extracellular polymeric substances were precipitated and were subjected to dialysed in a cellulose membrane at a molecular weight cut of- 10,000 against distilled water overnight. The amount of sugar in the exopolysaccharide was calculated using the phenol sulfuric acid method, and glucose was used as the standard [8].

Extracted exo-polysaccharides were estimated for their glucan profile following the methodology described in the Yeast β-Glucan Assay Kit (Megazyme Institute Wicklow, Ireland). For FT-IR analysis, Chrysosporine was prepared with KBr (SRL, India) to form pellets, and the pellets were checked at the frequency of 400–4000 cm⁻¹. Gel permeation chromatography was adopted to detect the molecular weight of Chrysosporine. Sepharose 6B column was prepared and distilled water served as eluent (4 mL min⁻¹). Dextrans with different concentrations (40 kDa, 70 kDa, 200 kDa) were used as the standard of sugar [33]. The composition of monosaccharides was analysed through chromatographic techniques [50]. 100 mg of dried Chrysosporine was taken and 1 mL of GC grade methanol, 20 uL of internal standard ribitol, and 20 uL of DL norleucine were mixed together. This solution was placed in a water bath for 15 min at a temperature of 70 °C. After heating the solution, it was centrifuged at 10,000 rpm for 5 min. After centrifugation, the obtained supernatant was dried and 20 uL of methoxyamine HCL was added. Again, the acidic mixture was incubated for 120 min at 37 °C. At last, 40 uL of derivatising agent Tri methyl siloxane (TMS) was added and finally, 1 µL of the derivatized Chrysosporine was injected into the GC–MS. A DB-5 ultra-inert column was used in the system and the column was 30 m × 0.25 mm long. The sample was injected in a split mode of 25:1. Both the temperature of the inlet and MS transfer line were set at 230 °C and 250 °C respectively. The column was set to a steady flow mode of 1.3 mL min⁻¹ and an average velocity was set at 39 cm sec⁻¹. Helium was set as the carrier gas, and ZB-1701 was taken as the guard column. The isothermal mode was adopted. There was a temperature hold at 70 °C for 1 min, then the temperature was increased up to 190 °C with a per minute increase of 20 °C. At 190 °C, there was a hold for 1 min, and again at a speed of 20 °C per minute, the temperature was raised to 230 °C. At this point, a 1-min hold was provided, and again, a ramp of 2.5 °C per minute was set, which helped to reach 265 °C. Again, a 1-min hold was given, followed by a ramp of 10 °C per minute, and the temperature reached 295 °C. The mass spectrometric scan range was set between 40–650 amu with a 100 ion counts detection sensitivity and the detector was set at positive ion mode.

Evaluation of In Vitro Free Radical Scavenging Potentialities of Chrysosporine

DPPH (diphenyl picryl hydrazyl), ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), H₂O₂, and FRAP (Ferric ion reducing antioxidant power) were utilised to evaluate the free radical scavengability of Chrysosporine. Ascorbic acid and Ethylenediamine tetra-acetic acid were taken as the internal standards.

Chrysosporine was tested against DPPH following the methodologies described by Blois (1958). 200 μL of methanolic DPPH solution (0.1 mM) was added to different strengths- 0, 10, 25, 50, and 100 μg mL⁻¹ of Chrysosporine and mixed well. The solution was incubated in the dark for not more than half an hour at room temperature and then spectrophotometric analysis (517 nm) was performed. Only methanolic DPPH was taken as blank. The radical scavenging potential was presented in percentages adopting the formula = [1 − (Abs S₁/Abs S₂)] × 100. Abs S₁ = absorbance_517nm of Chrysosporine; Abs S₂ = absorbance_517nm of control. Ascorbic acid served as a positive control.

Aqueous ABTS (2 mM) was mixed with potassium persulfate (2.45 mM) and kept overnight in the dark. Next, the absorbance (at 734 nm) of the solution is set at 0.750 ± 0.025 by diluting using sodium phosphate buffer (0.1 M, pH 7.4). Different strengths-12.5–100 µg mL⁻¹ of Chrysosporine was used and the ABTS scavengability was detected through the equation- (1 − (A₁/A₂) × 100) where A₁, and A₂ were the initial and remaining concentrations of the ABTS in the presence or absence of Chrysosporine respectively [14].

Different strengths- 20 µg mL⁻¹–100 µg mL⁻¹ of Chrysosporine was first mixed with phosphate buffer (0.1 M, pH 7.4) and then mixed with H₂O₂ (43 mM) to detect the H₂O₂ scavenging assay. The absorbance of the individual solution was measured at 230 nm, and the scavenging percentage was calculated following the Eq. (1 − A₁/A₂) × 100 here, A₁ and A₂ represent the absorbance of the control and Chrysosporine [10].

Different strengths of Chrysosporine (25–400 µg mL⁻¹) were added to sodium phosphate buffer (0.2 M, pH 6.6), 1% potassium ferricyanide [K₃Fe(CN)₆] and kept at 50 °C for 20 min. Next, trichloroacetic acid (10%), FeCl₃ (0.1%) were added and absorbance was taken at 700 nm. Higher optical densities mean higher Ferric Reducing Antioxidant Power Assay (FRAP) and vice versa [14].

Detection of Total Antioxidant Capacity (TAC)

The phosphomolybdenum method, as described by Prieto et al. (1999), was followed for this test. Sulfuric acid (0.6 M), sodium sulfate (28 mM), and ammonium molybdate (4 mM) were mixed together to prepare the reagent. 3 mL of this functional reagent was taken and 0.3 mL of Chrysosporine was added further. Different sets were prepared using different strengths of Chrysosporine. The solution was then kept at 95 °C for one hour and thirty minutes and the final absorbance was measured at a wavelength of 695 nm against blank. Various concentrations of ascorbic acid were evaluated to prepare a standard curve and the total antioxidant capacity is calculated in the units of gm equivalent of ascorbic acid.

In Vitro Antioxidative Potentialities of Chrysosporine

Peritoneal macrophage cells of Wistar rats were utilised to detect the effect of Chrysosporine on modifying both the selected enzymatic antioxidant markers and lipid peroxidation. At first, the macrophages were divided into seven sets, of which set-1 did not receive any treatment, whereas the other sets received Chrysosporine with different concentrations starting from 1 to 1000 µg mL⁻¹. All seven sets also receive macrophage cells with an approximate count of 4 × 10⁸. Lipid peroxidation, glutathione peroxidase, and superoxide dismutase activity, levels of oxidised and reduced glutathione were measured following the instructions written on the Kits MAK085, CGP1, CS1000, CS0260, and MAK364, respectively, purchased from Sigma Aldrich, USA.

Maintenance of Lab Animals

The in vivo experiments were performed using common lab animals. Male Wistar rats with an initial weight of 110–130 g were chosen for the Chrysosporine-related experiment and were provided seven days of pre-experiment time for acclimatisation. Healthy rats were fed a standard approved diet in the form of pellets, supplied water in ad libitum and the animals were kept in a polypropylene cage. The cages were kept in a congenial environment with 12 h of light and dark cycle following the institutional ethical guidelines (VU/IAEC/CPCSEA/12/6/2022).

Determination of Prebiotic Ability

To evaluate the prebiotic activities of Chrysosporine, rats were first divided into five groups, each with five animals. Group I did not receive any Chrysosporine treatment and groups II-V received different doses (100–500 mg/kg of body weight) of Chrysosporine for 30 days. Then fecal material was collected from each treated animal in a clean tissue paper placed at the bottom of the animal cage. The procedures described by Mahapatra and Banerjee [34] were followed to analyse the population of intestinal bacterial flora. Specific media were used to isolate and enumerate aerobic and anaerobic bacteria, Lactobacillus sp., Escherichia coli, and Clostridium perfringens throughout one month from 0 to 30 days till the end of the procedure.

Statistical Analysis

Each experimental procedure was performed three times. The obtained results were represented as means ± standard errors (SE). Prism GraphPad software version 9.2.0 (332) was used for the data analysis. BBD and RSM-related data were analysed using a statistical software- Minitab (version 20.2).

Results

Identification of the Potent Chrysosporine-Producing Strain

KTL2 was isolated as endophytic fungi from healthy leaf samples of Ocimum sanctum L. and was identified as Chrysosporium articulatum (OQ372916) depending on their phylogenetic information. The isolate was white in colour and sterile in nature (Fig. 1a). The ITS sequence (594 bp of DNA) of this isolate was allied with different species of Chrysosporium but it was similar to C. articulatum, confirmed through BLAST analysis (by comparing with NCBI database) (Fig. 1b). To construct the phylogenetic tree, the bootstrap data was utilised and the prepared phylogram assures the identity of the isolate as C. articulatum (a high bootstrap value of 100). Five hundred bootstrap repetitions were used to construct the cladogram and sixteen isolates were grouped into five sections to construct the phylogram. Different members of five taxa of the isolate were placed in the phylogenetic tree: Chrysosporium articulatum, C. reticulisporus, C. tropicum, and A. verrucosus. Mucor circinelloides was used to prepare the tree as an outgroup. KP147968 Aphanoascus verrucosus along with three other taxa SOIL-TUITS12, SOIL-TUITS11, and MASR CT, were placed in the first group. All these form group 1. Group- 2 and Group 3 have four and three strains of C. tropicum, and C. reticulisporus, respectively. Group 4 consists of four strains of C. articulatum accompanied by the endophytic fungal isolate KTL2. The overall branch length was reported to be 0.2745659871. EPS (Chrysosporine) producing isolate KTL2 completely matches (94.74% query cover) with C. articulatum GQ376097 and, hence, can be concluded as C. articulatum KTL2.

Fig. 1.

a Plate morphology of KTL2, b Phylogenetic relationship of KTL2 with other related and unrelated fungi

Composition of Chrysosporine

Chilled ethanol was added to the culture broth of KTL2 and it led to the precipitation of Chrysosporine. The crude Chrysosporine was subjected to purification through dialysis, and further purification was done through gel filtration chromatography using a Sepharose-6B column. Only one fraction with high carbohydrate concentration was obtained between 31 and 35 tubes (Fig. 2a). Colorimetric tests confirm that the protein is not present in this fraction. The glucan profile of the Chrysosporine was composed of β-glucan—41.13 ± 2.01 g 100 g⁻¹ of Chrysosporine and total glucan, α-glucan were 48.14 ± 2.13 g 100 g⁻¹, and 07.23 ± 0.12 g 100 g⁻¹ of Chrysosporine, respectively.

Fig. 2.

Structural characterisation of Chrysosporine a Estimation of molecular weight, b FT-IR analysis, and c GC–MS chromatogram of the Chrysosporine showing different monosaccharides- arabinose, glucose, galactose, fucose, d H¹ NMR spectrum of Chrysosporine

Analysis of FT-IR spectra of Chrysosporine revealed the presence of strong vibrations at selected wavelengths of 3375.78, 2851.37, 1490.57, 125.10, 875.61, 467.69 which indicates some basic characteristics (i.e., asymmetric stretching of C-H, O–H, and C-O) and purity of the carbohydrate moiety (Fig. 2b).

Arabinose, glucose, galactose, and fucose were the monomer present in Chrysosporine with a molar ratio of 1:5:2:2. Chrysosporine attains a molecular weight of ~ 2.95 × 10⁵ Da (Fig. 2c). Each repeating unit of Chrysosporine has five glucose units, two galactose and fucose units, and one unit of arabinose. So, in total, 169 repeating units are there in Chrysosporine.

The ¹H NMR spectrum of Chrysosporine had protons within chemical shifts (δH) between 3.40 and 4.0 frequencies, which confirms that the compound possesses –CH and –CH₂ functional groups, that resonate with the oxygen atom of the –OH group. The occurrence of anomeric protons at δH 3.44, 3.59, and 3.83 (Fig. 2d) regions assures that it is of polysaccharide nature [53].

Optimisation of Chrysosporine Production from Chrysosporium sp. KTL2

Chrysosporine production was first optimised using the OVAT technique, and the organism synthesised maximum Chrysosporine at a glucose and tryptone concentration of 12 and 1.75 g L⁻¹; sodium chloride- 0.05 g L⁻¹; with medium pH 6.5. (Tables 1, 2). Utilising the four most influential parameters i.e.,- glucose concentration- 12 g L⁻¹, tryptone concentration- 1.75 g L⁻¹, sodium chloride- 0.05 g L⁻¹, fermentation temperature- 28 °C, incubation days- 14, medium pH- 6.5 Chrysosporine synthesis was further statistically validated using the RSM technique by adopting a three-level BBD (Box-Behnken Design). Here, optimum Chrysosporine synthesis was reported at major central points (five in this case) (Supplementary Table 1) following the equation Y_{Chrysosporine} = 2.1479- 1.2340x₁ -1.4512x_2—1.1194x₂ –1.2144x₃ –1.1759x₄ –1.1029x₁x₂ –1.4579x₁x₃ + 1.1347x₁x₄ –1.1645x₂x₃ + 1.1810x₂x₄– 1.1037x₃x₄ –1.4568x₁²– 1.0627x₂² –1.0751x₃² –1.1475x₄²; x₁, x₂, x₃, and x₄ are the coded ones for the concentration of glucose, and tryptone, initial pH of the medium, and time needed for the fermentation process, respectively, suggested by the statistical model. A regression analysis performed by the statistical system assesses the goodness of fit of both the RSM data and the outcomes of the experiment (Supplementary Table 2). The proper significance of the model is demonstrated by the high value of F- 3969.18. Both the values related to the observational and exceptional cases are strongly correlated with each other and it is evident by the adjusted R² Adj of 0.991. This value also confirms that the response of the model could be accurately predicted by the second-order polynomial equation.

Table 1.

Effect of different physical conditions and chemical supplements, on biomass and Chrysosporine production by endophytic fungi Chrysosporium sp. KTL2 in submerged fermentation conditions

Parameters tested	Effectors	The concentration of the effectors (g L−1)	Biomass (g L−1)	Chrysosporine (g L−1)
Fermentation time (h)	10 days	–	0.6 ± 0.002a	1.42 ± 0.01a
	12 days	–	1.06 ± 0.03b	2.9 ± 0.03b
	14 days	–	1.85 ± 0.04c	4.94 ± 0.06c
	16 days	–	1.96 ± 0.04c	3.33 ± 0.04d
Fermentation temperature (°C)	22	–	1.93 ± 0.07a	4.01 ± 0.06a
	24	–	1.99 ± 0.05a	4.91 ± 0.07b
	26	–	2.41 ± 0.04b	4.98 ± 0.08c
	28	–	2.46 ± 0.04b	4.05 ± 0.03a
Initial medium pH	6	–	2.09 ± 0.01a	4.01 ± 0.02a
	6.5	–	2.42 ± 0.02b	5.71 ± 0.09b
	7	–	2.48 ± 0.04b	4.97 ± 0.04c
	7.5	–	2.51 ± 0.01c	3.93 ± 0.07a
Additional carbon sources	Fructose	–	2.50 ± 0.01a	5.77 ± 0.02a
	Glucose	–	2.65 ± 0.04b	6.03 ± 0.04b
	Maltose	–	2.49 ± 0.04a	5.32 ± 0.08c
	Starch	–	2.38 ± 0.01c	4.90 ± 0.02d
	Rhamnose	–	2.37 ± 0.02c	5.34 ± 0.03c
	Raffinose	–	2.17 ± 0.01d	5.71 ± 0.04a
	Glycerol	–	2.18 ± 0.01d	5.78 ± 0.05a
Additional nitrogen sources	Peptone	0.4	2.21 ± 0.04a	6.51 ± 0.09a
	(NH4)2SO4	0.4	2.39 ± 0.01b	6.94 ± 0.04b
	NH4NO3	0.4	2.19 ± 0.01a	6.41 ± 0.05a
	Urea	0.4	2.13 ± 0.03a	6.08 ± 0.05c
	NH4Cl	0.4	2.31 ± 0.04b	6.90 ± 0.03b
	Tryptone	0.4	2.69 ± 0.04c	7.00 ± 0.09b
	Yeast Extract	0.4	2.59 ± 0.02d	6.89 ± 0.04b
Glucose concentration	Glucose	8	2.61 ± 0.02a	6.9 ± 0.01a
		10	2.67 ± 0.03a	7.28 ± 0.09b
		12	2.84 ± 0.02b	7.87 ± 0.08c
		14	2.83 ± 0.02b	7.39 ± 0.05b
Tryptone concentration	Tryptone extract	1.5	2.69 ± 0.03a	7.85 ± 0.04a
		1.75	2.83 ± 0.04b	8.08 ± 0.01b
		2	2.87 ± 0.04b	7.89 ± 0.01a
		2.25	2.89 ± 0.05b	7.82 ± 0.01a
Different metal ions	MgCl2	0.1	2.19 ± 0.01a	7.52 ± 0.01a
	FeCl3	0.1	2.07 ± 0.02b	7.01 ± 0.04b
	KCl	0.1	2.29 ± 0.02c	7.55 ± 0.01a
	NaCl	0.1	2.98 ± 0.04d	8.18 ± 0.04c
	NaH2PO4	0.1	2.58 ± 0.07e	7.51 ± 0.02a
	K2HPO4	0.1	2.40 ± 0.01f	7.92 ± 0.01d
	KH2PO4	0.1	2.01 ± 0.01b	7.95 ± 0.01d

One-way ANOVA (Tukey’s Multiple Comparison tests) was performed to check the potential statistical differences in the case of the biomass (g L⁻¹) and Chrysosporine production (g L⁻¹) in different fermentation conditions (incubation time, temperature, the concentration of sugar, and nitrogen sources, etc.). There were valid statistical differences in most of the cases (P < 0.05), the different letters a, b, and c indicate a significant difference, and the same letter at two positions indicates no statistical differences

Table 2.

Effect of medium volume, headspace volume, and medium depth on the dissolved oxygen level in the fermentation medium and their effect on Chrysosporine production

Medium volume (mL)	The total volume of the flask (mL)	Headspace volume (mL)	Medium depth (cm)	Surface area (cm)	Biomass (g L−1)	Chrysosporine (g L−1)
25	320	295	1	2.5	03.08 ± 0.01a	7.45 ± 0.03a
50	320	270	1.5	2.58	3.82 ± 0.04b	8.91 ± 0.03b
75	320	245	2	2.53	3.92 ± 0.01b	6.78 ± 0.07c
100	320	220	2.5	2.41	3.96 ± 0.04b	7.04 ± 0.02d

One-way ANOVA (Tukey’s Multiple Comparison tests) was performed to check the potential statistical differences between the data (column-wise) of biomass (g L⁻¹), and Chrysosporine production (g L⁻¹) in different medium volumes (mL). There was a valid statistical difference in most cases indicated by different letters (P < 0.05)

The model suitability is also confirmed by the Adeq precision value of 81.45. The model’s fitness is appreciable and is assured by the lack of fit F-value of 1.37 and P-value of (P > 0.0001). It also indicates that the lack of fit F value has no critical effect on the value of model error. The P value for lack of fit was 0.8967 (> 0.05), and the P value for likelihood was larger than or equal to 0.05, indicating that the investigative results were highly precise and consistent. The statistical outputs also suggest that impacts of glucose conc., tryptone conc., medium pH, and fermentation time were linear, quadratic, and meaningful (P > 0.0001). The overall statistical data confirms that the model is acceptable and can be applied in automated bioreactors/fermenters for the pharmaceutical and commercial synthesis of Chrysosporine. The model predicted fermentation parameters- glucose- 11.679 g L⁻¹, tryptone- 1.6 g L⁻¹, pH- 6.47, fermentation time- 13.5 days were tested in laboratory condition in a 2.5 L bioreactor (Eyela, Japan) and a Chrysosporine production of 10.32 ± 0.03 g L⁻¹ was obtained which resembles the model predicted value of 10.35 ± 0.01 g L⁻¹. Further elevation of Chrysosporine production (17.07 ± 0.10 g L⁻¹) was obtained after adding 100 nm of BRD4770 (Fig. 3).

Fig. 3.

Optimisation of Chrysosporine production using three different techniques

Chrysosporine Exhibits Potent In Vitro Antioxidative Potentialities

Chrysosporine exhibited an IC₅₀ value of 37.11 ± 1.08, 42.21 ± 1.71, 51.03 ± 2.01, 62.10 ± 3.54 µg mL⁻¹ against DPPH, ABTS, FRAP, and H₂O₂ free radical generator, respectively. Ascorbic acid was used as a standard and had an IC₅₀ value of 09.12 ± 0.69, 17.88 ± 1.25, 20.31 ± 1.65, 28.11 ± 2.01 µg mL⁻¹ towards DPPH, ABTS, FRAP, and H₂O₂ free radical generators. TAC was found to be 38.78 ± 2.05 µg mL⁻¹, which indicates that 1 mg of Chrysosporine and 38.78 ± 22.05 µg mL⁻¹ of ascorbic acid have equal levels of antioxidative activity.

Levels of GSSG (oxidized glutathione) and MDA (Malondialdehyde) in Chrysosporine-treated peritoneal macrophage cells reduced in a dose-based manner (Fig. 4a, b). The events of lipid peroxidation were maximally inhibited after treatment with 500 µg mL⁻¹ of Chrysosporine. Also, a reduction in the amount of oxidised glutathione level was detected at the exact dosage (Fig. 4c). The activity of GPx and SOD was supreme when they were treated with Chrysosporine (Fig. 4d, e). A similar type of treatment also improves the levels of reduced glutathione concerning control.

Fig. 4.

Levels of a Oxidized glutathione (GSSG) contents, b malondialdehyde (MDA) contents, c glutathione peroxidase activity, d superoxide dismutase (SOD) activity, e reduced glutathione (GSH) contents in control and different concentrations of Chrysosporine treated peritoneal macrophages. Values in the graphs are the means ± standard error (SE) of the three replicates. One-way ANOVA (Tukey’s Multiple Comparison tests) was performed to check the potential statistical differences in the case of the control and different Chrysosporine-treated groups. The different letters indicate a significant difference (P < 0.05), and the same letter at two positions indicates no valid statistical differences

Chrysosporine Influences the Gut Microbial Flora

Total anaerobes, total aerobes, Lactobacillus sp., Clostridium perfringens, Enterobacteria, and E. coli, were counted in log10 CFU/g wet faecal samples from both the treated and untreated rats. Initially, the difference in bacterial load between Chrysosporine-treated and untreated rats after 1–7 days was ignorable; however, after two weeks, minor changes were reflected and a notable change was recorded after 30 days of treatment. The untreated rats had more or less constant microbial flora. After a month of treatment, Chrysosporine-administered rats had an impactful rise in the Lactobacillus sp. population whereas the population of C. perfringens has been diminished. The animals that received 300 mg/kg body weight Chrysosporine resulted in a prominent decrease of C. perfringens, and, on the other hand, Lactobacillus, total anaerobes increased (Table 3). Throughout the trial, the number of E. coli, total aerobes, and other Enterobacteria remained constant in all the groups.

Table 3.

Enumeration of different taxa of bacterial populations of faecal matter of the Chrysosporine treated (after 30 days) rats

Treatment groups (Chrysosporine-mg/kg body weight)	Bacterial enumeration (log10 CFU/g wet faecal sample)
	Total aerobic microbes	Total anaerobic microbes	Lactobacillus sp.	Escherichia coli	Other enterobacteria	Clostridium perfringens
Control	6.15 ± 0.003	11.01 ± 0.003 a	4.24 ± 0.011a	5.12 ± 0.010	5.17 ± 0.020	6.36 ± 0.028a
100	6.26 ± 0.001	11.35 ± 0.009 b	4.79 ± 0.014b	5.17 ± 0.023	5.19 ± 0.019	5.78 ± 0.024b
200	6.33 ± 0.007	11.61 ± 0.004 c	5.27 ± 0.009c	5.19 ± 0.029	5.27 ± 0.029	5.11 ± 0.019c
300	6.47 ± 0.002	12.26 ± 0.010 d	5.97 ± 0.007d	5.15 ± 0.021	5.26 ± 0.022	4.87 ± 0.021d
500	6.59 ± 0.003	12.89 ± 0.011 e	4.87 ± 0.004e	5.26 ± 0.022	5.24 ± 0.021	4.49 ± 0.016e

Values on the table are the means ± Standard error (SE) of the three replicates. One-way ANOVA (Tukey’s Multiple Comparison tests) was performed to check the potential statistical differences in the case of the control and different Chrysosporine-treated groups. The different letters a, b, c, d, and e indicates significant difference (P < 0.05), and the same letter at two positions indicate no valid statistical differences

Discussions

Polysaccharides are highly hydrated polymers of monosaccharides joined by glycosidic linkages. Its composition, as well as internal bonding, varies widely. Recently, growing interest has been in producing exopolysaccharides (EPS) by microbes for pharmaceutical, food, and agricultural exploitation. Endophytic microbes, in particular, have garnered attention as they can produce a wide range of bioactive compounds such as antiviral, anticancer, antioxidant, anti-proliferative, immunomodulator, antibiotics, and antidiabetic agents [48].

Microorganisms from several taxonomic groups are known to produce exopolysaccharides with intriguing physical and chemical characteristics, including high viscosity, excellent gelation qualities, and a synergistic impact when combined with other bio-active polymers. From the perspective of monosaccharide contents, both homo, and heteropolysaccharides are made up of identical or varied sugars, respectively, e.g., xanthan gum, mannans, phosphomannans, glucans, and glucomannans are equally bioactive. Exopolysaccharides from endophytes hold tremendous promise in pharmaceuticals and are potential alternatives to synthetic chemotherapeutic agents. Polysaccharides from microbes residing in varied habitats draw strict attention and are preferred over plant or animal sources from both agricultural and medicinal perspectives [4, 63]. High production, easy alteration of growth parameters, economic feasibility, and linear/branched structure have made them even more industry-friendly [46, 54]. In the present investigation, Chrysosporine, synthesised from Chrysosporium articulatum KTL2, has been optimised for its production and is found to possess bioactivities- in vitro antioxidative action, and prebiotic utilities.

Chrysosporine accelerates the antioxidant defense in the macrophage cells of male Wistar rats and prevents the generation of peroxy radicals, thus reducing the MDA concentration in a dose-dependent manner. Healthy cellular activities regularly generate free radicals which are eventually wiped out by cellular defense but inappropriate diet faults the process. That’s why exogenous antioxidants are required to prevent the synthesis and buildup of various types of aldehydes which are generated through lipid peroxidation. Both intracellular and intercellular autooxidation generate superoxide anion, singlet oxygen, hydroxyl radicals, and hydrogen peroxides which are routinely removed by superoxide dismutase. As macrophages produce a large pool of these harmful ions during phagocytosis it is immensely needed to improve SOD activity for a strong immunity. Chrysosporine enhances SOD activity and another prime component of antioxidant defense is a glutathione-dependent biocatalyst, glutathione peroxidase, which reduces the oxidised glutathione, and transforms peroxides into alcohols. This is a must-need recovery for the healthy redox balance of the cell. Chrysosporine treatment increases the glutathione peroxidase activity and causes an upliftment of the levels of reduced glutathione. So, there is a reduced incidence of ‘glutathione disulfide formation’ and ‘decreased GSSG levels’ which reports the critical oxidative damage in cells/tissues. So, the investigated EPS-Chrysosporine has a significant antioxidant action. Recent findings intensify that the exopolysaccharides exhibit effective free radical scavenging and antioxidant activities in vitro, and their biological activities are allied to their structures, such as both chemical and monosaccharide composition, molecular weight, and process for optimisation [66]. Microbial EPSs are favoured in fields like food, pharmaceutical, agriculture, and environmental protection due to their cost and time-efficient production, easy extraction/separation procedures, and safe biological applications [25]. In vitro antioxidative potentialities of EPS of Sporidiobolus pararoseus PFY‑Z1, Rhodococcus qingshengii are nearly equal to the antioxidative activities of Chrysosporine [28, 30].

The structural elucidation of EPSs is highly needed as it is the fundamental basis for sketching the structure–function relationship. Chrysosporine is a β-glucan-rich EPS, and β-glucans have been enlisted as one of the vital bioactive polymers from the plant, macrofungal, and microbial sources with pharmaceutical utilities (Maity et al., 2021, [21, 50]). The molecular weight (Mw) of EPS can classify its functional attributes, i.e., biological potentialities. The GPC analysis reveals the Mw of Chrysosporine as 295 kDa, which was contemporary to the findings on Rhodotorula mucilaginosa sp. GUMS16 (84 kD), R. mucilaginosa CICC (7.13 × 10⁶ Da) and ultimately concludes that polysaccharides with low molecular weight are effective antioxidants [16, 18, 60]. Mathivanan et al. [37] and Asianezhad et al. [2] reported vital antioxidant activities of exopolysaccharide produced by Lysinibacillus fusiformis, and Bacillus coagulans respectively.

EPSs are required in bulk quantities for pharmaceutical applicability. That’s why multiple optimisation strategies are adopted to accelerate Chrysosporine output. The growth and production pattern of secondary metabolites from microbial cells varies abruptly from situation to situation based on available resources and given fermentation conditions [41]. That’s why production parameters must be optimised for EPS-related studies. Different approaches, i.e., genetic engineering, epigenetic modulation, and fermentation conditions are combined for this purpose. EPS production from other strains (KX041, and ATCC8014, YM2) of Lactobacillus plantarum increased to threefold to sixteen folds in post-optimised conditions.

The fermentation time is the basic need to ensure an efficient production strategy and maximum Chrysosporine production was seen after seven days of incubation in the current study. The fermentation medium's pH affects the cell's redox potential, significantly altering the enzyme cascades involved in metabolite synthesis and causing the metabolic channels to flip on and off [28]. Chrysosporine synthesis by C. articulatum KTL 2 was evaluated at medium pH values ranging from 4 to 8. Maximum production of biomass and Chrysosporine (8.91 ± 0.03 g L⁻¹) was detected at pH 6.5. Alkaline pH (pH 7.2–8.0) reduced biomass synthesis and Chrysosporine formation. EPS production from the submerged culture of Ganoderma cantharelloideum and Lactobacillus plantarum SP8 formation was optimised through the OVAT technique and partly coincided with the present investigational outcome [31].

Carbon sources, i.e., carbohydrates in most cases, are the principal source of energy and nutrients for most fungi, and they have been linked to both production of secondary metabolites and organisms’ growth [12]. A single organism reacts differently to various sugars regarding growth and production, whereas different species prefer various carbon sources. In the case of Chrysosporium sp. KTL2 glucose had a more substantial impact on biomass and Chrysosporine production (6.03 ± 0.04 g L⁻¹) than the other sugars (fructose, maltose, rhamnose, and raffinose) tested. Also, glucose was preferred due to its cost efficiency. EPS production from Lactobacillus plantarum SP8, and L. delbrueckii subsp. Bulgaricus also exhibited the highest EPS production when glucose was used [44, 65]. Organic nitrogen influences the output of Chrysosporine, and tryptone exhibits comparatively better production from C. articulatum KTL2 (7.00 ± 0.09 g L⁻¹). Different glucose concentrations were applied to evaluate their impact on Chrysosporine production, and 12 g L⁻¹ was detected to be the most suitable one from the Chrysosporine production perspective (7.87 ± 0.08 g L⁻¹). C. articulatum KTL2 produced the maximum quantities of Chrysosporine (8.08 ± 0.01 g L⁻¹) and biomass when 1.75 g L⁻¹ tryptone was added to the culture medium. Several other investigators working on different fungal products have revealed similar effects of extra nitrogen sources [1]. After a certain level of increase in both these parameters, the production of Chrysosporine decreased for both the glucose and tryptone.

Microbial metabolism is influenced by both mineral salts and metal ions [43]. NaCl had the most significant impact on biomass formation and Chrysosporine (8.18 ± 0.04 g L⁻¹) production of all the salts examined. Although the particular process is unknown, it is conceivable that this ionic salt increased cell membrane permeability, affecting Chrysosporine production [3, 22]. The effect of various supplemental phosphate sources on Chrysosporine production was studied, and it was reported that phosphates do not impact its production.

There was an interaction of available oxygen between the fermentation media and the headspace portion of the Erlenmeyer flask [59]. Because the mechanism constantly favoured balance, oxygen was given to the liquid phase. A cotton stopper in a culture flask did not prevent air penetration but limited the flow of free air from the external environment into the Erlenmeyer flask. As a result, the headspace air volume may influence the oxygen concentration in a liquid medium. In addition, the surface area of a liquid can be used to influence oxygen transport. The increase in the surface area generates oxygen diffusion in liquid media. Medium depth is another important factor influencing the amount of oxygen in liquids. As medium depth grew, oxygen diffusion commonly decreased.

We used four different volumes (25 mL, 50 mL, 75 mL, and 100 mL) in 250 mL Erlenmeyer flasks to analyse the impact of oxygen on Chrysosporine production. When evaluating the surface area, headspace volume, and medium depth, it is plausible to assume that lower medium volume correlates to more dissolved oxygen and vice-versa. The results showed that when grown in 50 mL of medium, C. articulatum KTL2 produced the most Chrysosporine (8.91 ± 0.03 g L⁻¹). Growth and Chrysosporine production decreased rapidly as medium volume dropped below or over 50 mL. OVAT analysis was used to choose various physical circumstances and beneficial nutrients for optimum growth of C. articulatum KTL2.

Besides these, adding an epigenetic modulator into the optimised culture medium remarkably accelerates the Chrysosporine production. Actually, several gene clusters are present in endophytic fungi responsible for synthesizing secondary metabolites, but most of them remain silent generally [47]. These silent genes, majorly, are primarily responsible for producing valuable compounds or can induce the rapid production of any selected component. A particular type of chemical, called epigenetic modulators, can switch on these cryptic genes and stimulate the endophytic fungi for improved production of novel compounds [52]. Varieties of epigenetic modulators are there i.e., histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid- SAHA), sodium butyrate, valproic acid, and DNA methyltransferase (DNMT) inhibitors (5-aza-20-deoxycytidine 5-azacytidine, procainamide, procaine, and hydralazine), that can improvise the organisms’ biosynthetic potentials. Here, we have evaluated the effects of Histone Methyl Transferase Inhibitor (HMTI) specific probe BRD4770 on the acceleration of Chrysosporine production from KTL2. These modifiers can potently reduce cellular levels of H3K9me2 and H3K9me3 and parallelly increase the levels of H3K9me1 by modifying histones and altering DNA methylation [38].

Chrysosporine-fed rats influence the gut microbial flora and maintain a healthy gut environment, thus acting as a prebiotic. Some particular types of metabolites, i.e., organic acids, bacteriocins, and exopolysaccharides, can influence the health of our gut, the second brain, by affecting the intestinal microflora. Exopolysaccharides or long-chain sugar polymers (e.g.- fructans, starch, galactooligosaccharides, glucose-derived and non-carbohydrate oligosaccharides) possess all square effectivity in human health and act as a hypo-cholesteric agent, anti-inflammatory, immunomodulator, and antioxidant agent. EPSs are ‘dietary prebiotics,’ which are ‘specifically fermented ingredients that result in specific changes in the composition and activity of the gastrointestinal microbiota,’ ultimately beneficiating host health (Davani-Davari et al., 2019). Colonic and intestinal microflora ferments non-digestible dietary products and synthesises SFAs (short-chain fatty acids-lactic acid, butyric acid, propionic acid) which improve the absorption of necessary ions, i.e.,—calcium, magnesium, and iron, and aid vitamin production. These influence the large and small bowels by controlling the growth and differentiation of epithelial cells. The levels of propionic acid impact the activity of TH2 helper cells, dendritic cells, and macrophages (Davani-Davari et al., 2019). Also, the distant organ sites and the immune system are modulated by the SFAs. They reduce the gut pH and influence the gut microbial communities, affecting the acid-sensitive species (Bacteroides) [9, 57]. On the other hand, butyrate, necessitated for intestinal cell growth, is produced at enhanced rates by Firmicutes. Prebiotics act as a potent source of metabolic energy for the gut microflora and can improve their composition and functional attributes. Galacto-oligosaccharides enhance the population of Bifidobacteria and Lactobacilli and suppress the growth of Bacteroidetes, Firmicutes, and Enterobacteria [32]. Also, sometimes complex dietary polymers induce the cross-feeding mechanism where one organism breaks down the complex polymer, and the by-products serve as the source of several other gut-beneficial microorganisms. The breakdown of resistant starch by Ruminococcus bromii is a classic example of that fact [64]. The presently studied exopolysaccharide Chrysosporine is majorly constituted with Galactose and improves gut health. Endophytic Fusarium sp. A14 (isolate of Fritillaria unibracteata) produces heteropolysaccharide with a composition of mannose, rhamnose, glucose, galactose, xylose and arabinose with a molecular weight of ∼ 0.5 × 10⁴ Da and exhibits in vitro antioxidative potentiality, and antiproliferation effect towards hepatocellular carcinoma HepG2 cells [42]. Another endophytic fungus Penicillium javanicum (an isolate of Millettia speciosa Champ), produces acidic heteropolysaccharide with a composition of arabinose, galactose, glucose, mannose (molecular weight of 37.8 kDa) and is a potent anti-inflammatory agent by modifying tumor necrosis factor-α, interleukin-6 and nitric oxide release [24]. Neopestalotiopsis clavispora AUMC15969 isolated from avocado fruits produces optimum exopolysaccharide of 7.86 g/L utilising lactose and peptone as the carbon and nitrogen source and exhibited strong antioxidative action with an IC₅₀ value of 3.89 mg/mL [23]. Bacterial endophytes are also dominant contributors in the field of pharmaceutically valuable exopolysaccharide production [6, 61]. Talaromyces purpureogenus is reported to produce bioactive exopolysaccharide with strong antioxidative, wound healing activity and the heteropolysaccharide is composed of mannose, ribose, glucose, and galactose [17].

Previous studies have demonstrated that an increase in the population of Lactobacillus and a decrease in the count of C. perfringens characterise healthy intestinal physiological conditions [36]. Beneficial microbes act as the first line of chemical defence against the colonisation of pathogenic microorganisms [13]. Based on the obtained coliform (E. coli) count data, it could be summarised that the enhancement was ignorable in all the tested parameters. It was also noted that there was almost no difference between the initial count of other Enterobacteria (non-coli forms) and the count following the same treatment. There were no significant differences in the counts of E. coli and other Enterobacteria in any of the tested doses, and Enterobacteria other than E. coli were frequently pathogens or opportunistic, indicating that the gastrointestinal tract was in good health. Treatments utilising Chrysosporine at doses higher than 200 mg/kg body weight did not significantly change the microbial count. This might be the effect of the intestinal flora tackling self-growth endogenously or the gut environment favouring the survival of microbes until it is advantageous to the host. Compared to the control groups, there was a noticeable improvement in each group that got Chrysosporine treatment. The current findings demonstrated that Chrysosporine has a beneficial effect on intestinal health by inducing, blocking, and also minimising the proliferation of a few specific bacteria in the colon that have a significant impact on the health of the host. Chrysosporine holds pharmaceutical interest and may work as a potent exogenous therapeutic agent.

Conclusion

Strong and potent antioxidants, and prebiotics are currently in extremely short supply, a problem for pharmaceutical sectors. There are microbial consortiums and plant-based formulations, but an exponential requirement for fresh and adaptable pairings is still there. In the present investigation, different culture conditions were adopted to maximise the Chrysosporine synthesis, and the addition of epigenetic modulator- Histone Methyltransferase Specific Probe BRD4770 accelerated it from 1.64-fold. This fungal exopolysaccharide is an effective antioxidative agent and has demonstrated prebiotic actions. There was a reduction in the count of putrefying C. perfringens and simultaneously a surge of beneficial Lactobacillus sp. The current research demonstrates that oral treatment of Chrysosporine significantly improves gut health and has the potential for in vitro antioxidant activity. This promotes cutting-edge alternatives, including prebiotics, antioxidants medications like Chrysosporine.

Supplementary Information

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