Bioconversion of agro-residues to make extracellular polysaccharides in solid state fermentation via Trichoderma hamatum using response surface methodology: Antioxidant and α-glucosidase inhibitor activity

Abstract

Agro-residues, including banana peel, pineapple peel, mango peel, apple peel, and coconut husk, were utilized to produce extracellular polysaccharide (EPS) via solid state fermentation(SSF) by the fungus, Trichoderma hamatum. EPS production was highest in pineapple peel (84.2 ± 0.4 mg/g), followed by mango peel (70.3 ± 0.41 mg/g) (p<0.01). The SSF bioprocess was optimized via a two-level full factorial design and response surface methodology. The effects of five selected variables on EPS biosynthesis, namely, the concentrations of glucose (10 to 30%), ammonium sulphate (0.1 to 1%), yeast extract (0.5 to 2%), MgSO₄ (0.01 to 0.1%), and medium pH (4.5 to 6.5), were analyzed via a full factorial design (FFD). The EPS production ranged widely from 15.3 to 576.2 mg/g substrate. Three significant variables affecting EPS production were assessed in central composite design (CCD) to optimize concentrations of MgSO₄, and glucose, and the pH. The designed CCD model was fitted to the quadratic model and was significant (p<0.0001). For 50 μL of EPS, the scavenging ratio was 43.4 ± 4.1% at a concentration of 200 μL (78.5 ± 6.9 μL) (p<0.01). The extracted EPS exhibited an alpha-glucosidase inhibitory effect (p<0.001). Solid-state fermentation allows the utilization of low-cost biomass for EPS production and the application of T. hamatum EPS as a natural antioxidant and α-glucosidase inhibitor.

Full Article

Bioconversion of Agro-residues to Make Extracellular Polysaccharides in Solid State Fermentation via Trichoderma hamatum Using Response Surface Methodology: Antioxidant and α-Glucosidase Inhibitor Activity

Prakash Shoba Savariyar Adimy,^a Mohamed S. Elshikh,^b Mohammad Ajmal Ali,^b and Gurupatham Devadhasan Biji ^c,*

Agro-residues, including banana peel, pineapple peel, mango peel, apple peel, and coconut husk, were utilized to produce extracellular polysaccharide (EPS) via solid state fermentation(SSF) by the fungus, Trichoderma hamatum. EPS production was highest in pineapple peel (84.2 ± 0.4 mg/g), followed by mango peel (70.3 ± 0.41 mg/g) (p<0.01). The SSF bioprocess was optimized via a two-level full factorial design and response surface methodology. The effects of five selected variables on EPS biosynthesis, namely, the concentrations of glucose (10 to 30%), ammonium sulphate (0.1 to 1%), yeast extract (0.5 to 2%), MgSO₄ (0.01 to 0.1%), and medium pH (4.5 to 6.5), were analyzed via a full factorial design (FFD). The EPS production ranged widely from 15.3 to 576.2 mg/g substrate. Three significant variables affecting EPS production were assessed in central composite design (CCD) to optimize concentrations of MgSO₄, and glucose, and the pH. The designed CCD model was fitted to the quadratic model and was significant (p<0.0001). For 50 μL of EPS, the scavenging ratio was 43.4 ± 4.1% at a concentration of 200 μL (78.5 ± 6.9 μL) (p<0.01). The extracted EPS exhibited an alpha-glucosidase inhibitory effect (p<0.001). Solid-state fermentation allows the utilization of low-cost biomass for EPS production and the application of T. hamatum EPS as a natural antioxidant and α-glucosidase inhibitor.

DOI: 10.15376/biores.19.4.8368-8387

Keywords: Pineapple peel; Solid-state fermentation; Central composite design; Extracellular polysaccharide; Antioxidant; α-Glucosidase inhibitor

Contact information: a: Holy Cross College (Autonomous), Nagercoil-629004, Affiliated to Manonmanium Sundaranar University, Tirunelveli, India; b: Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia 11451; c: Department of Zoology, Nesamony Memorial Christian College, Marthandam, Affiliated to Manonmaniam Sundaranar University, Tirunelveli, India;

*Corresponding author: gdbiji45@gmail.com

GRAPHICAL ABSTRACT

INTRODUCTION

Agro-industrial residues, which are sometimes called agro-wastes, are generally disposed into the environment and constitute an environmental risk. This has affected the environment and led to the generation of greenhouse gases and affect environmental and human health. Agro-residues, such as seeds, peels, and stones from vegetables and fruits, contain various phytochemical compounds, and these compounds can be utilised for the production of various products (Marraiki et al. 2020; Sathya et al. 2023). The culture of microbes on types of agro-residues to produce biochemical compounds in solid state fermentation (SSF) has increased in recent years as an alternative approach to costly synthetic media (Sathya et al. 2024; Vijayaraghavan et al. 2024). Microorganisms, especially fungi, utilise agro-residues and produce enzymes, extracellular polysaccharides, bioactive compounds, organic acids, and nutritional supplements. The lignocellulosic biomass is composed of lignin, hemicelluloses, and celluloses, which can be utilised by microbes to produce various products. These residues, especially renewable biomasses, are desirable alternative sources for the production of pharmaceuticals, biofuels, and functional compounds (Vijayaraghavan et al. 2014; Arokiyaraj et al. 2024). Trichoderma is one of the largest groups of fungi. It is a member of the Ascomycota. Asexual reproduction occurs via conidia. It has been exploited to produce various biomolecules, and has been applied in textiles, paper, agriculture, and biofuels because of its ability to produce biomolecules (Chaverri et al. 2003; Giraldo et al. 2007).

To date, only a few studies have optimized bioprocesses to produce EPS from Trichoderma sp. using response surface methodology. The extracellular polysaccharide-producing strains, such as Trichoderma erinaceum DG-312 (Ji-Hoon and won 2005), Trichoderma sp. KK19L1 (Li et al. 2017), and Trichoderma harzianum (Saravanakumar et al. 2021), were previously reported. Fungi produce polysaccharides, primarily extracellular polysaccharides (EPSs),which present novel therapeutic characteristics such as antioxidant, antimicrobial, and anticancer properties. EPSs are applied in various processes in various industries, including, the food, cosmeceutical, and pharmaceutical industries (Farraj et al. 2024; Rajaselvam et al. 2024). Considering the significance and application of EPS for biotechnological processes, EPS can be produced either by submerged fermentation or solid-state fermentation. The process conditions, such as the medium, temperature, and pH, are very significant factors influencing fungal growth and product production (Biji et al. 2016; El-Sheikh et al. 2020). In the fermentation process, the optimization of bioprocess conditions is a significant approach for improving product yield; hence statistical approach is used to optimize the medium components. Response surface methodology is an important statistical tool for predicting the optimum response of medium components (Al-Farraj et al. 2020; Kalaiyarasi et al. 2020). In this study, the agro-residues banana peel, pineapple peel, mango peel, apple peel, and coconut husk were used for EPS production. The antioxidant, and α-glucosidase inhibitor properties of EPS were determined.

EXPERIMENTAL

Isolation of Fungi from Sugarcane Bagasse

The sugarcane bagasse sample (0.5 g) was ground using a pestle and mortar, and it was serially diluted with sterile double-distilled water. The diluted sample was spread on potato dextrose agar (PDA) medium and incubated at 28 °C for 4 days. The morphologically dissimilar colonies were preserved on PDA slants at 4 °C and the strain was subcultured every four months. Fungi isolated from sugarcane bagasse were used for extracellular polysaccharide production.

Inoculum

A total of 58 fungal strains were isolated from sugarcane bagasse, and 11 isolates (EP2, EP5, EP13, EP15, EP20, EP28, EP31, EP39, EP48, EP54, and EP58) were selected for EPS production. These 11 isolates grew well on PDA medium within 96 h of incubation. The isolated 11 strains were cultivated in Sabouraud’s broth medium composed of peptone (10 g/L), glucose (40 g/L), MgSO₄·7H₂O (0.5 g/L), KH₂PO₄ (1.0 g/L), FeSO₄·7H₂O (0.05 g/L), and were incubated in an orbital shaker incubator at 175 rpm at 28 °C for 5 days. The seed cultures (EP2, EP5, EP13, EP15, EP20, EP28, EP31, EP39, EP48, EP54, and EP58) were inoculated into a basic mineral salt medium (g/L) composed of dipotassium phosphate 0.700, ammonium chloride 0.800, disodium EDTA 0.0092, magnesium sulphate. heptahydrate 0.010, calcium sulphate, dihydrate 0.002, ferrous sulphate heptahydrate 0.007, zinc sulphate, heptahydrate 0.0001, boric acid 0.0001, cobalt nitrate 0.00001, sodium molybdate dihydrate 0.00001, manganese sulphate, quadrahydrate 0.00002, and copper sulphate pentahydrate 0.0005. In the Erlenmeyer flask, 100 mL culture medium was added and was inoculated with 2% inoculum. The mixture was cultured in an incubator for 10 days at 28 °C.

Extraction of Extracellular Polysaccharide and Assay

The broth culture was centrifuged at 5000×g for 10 min, and the cell-free extract was retained. The supernatant was concentrated via a speed vacuum at 40 °C using a rotary evaporator. It was further mixed with 95% ethanol (double volume) and incubated for 24 h at 28°C. The mixture was subsequently centrifuged at 5000×g for 10 min and resuspended in a small volume of ethanol (0.5 mL). It was deproteinized using Sevag reagent (chloroform-n-butanol at 4:1, v/v), decolourized with 10% (v/v) H₂O₂for 2 h under stirring, and dialyzed for 4 h. The sample was concentrated with ethanol (95%) and lyophilized. The final yield was weighed, and the sample was considered EPS. The carbohydrate content of each sample was assayed using the anthrone-sulfuric acid method. Approximately 100 μL of sample was hydrolyzed with acid and incubated with anthrone reagent. The absorbance of each sample was read at 620 nm. Glucose (10to100 μg/mL) was used as a reference. The amount of EPS produced was determined by the carbohydrate content (μg) of the EPS/L production medium.

Solid State Fermentation (SSF)

Approximately 5 g of agro-residue (banana peel, pineapple peel, mango peel, apple peel, or coconut husk) was transferred to a 100-mL Erlenmeyer flask, and the moisture content was adjusted to 65%. The substrates were sterilized for 30 min for 1 h at 121 °C. The maximum polysaccharide-producing fungal strain (EP20) was subsequently inoculated into the medium and incubated for 8 days. After 8 days of incubation, the extracellular polysaccharide was extracted and assayed.

Screening of Variables to Produce EPS

The effects of MgSO₄, glucose, yeast extract, and ammonium sulphate on EPS production were determined. Pineapple peel (5 g) was mixed with sodium phosphate buffer (3 mL) at pH 6.0. To optimize the MgSO₄ concentration, 0.02 to 0.1% MgSO₄was added to the substrate. Similarly, the culture medium was supplemented with glucose (20 to 80%), yeast extract (0.25 to 1.25%), and ammonium sulphate (0.2 to 1%) at various concentrations and the optimum concentrations were analyzed.

Screening of Variables for Extracellular Polysaccharide Production

A two-level full factorial design (FFD) was used to screen the significant variables affecting EPS production in SSF. Pineapple peel was used as a solid substrate until otherwise stated. The FFD was used to screen the medium components influencing EPS production. The selected major medium supplements were, glucose (10 to 30%), ammonium sulphate (0.1 to 1%), yeast extract (0.5 to 2%), medium pH (4.5 to 6.5), and MgSO₄ (0.01 to 0.1%), and the FFD experiment was designed using the Design Expert software (Version 8.1, Stat-Ease, Inc., Minneapolis, MN, USA). The selected factors were set at low and high levels and are illustrated in Table 1. The FFD matrix design comprises 32 experiments for five variables.

Table 1. Low and High Values of the Selected Variables for Screening Experiments in a Two-Level Full Factorial Design

Central Composite Design (CCD) and Response Surface Methodology (RSM)

Three significant variables affecting EPS production were assessed via CCD design to optimize the concentrations of MgSO₄, glucose and the pH of the culture medium. A total of 20 experiments were performed based on the Design-Expert matrix. In recent years, CCD has been widely used to optimize culture medium components and environmental factors for product development. This method is considered as precise and has proved to be an efficient method for designing, predicting, and optimizing variables. The selected variables were screened at five different levels (−1.682, −1, 0, +1, +1.682) as depicted in Table 2.

Table 2.Variables and Levels in Central Composite Design and Response Surface Methodology

Antioxidant Activities

The free radical scavenging ratio of EPS to1-diphenyl-2-picryl-hydrazyl (DPPH) was tested as described previously (Shimada et al. 1992). The DPPH was prepared in ethanol at 0.2 mmol/L concentration. A total of a 2 mL of DPPH solution was mixed with EPS at various concentrations (50 to 250 μL) and incubated at 30 ± 1 °C for 30 min. After 30 min of incubation, the optical density of the sample was evaluated at 517 nm against the reagent blank. The free radical scavenging activity was calculated via Eq.1,

Free radical scavenging ratio = (1 − A₁/A₀) × 100 (1)

where A₀ is the absorbance of the blank and A₁is the absorbance for the test condition.

Determination of Alpha-Glucosidase Inhibition Activity

The α-glucosidase inhibitory activity of EPS was determined. Alpha-glucosidase was prepared at a 1 U/mL concentration in 0.1 M PBS (pH 6.5) and EPS was prepared at various concentrations (2, 4, 6, 8, and 10 mg/mL). Approximately 50 μL of the diluted sample was mixed with 100 μL of the diluted alpha-glucosidase solution and incubated for 10 min at 28 ± 1 °C. After 10 min incubation, 50 μL of 4-nitrophenyl-β-D-glucopyranoside (5 mM) was added and the mixture was incubated for 5 min at 28 ± 1 °C. The absorbance of each sample was read at 405 nm before and after the final incubation. Acarbose (0.01 mg/mL) was used as the positive control and the results are expressed as follows,

α-glucosidase inhibition (%)= (1 − Δ𝐴_{𝑠𝑎𝑚𝑝𝑙𝑒}/Δ𝐴_{𝑐𝑜𝑛𝑡𝑟𝑜𝑙}) × 100      (2)

where A_sample is the absorbance of the sample and A_control is the absorbance of the control.

Statistical Analysis

The experimental procedures were done three times for each condition, and the final results are expressed as mean ± standard deviation. A p-value<0.05 was considered a statistically significant result.

RESULTS AND DISCUSSION

Screening of Extracellular Polysaccharide-Producing Fungi

The fungal strains (EP2, EP5, EP13, EP15, EP20, EP28, EP31, EP39, EP48, EP54, and EP58) screened from the sugarcane bagasse produced extracellular polysaccharides. The isolated strains were cultivated in a Sabouraud’s broth medium and the yield varied on the basis of the isolate. Compared with the other isolates, EP20 produced a significant amount of EPS (51.2 ± 1.6 mg/mL) (p<0.01) (Fig. 1). Fungi and bacteria produce EPS for industrial applications. Considering the significance of EPS, the isolated fungi were screened for EPS production. Microbial EPS are the subject of research in various fields in science and technology.

Fig. 1. Extracellular polysaccharide-producing fungi isolated from sugarcane bagasse

The major research direction is focused on determining variables responsible for synthesis, optimization, and application of EPSs in various fields. The bioprocess of EPS derivation involves selecting suitable fungi, culture (solid state fermentation or submerged fermentation) and suitable extraction methods (Osińska-Jaroszuk et al. 2015).

Agro-residues as a Cheap Substrate for Extracellular Polysaccharide Production

To screen agro-residues to produce EPS, various biomass, including banana peel, pineapple peel, mango peel, apple peel, and coconut husk were used. The fungus T. hamatum EP20 utilized these substrates and produced EPS in SSF. Moreover, the amount of EPS produced varied among substrates (Fig. 2). The EPS production was high in the pineapple peel (84.2 ± 0.4 mg/g), followed by mango peel (70.3 ± 0.41 mg/g). Among the biomass, coconut husk showed the least EPS production (12.1 ± 0.2 mg/g) (p<0.01). Fungi from the Ascomycota and Basidiomycota groups presented potent biological and industrial properties. The selected T. hamatum EP20 was an Ascomycota group fungus that may be highly applicable in industry. Similarly, fungi from the Basidiomycetes group utilize solid substrates, especially broadbean seed capsules, cane bagasse, and the strain Kosakonia cowanii LT-1 which utilize a mixture of these two substrates and produce EPS (Montoya et al. 2013). Moreover, the amount of EPS produced by the strain K. cowanii LT-1 was lower than that in this study. EPS producing basidiomycetes species, including Lentinula edodes, T. versicolor, and Pleurotus ostreatus were used,and they utilized lignocellulosic residues such as coconut fiber, oak sawdust, corn bran, coffee husks, and soybean oil (Sánchez and Montoya 2020). The variation in the biomass composition positively regulated EPS production in SSF. In this study, T. hamatum EP20 utilized peels and coconut husk, and EPS production varied with substrate. The lignocellulose biomass is considered the substrate for the production of extracellular polysaccharides. Lignocelluloses are composed of lignin, hemicelluloses, and cellulose, among which sugar polymers account for a large proportion of the biomass. Microorganisms utilise these sugars for the conversion of polysaccharides (Manivasagan et al. 2013). Thus, agro-residues, preferably lignocellulose biomass can be utilized for polysaccharide production.

Fig. 2. Agro-residues to produce extracellular polysaccharides in solid state fermentation

Preliminary Screening of Nutrient Factors for Improved Production Extracellular Polysaccharide

In EPS production, nutrient factors (MgSO₄, glucose, and ammonium sulphate) significantly influenced product yield. MgSO₄ was tested at various concentrations, and maximum EPS production was achieved at 0.04% MgSO₄ concentration (189.2 ± 10.2 mg/g) (Fig. 3a). The effects of glucose concentration on EPS production were tested (Fig. 3b). Higher EPS production (184.2 ± 7.4 mg/g substrate) was obtained at 60% glucose concentration. Furthermore, most microorganisms have been reported to utilize sucrose or glucose to produce extracellular polysaccharides (Zhang et al. 2020);therefore glucose was selected as the preferred carbon source. In this study, EPS production improved at increasing concentrations of glucose up to 60% level. The effects of the yeast extract concentrations on EPS production were tested (Fig. 3c). The higher EPS production (154.3 ± 1.7 mg/g substrate) was obtained at 0.75% yeast extract. Various ammonium sulphate concentrations were tested in the present study to determine their effects on EPS production and maximum EPS production was obtained at 0.6% ammonium sulphate (182.5 ± 4.5 mg/g substrate) (Fig. 3d). Several nitrogen sources have been screened for EPS production; however, the yield varies with nitrogen source and organism. The supplemented nitrogen sources improved the biosynthesis of polysaccharides, and this finding was similar to results of Cui et al. (2017), in which available nitrogen sources improved the growth of bacteria and EPS production. The optimum carbon/nitrogen ratio is critical for EPS production; thus optimization of process parameter is warranted for the improvement of EPS yield. In the current study, EPS production was affected at higher ammonium sulphate concentrations, and these results are in accordance with the previous reports. Lo et al. (2007) reported the inhibitory effect of nitrogen sources on EPS yield in P. acidipropionici culture, whereas EPS production was improved in S. thermophilus. This previous study revealed that the requirements of nitrogen sources vary among organisms. Hence, optimization of culture conditions for any new isolate is needed to analyse nutrient requirements to improve product yield.

A

B

C

D

Fig. 3. Effects of MgSO₄ (A), Glucose (B),Yeast extract (C), and Ammonium sulphate (D) on EPS production. Pineapple peel (5 g) was mixed with various concentrations of MgSO₄, glucose, yeast extract, and ammonium sulphate. Then, it was inoculated with 2% inoculums and incubated for 8 days at 32 ± 1 °C.

Two-Level Full Factorial Design and Analyses

A full factorial design enables the determination of the main effect of each factor, and it allows screening of the interactive effect of factors using low and high levels. The effects of the five variables on EPS biosynthesis, i.e., the concentrations of glucose (10to30%), ammonium sulphate (0.1to1%), yeast extract (0.5to2%), MgSO₄(0.01to0.1%), and the pH of the medium (4.5to6.5) were analysed via FFD. The response of variables (EPS production) varied widely from 15.3 to 576.2 mg/g substrate (Table 3).

Table 3. Two Level Full Factorial Experimental Design and Extracellular Polysaccharides Production

The variation in EPS yield revealed the significance of the screening method for analysing the interactive effect between the selected variables. The analysis of variance of the full factorial experiments is depicted in Table 4. The concentrations of MgSO₄, yeast extract, and glucose significantly influenced EPS production and the p-values were0.01, 0.00032, and 0.002, respectively. The F-test analysis revealed that pH significantly influenced EPS production (p=0.0003). Balasubramanian et al. (2019) screened medium components to produce EPS by Aspergillus sp. The supplementation with gelatine, maltose, and divalent ions influenced EPS production in SSF. The EPS yield ranged between 3.18 and 11.65 mg/g substrate, and the yield obtained in the present study was greater than that reported previously. The two-level full factorial design and Plackett–Burman design are frequently used to screen process variables before optimising the concentrations of variables or factors involved in bioprocesses (Al-Ansari et al. 2020; Marraiki et al. 2020). In the present study, a two-level full factorial design was employed to screen the selected variables to determine the optimum response and the Plackett–Burman design was employed to screen a large number of variables. The two-level factorial design was best suited to screen a small number of variables, because a large number of experimental runs are needed.

Table 4. Analysis of Variance of the Two Level Full Factorial Design Experiments to Produce Extracellular Polysaccharide

Five variables were selected, and it comprised a total of 32 experimental runs; hence, this method was selected. Manivasagan et al. (2013) screened variables, including pH, NaCl concentration, fructose, tryptone, glucose, and yeast extract via Plackett-Burman design for EPS production by Streptomyces violaceus MM72, and central composite design was preferred for the determination of optimum response. In the present study, central composite design was utilized to determine the optimum medium components for EPS production in the SSF.

Central Composite Design and Response Surface Methodology

Based on a two-level full factorial experimental design, three variables (pH, MgSO₄, and glucose) were selected for optimizing the variable concentrations. The middle value of the variables was selected based on two-level full factorial design experiments. The CCD model analyzes the effects of variables at five different levels.The experiment was designed via Design-Expert software. A total of 20 experimental runs were used to determine the optimum response, and the results of CCD are summarized in Table 5.

Table 5. Matrix of CCD and Extracellular Polysaccharide