INTRODUCTION:

Microorganisms are rich sources of bioactive compounds such as antibiotics, industrially enzymes, antioxidants, and other pharmacologically important molecules. Numerous studies indicate that, among all the microbes, fungi and actinomycetes have been found as the intense producers of a vast variety of bioactive metabolites¹.

These molecules are commonly referred to as secondary metabolites which vary structurally and functionally, and they range from intra- and extra-cellular low molecular weight compounds, proteins, polysaccharides or polysaccharide-protein complexes².Because of their diversity in nutritional and biochemical features, the production of metabolites varies with a slight change in their surrounding environment in terms of both quality and quantity³. Antioxidant compounds from various fungi have contributed immensely to the discovery of novel and potent drugs⁴.

Aspergillus terreus is a common fungus grows in soil and plants. It is brownish in color and gets darker as it ages on culture media. Secondary metabolic machinery of Aspergillus species is a rich source of novel drug candidates and other industrially relevant enzymes and biomolecules⁵. The fungus is known for its production of a secondary metabolite, lovastatin, which is used as a drug for lowering blood cholesterol levels. Earlier studies indicated that A. terreus is a rapid producer of many bioactive molecules such as terpenes⁶, statins⁷, antifungals⁸, anticancer agents⁷ and polyketides⁹. Biologically relevant compounds have been isolated from the fungus, including sulochrin, terretonin, asterriquinone and butyrolactone terreineol⁷, terreulactone A¹⁰, terrain¹¹, terreic acid¹² and aspulvinones¹³. A. terreus is a well-known industrially explored fungus for the production of bioactive secondary metabolites¹⁴. Large scale industrial production of these compounds requires an extensive evaluation of nutritional and environmental parameters for the optimum growth of A. terreus in the culture lab¹⁵. However, selection of nutrient medium and providing physicochemical conditions depend upon the type of metabolite to be exploited. Both submerged fermentation (SmF) and solid substrate fermentation (SSF) are widely employed for large scale production of antioxidant metabolites¹⁶. Traditional optimization procedures of media components involve supplementation of inexpensively available different solid substrates with various C/N sources¹⁷. Industrial scale productions of enzymes from various Aspergullus spp. are carried out using inexpensive agro-bio wastes. Agro-bio wastes such as sugarcane bagasse¹⁸, rice husk and rice bran¹⁹, wheat bran²⁰, potato skin²¹, sweet potato²² and groundnut oil cake²³ have been utilized for the production of various carbohydrate hydrolyzing and proteolytic enzymes from the fungal species.

Optimal culture condition has a major role in the growth and fungal biomass accumulation, favoring the maximal secretion of metabolites²⁴. The product yield can be increased by optimization of process parameters viz., physical (temperature, salinity, pH and light, incubation time) and chemical factors (media components, carbon or nitrogen sources, trace elements)²⁵. It can be achieved either by employing statistical approaches like Plackett-Burman design²⁶, Response Surface Methodology^27,28, Central Composite Design²⁹ or by traditional methods. The present study was carried out to evaluate various environmental and biochemical parameters to establish optimum growth conditions for the maximal production of bioactive secondary metabolites by A. terreus.

MATERIAL AND METHODS:

Screening of selected microorganism:

A. terreus was procured from MTCC, Institute of Microbial Technology; Chandigarh, India. The culture was revived on potato dextrose agar (PDA) slants and stored at 4°C. Media components such as PDA, dextrose, galactose, sucrose, lactose, maltose, fructose, peptone, yeast extract, malt extract, urea, ammonium chloride, ammonium sulphate, were purchased from Hi-Media, India and Merck, India. Ethanol and ethyl acetate were purchased from Rankem, New Delhi, India. All the chemicals used were of analytical grade.

A. terreus was grown for fourteen days at room temperature. The secondary metabolite production was indicated by the change in broth color from pale yellow to dark brown. The broth surface was covered by the thick mycelial mat. At the end of the incubation period, the broth was filtered and the mycelial mat was harvested. The thick mycelial mat was washed thrice with distilled water and allowed to dry at 50°C in a hot air oven for 48 hours, and the yield was expressed as dry weight of mycelia (mg/25ml)³⁰.

DPPH radical scavenging assay:

The free radicals scavenging activity of the filtrate was analyzed by using 1, 1-diphenyl-2-picryl-hydrazyl (DPPH). 3ml of the filtrate was mixed with 1 ml of a methanolic solution containing 0.1mM DPPH solution. The mixture was kept in dark for 30 min after homogenizing and the absorbance was measured at 517 nm against the blank and the percentage DPPH radical scavenging activity was calculated. Ascorbic acid was used as a reference standard.

DPPH scavenging effect (%) = [(A0-A1/ A0) × 100]

A0 = absorbance of the control,

A1= absorbance of the sample.

Optimization of growth and bioactive metabolite production:

Optimization of the physicochemical parameters for the production of bioactive secondary metabolites was performed in potato dextrose broth.

Effect of different culture media:

In order to evaluate a suitable growth medium, A. terreus was grown in different culture media such as Potato dextrose broth, Czapek’s Dox broth, Sabouraud dextrose broth, Potato Carrot broth and malt extract broth. The medium which showed maximum mycelial biomass accumulation and bioactive metabolite (antioxidant) production in terms of DPPH activity was used as the optimized medium for further study.

Effect of salinity:

The effect of salinity on mycelia growth and bioactive metabolite production was carried out in potato dextrose broth with different concentrations of NaCl (3%-7%). A. terreus was grown in PDB at room temperature for 14 days. The biomass and bioactive metabolite production were determined by quantifying the dry weight and DPPH assay, respectively.

Effect of pH:

25ml of potato dextrose broth with varying pH levels (pH 4.5-7.5) were inoculated with actively growing 5 mm mycelial discs of seven days old fungal culture under aseptic condition and incubated at optimal physical parameters. After 14 days of incubation, the broth was filtered using Whatman No.1 filter paper and mycelial dry weight and radical scavenging activity was recorded.

Effect of temperature:

The fungus was allowed to grow under different temperatures ranging from 23°C to 42°C keeping all other parameters constant. After 14 days of incubation, the mycelial dry weight and radical scavenging activity were determined.

Effect of carbon sources:

Different carbon sources such as dextrose, lactose, sucrose, fructose, maltose, starch, cellulose, carboxy methyl cellulose were used to assess their influence on the growth of A. terreus and metabolite production. 25ml basal medium was supplemented with 1% of each carbon source. The flasks were inoculated with the single mycelial disc (5mm) and incubated keeping optimal growth parameters constant. After incubation period mycelia weight and radical scavenging activity were determined.

Effect of nitrogen sources:

To study the effect of nitrogen source, 1% of different nitrogen sources such as peptone, yeast extract, malt extract, ammonium chloride, ammonium sulphate and urea were supplemented to 25ml of basal PDB medium individually and inoculated with 5mm mycelial disks of seven days old fungal culture under aseptic condition and incubated for 14 days. The mycelial weight and antioxidant activity were recorded at the end of the incubation period.

Effect of combined optimized conditions:

The effect of all the optimized conditions was also studied and determined biomass yield in conjunction with antioxidant activity. The PDB was supplemented with sucrose and ammonium sulfate as carbon and nitrogen sources respectively and the fungus was grown at a temperature of 28°C; pH 5.5 with 7% NaCl. At the end of incubation, the filtrate was checked for DPPH activity and dry weight of mycelia.

Statistical analysis:

All experiments were carried out in triplicate using the aforesaid variables. The data were analyzed and expressed as mean±standard deviation (SD). Statistical analysis was conducted using Analysis of Variance (ANOVA) for unpaired observations between the control and experimental samples, followed by Dunnett’s multiple comparison tests using GraphPad Prism - version 5.

Fig (3): Effect of different culture media on (a)Mycelial dry weight (mg/25ml) and (b) Percentage scavenging activity; *p < 0.05

PDA-Potato dextrose agar, SDA- Sabouraud’s dextrose agar, CDA-Czepek Dox agar, MEA-Malt extract agar, PCA- Potato Carrot agar

Fig (4): Effect of carbon sources on (a) Mycelial dry weight (mg/25ml) and (b) Percentage scavenging activity

***p<0.001; **p<0.01;*p<0.05

Fig (5): Effect of nitrogen sources on (a)Mycelial dry weight (mg/25ml) (b) Percentage scavenging activity;

*p< 0.05; **p<0.01; ***p<0.001

Table (1): Effect of pH, temperature and salinity on mycelial dry weight and Percentage scavenging activity

Parameters		Mycelial dry weight (mg/25ml)	% Scavenging activity
pH	Control (5.4)	170±1.41	65.45±3.03
	4.5	160 ±2.82	78.03±0.39^***
	5.0	164±1.41	79.02±1.78^***
	5.5	171±1.41^***	82.47±0.31^***
	6.0	167±2.83*	67.90±0.31
	6.5	156±1.41	69.28±3.79
	7.0	155±0.71	68.50±4.11
	7.5	152±4.24	73.49±0.16^**
Temperature	Control (25)	173±2.83	62.97±1.55
	23	169±8.48	53.19±3.78
	28	267.5±4.94***	68.49±0.89*
	37	189±2.83*	63.31±0.66
	42	137.5±2.12	56.21±0.73
Salinity	Control (1)	173±2.83	65.45±0.70
	3	167±1.41	60.08±0.23
	4	389±2.83	63.30±0.93
	5	321±1.41	70.10±0.62^**
	6	361±1.41	70.31±0.15^**
	7	147±2.83	70.47±3.02^**
Combination of the optimized parameters (28°C; pH 5.5 with 7% NaCl)	Control	173±1.41	65.32±2.03
	Modified media	253.6±3.92***	78.5202±1.78**

*p< 0.05; **p<0.01; ***p<0.001

Nutrient media and physicochemical parameters for growth of fungal species play a significant role in deriving the large-scale production of biomass and high yield of active metabolites^31,32. In this context, the present study was conducted to optimize the physicochemical parameters for a high yield biomass production of A. terreus and its antioxidant potency.

Morphological identification and microscopic evaluation of the selected fungal cultures is the first and foremost requirement which helps to assess viability and growth patterns of the fungi. The colonies were observed to be typically leathery and cinnamon-buff to sand brown in color with brownish exudates and dark brownish on the reverse side. The colony characteristics and microscopic features of A. terreus on PDA medium were shown in Fig. 1. The secondary metabolite production was indicted by a change in the broth color from yellow to cinnamon brown as shown in Fig. 2.

In the present study, the crude extract of A. terreus cultured in different growth media showed a substantial antioxidant property in terms of DPPH free radical scavenging activity (Fig. 3b). Comparatively, the extract derived from the fungus grown in PDA medium exhibited the highest antioxidant activity ((84%; p<0.05). The DPPH free radical scavenging activity of different fractions of A. terreus cultured in PDA medium has been reported in earlier studies³³. The influence of different culture media on biomass production of A. terreus in terms of mycelial dry weight is shown in figure 3a. A. terreus exhibited a profuse growth and highest metabolite production in the PDA broth when compared with the other tested media. Moderate growth and DPPH scavenging activity was observed in potato carrot broth, malt extract broth, Sabouraud dextrose broth, and Czapek’s Dox broth, respectively. Thus, among different nutrient media, the PDA medium was found to be the best one for a high yield biomass production and exhibit antioxidant activity. The current observation is in parallel with the previous report³⁴, where PDB and Sabouraud's dextrose were found to be better nutrient media for growth and metabolite production of A. terreus. PDB has been shown to exhibit the maximum results for the biomass and pigment production of A. terreus KMBF1501 in the study conducted by Akilandeswari and Pradeep³⁵.

Among all the tested carbon sources, sucrose was found to be the best carbon source for antioxidant metabolite production (Fig.4b, p˂0.001), which was followed by cellulose and maltose. Biomass production was found to be high in the medium supplemented with cellulose followed by sucrose and dextrose (Fig 4a). Carboxy methylcellulose was the least utilized compound by the fungus.

Mathan et al.³⁰and Bhattacharyya & Jha³⁶ reported a profuse growth and maximal metabolite production by Aspergillus on all the carbon sources and sucrose-supplemented media. According to the reports by Arora and Chandra (2010)³⁷ on the assay of antioxidant potential of two Aspergillus isolates by different methods under various physicochemical conditions, higher activity was observed in sucrose as carbon source followed by dextrose and maltose which are in consonance with the present study. However, it is contradictory to the general concept that glucose and starch are the best carbon source for fungal growth by Yen and Chang (1999)³².

Fig (5) presents the results obtained for the effect of different nitrogen sources on biomass and antioxidant metabolite production. Maximal biomass production was noted in the culture medium supplemented with urea, followed by (NH₄)₂SO₄ and NH₄Cl. Statistically, all these exhibited a significant biomass production (mycelial dry weight) compared to the control (p<0.001). Yeast extract also showed a substantial high yield biomass production (p<0.01). With reference to the DPPH scavenging activity, supplementation of ((NH₄)₂SO₄ and NH₄Cl, gave the maximum results (p<0.001), followed by the malt extract (p<0.01). It was noted that there was no clear-cut correlation between biomass production and antioxidant activity of the crude extract of the fungus. This indicates that the production of metabolites with potential antioxidant activity varies depending upon the supplemented nitrogen sources.

From the results, it was evident that the fungi exhibited growth at its maximal level and metabolite production in the acidic pH (Table 1). pH 5.5 was found to be the best for both metabolites and biomass production, but the fungal biomass and bioactive compound synthesis was found to be the least in the media with alkaline pH. The influence of temperature on mycelial growth and bioactive antioxidant metabolite production by A. terreus was presented in the table 1. The maximal growth and DPPH activity was observed at 28°C followed by 37°C. There was a decrease in both biomass and antioxidant bioactive production at 23°C and 42°C. The results obtained are in parallel with the earlier reports; incubation temperatures ranges from 25 to 30°C enhanced the growth and synthesis of bioactive fractions by certain Aspergillus species in a study carried out by Bhattacharyya and Jha³⁶ & Jain and Pundir³⁸. A. terreus showed a profuse growth and biomass production at salinity 4 % to 6% with optimal growth at 4% (Table 1). The secondary metabolite production was higher at a salinity range of 5% to 7% with the best result at 7% NaCl concentration. The results were in parallel with the study conducted by Masuma et al.⁶on the effect of increasing salinity in seawater on the growth of certain Aspergillus and other marine fungi.

The results of the experiment carried out for the combined optimal conditions (28°C; pH 5.5 with 7% NaCl), indicated there was a positive changes. The yield of mycelial dry weight (253.6±3.92) was found to be significantly higher compared to the control (p<0.001). The DPPH scavenging activity (78.5202±1.78) was also found to be significantly higher at the level of p<0.01 compared to the controlled conditions. Thus, the optimized conditions such as temperature, pH, and salt concentration in addition to the PDB supplemented with sucrose and ammonium sulfate as carbon and nitrogen sources, provide ideal conditions for high-yield biomass production of secondary metabolites and free radical-scavenging activity from A. terreus in laboratories.

CONCLUSION:

From the present study, it is evident that A. terreus is a potent source of antioxidant compounds which can be exploited widely in pharmaceutical industries by employing different kinds of optimized physicochemical conditions. The findings of the present work suggest that the formulation of growth media and optimized physical and chemical parameters conditions will be helpful in the mass cultivation of the fungus for its industrial-scale production of bioactive molecules. Further studies like identification of bioactive compounds, statistical approaches for media optimization and gene expression mechanism of hyper activation of metabolite synthesis pathways is highly recommended for a better understanding of the downstream processing and economical production.

List of Symbols and Abbreviations:

A.terreus- Aspergillus terreus

C/N- carbon/nitrogen

CDA-Czepek Dox agar

CMC- carbodxy methyl cellulose

DPPH-1, 1-diphenyl-2-picryl-hydrazyl

MEA-malt Extract agar

PDA-potato dextrose agar

ROS -Reactive Oxygen Species

SDA-Sabouraud’s dextrose agar

SmF –submerged fermentation

SSF-solid substrate fermentation

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 01.04.2020 Modified on 10.08.2020

Research J. Pharm. and Tech. 2021; 14(6):2924-2930.

DOI: 10.52711/0974-360X.2021.00513