Zn Fortification Influential Impact on the Productivity of Calocybe indica Mycelium

Deepali¹, P. Dipti Rani², S.K. Jadhav¹ and Nagendra Kumar Chandrawanshi¹*,

¹School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India

²Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur

Abstract: Calocybe indica is an edible medicinal mushroom, preferably eaten for its culinary value. It was cultivated for its higher nutritional value, medicinal properties, and high polysaccharide content, especially glucan. Some particular minerals were enriched in food substitutes, an alternative to fighting against some targeted human ailments. Thus, mineral fortification is accessible in the submerged cultivation of mushrooms to produce bioactive compounds and fortified mushrooms. In this study, the submerged cultivation of C. indica was performed to make exopolysaccharides (EPS) using a supplemented medium of Zinc with varying concentrations. Thus, research revealed that the Zn fortification enhances the production of EPS and mycelial biomass after 21 days of incubation. The maximum mycelial biomass was 7.7133±0.30 g/L (dry weight), and the highest 0.3853±0.006EPS was produced in the 175mg/L mineral concentration, respectively. The present study revealed that the Zn supplementation gradually increased the mineral concentration and directly influenced the yield of mycelial biomass and EPS production. These EPS have various biological activities and can be helpful for fortified food or pharmaceutical product development in the medicinal and pharmaceutical sectors.

Keywords: Antioxidant, Fortification, Mycelial biomass, Submerged culture and Exopolysaccharides.

Introduction

C. indica, commonly known as a Milky mushroom, was first described by Purkayastha and Chandra in 1974. It belongs to the phylum Basidiomycetes and is a medicinal mushroom primarily consumed in West Bengal, India. Worldwide, it is recognized for the richness of its bioactive compounds (Balouiri et al., 2015; Ghosh et al., 2020). It is an umbrella-like mushroom and requires a hot, humid climate for its cultivation; the temperature is about 25^°C- 35^°C, and the stem is cylindrical and has no rings (Purkayastha et al., 1974; Subbiah et al., 2015). The cultivation of C. indica generally takes place on wheat straw and paddy straw as the substrate, such as sorghum stalks, groundnut hulls, soybean straw, and coconut coir, which are also used for cultivation (Rathore et al., 2020; Kosre et al., 2021; Chouhan et al. 2022). Milky mushroom consists of carbohydrates up to 6.8%, proteins 2.75%, lipids 0.6%, fibres 1.67%, water 87% and minerals 0.5-1 %, respectively (Gupta et al., 2012). The essential amino acids of C.indica consist of arginine, lysine, histidine, tryptophan, leucine, threonine, valine, isoleucine and methionine (Sumathy et al., 2015; Thejaswini et al., 2015). Therefore, it is used for cures or alternative food materials and combats many diseases like cardiovascular cancer and diabetes as it is rich in fibre, proteins and antioxidants (Balouiri et al., 2015). The polysaccharides of C.indica generally consist of rhamnose, arabinose, galactose, glucose xylose and mannose. However, β- glucan is known to present a massive amount in C.indica (Thejaswini et al., 2015). Solid-state cultivation of mushrooms takes several days for complete growth and fruiting body formation. In addition, it is a time-consuming and labour-intensive process, so it is unsuitable for metabolite production. Moreover, most research focused on submerged cultivation in the current scenario because it has many advantages over solid cultivation. Submerged cultivation involves low cost and high yield, and purification of pharmaceutically essential commodities or products is much easier. In the current generation, researchers believe and have found that submerged cultivation significantly affects productivity, leading to the excessive increase in biomasses greatly enriched bioactive yield (Kirsch et al., 2016; Koreti et al., 2023). The submerged fermentation of mushrooms involves a more significant response to its growth and production of mycelium. Many essential factors are optimized to yield higher (Subhadip et al., 2013; Bellettini et al., 2019; Wang et al., 2020). Mineral fortification is an alternative method for making functional foods and nutraceuticals. The fortification of mushrooms with elements enhances a specific property, such as the enrichment with selenium increases the anti-tumour activity, and it also has immunomodulatory properties of proteins (Zhang et al., 2006; Shang et al., 2011;  Shu et al., 2019);  thus, it has considered having medical nutrition therapy in food modification (Assuncao et al., 2012). The present study used the submerged cultivation method to focus on Zn fortification in C.indica mycelium. It tested various concentrations of Zn-containing medium employed for the experimentation and determined its influence in the production of the biomass, exopolysaccharides and fortification of cultured mushroom mycelia.

Materials and Methods

Microorganism collection and maintenance

The identified mushroom species of C. indica (DMRO-302) was procured from ICAR-Directorate of Mushroom Research, Chambaghat, Solan, Himachal Pradesh; for research purposes, the pure culture was maintained routinely in PDA slant for further experiment.

Submerged cultivation of C.indica

The experiment followed the procedure of Zhong and Tang (2004); Liu et al. (2018) with minor modifications. In this experiment, PDB media was used as cultivation media. The media was prepared in an Erlenmeyer flask containing 150ml of the medium, and it was autoclaved at 121^ºC and 15 psi. A Zn standard mineral stock solution was prepared in distilled water using ZnSO₄ at different ranges (0, 25, 50,100,125,150,175,200 and 225mg/L). After cooling, various concentrations of minerals were added to the media under laminar airflow and a 5mm disk from a 10-day-old culture of C. indica was inoculated. Furthermore, the inoculated flask was incubated at 28±30 ^ºC for 21 days in the incubator.

EPS extraction

After the incubation period, the medium with mycelium was successfully filtered using Whatman filter paper no.4. Mycelium and the filtrate medium were obtained. The wet and dry weights of the collected mycelium (dry biomass) were measured. Liu et al. (2018) protocol was followed with slight modifications for the EPS production. Add four volumes of 96% ethanol to each Erlenmeyer flask in the filtered liquid medium, mix vigorously, and keep it at 4°C for overnight incubation. The next day, each medium was transferred into the centrifugation tube and centrifuged at 4500 rpm for 15 minutes. The supernatant dried the pallet in the oven at 45°C overnight and washed the collected dried pallet with autoclaved distilled water. The remaining residue was lyophilized for the EPS production. The collected EPS was stored in a fresh eppendorf tube, maintained, and stored for further analysis at 4°C.

Data analysis

Statistical analysis and interpretations were made based on a comparison of factor treatment means, as well as the comparison between pH, production of biomass, and EPS. The collected data were subjected to analysis using the SPSS 16.0 version software package, and the graph was prepared using Origin Pro 8.5. 

 Results and Discussion

C. indica is one of the best edible medicinal mushrooms and has a variety of nutritional enrichments. In the present study, different concentrations of ZnSO₄ were used for supplementation, and the impact of biofortification on the production of EPS was gradually influenced.

Effect of Submerged culture

The production of EPS is generally extracted from the solid-state fermentation, but the submerged culture method provides a better culture condition for the mass production of the biomass. The pH, temperature and incubation days should be considered throughout the cultivation periods. The large-scale production in batch culture method provides better control over biomass production. Batch culture is supposed to produce secondary metabolites on a large scale. This current study showed the production of EPS from the submerged culture of C. indica. It revealed that the biofortification of a mineral such as Zinc influences the amount of a high yield of mycelia biomass as the bio enrichment technique. Thus, it enhances the biological activity and improves the total protein and carbohydrate content in the fruiting bodies. However, it is also observed that it enhances antioxidant activity and has potent anti-microbial activities against harmful microorganisms. The submerged culture for the EPS production provides better culture conditions. However, solid-state cultivation has huge technical constraints. Still, in vivo mushroom cultivation practices are labour-intensive and require only 2-3 months to cultivate fruiting bodies. Therefore, the submerging process is the solution for high mycelia biomass production to overcome the limitations of solid-state cultivation. Furthermore, submerged culture necessitates less space and is less prone to contamination (Zhong & Tang, 2004).

Effect of mineral on the production of mycelia biomass

Several experiments were studied for the production of mycelial biomass. Previously, researchers generally used different mediums for nutrient sources and reported that some minerals were also used to supplement and enhance biomass production. This study used Zn in varying concentrations as the mineral source for producing EPS. After the incubation, the dried weight of mycelia was observed. This study revealed that the fortification with Zn significantly increased the biomass yield compared to the control. The dry weight of biomass was observed at different concentration ranges. It was observed that the initial biomass concentration was increased with a particular range (175 mg/L) of mineral supplementation, and then after this range, the biomass concentration was decreased. There was an elevation in the result. The highest dried biomass was obtained at 7.7133±0.30g/L at 175 mg/L (shown in Table 1), and the lowest biomass was obtained in the control medium, which was 0.7778±0.10g/L was observed. The study revealed that the fortification directly influences biomass production at the tested range (25 to 175 mg/L) concentration; moreover, 200 mg/L has been shown to decrease biomass production caused by metabolic suppression of microbial growth of in vitro cultured C. indica. Similarly, the type of mushroom species and its growth cycle influenced biomass production. Some mushrooms, such as Pleurotus, are reported for their fast-growing capacity (Sen et al., 2020). C. indica mushroom is not a fast-growing species; it requires a long time compared to Pleurotus species. Limited research was carried out on mineral fortification in C. indica compared to other mushroom species. Lim et al. (2004) optimized the medium for EPS production in Collybia maculata. They reported that the maximum concentration of EPS in the 5-L stirred tank was 2.4 g/L. They studied different nitrogen, carbon, and mineral sources and found that the most suitable mineral sources for producing EPS were K₂HPO₄ and CaCl₂. In the same way, Singh et al. (2020) worked on P. eryngii mycelium submerge fermentation and achieved the maximum biomass at the concentration of 14.21± 0.25g/L in the 10 days of incubation. So, under the optimum growth condition, the biomass obtained at the concentration of 250ml, 500ml, and 1000ml in the incubation of 10 days was 13.8±0.5, 13.7±0.6, and 13.6±0.5g/L respectively.

Effect of pH of the filtrate

After the incubation of 21 days, the filtration process separated the mycelia biomass and the liquid media. The different concentrations of minerals were subjected to the measurement of pH. However, the pH increased in the various concentrations compared with the control. Somehow, the pH of all the concentrations was in the range between 4 to 6 in the acidic condition. The highest pH was observed in the concentration of 50µg/ml, which was 6.14±0.60. It was reported that EPS production by fungi requires low pH in the range of 3.0-6.5. Similarly, Chang et al. (2008) studied the initial pH effect on the submerged culture of Grifola umbellata. They analyzed the production of polysaccharides and optimized a different pH range from 3 to 8. They studied the initial pH in various pH ranges ranging from 3 to 8. They found that mycelial growth and EPS production were observed at pH 6 and 5. They reported that the EPS and the mycelial biomass were 0.571 g/L and 6.233 g/L, respectively. Similarly, Singh et al. (2020) studied the initial pH and temperature of mycelial production of P. eryngii under a submerged culture. They found that the initial pH of the medium did not change in the concentration of mycelia biomass. They reported the highest mycelia biomass at a concentration of 14.3 g/L. The present study revealed that the pH was decreased simultaneously with the increase in the concentration of Zn fortification in the mycelia.

Effect of EPS production

EPS are the external polysaccharides mainly present in the mycelium of the mushrooms. The EPS have various bioactive compounds comprising high nutritional value and medicinal properties. They possess medicinal properties such as anti-tumour, antioxidant, anti-microbial, anti-viral and anticancer activity. The present study of the extraction of EPS under submerged culture revealed the production of EPS of C. indica. The dried weight of EPS was observed at the different concentration ranges of mineral biofortification; similar to biomass concentration, the EPS production is also influenced by mineral supplementation, initially increasing with mineral treatment and then decreasing at higher concentrations, which is displayed in Table 2 and Figure 1. The highest EPS production was observed at 175mg/L (0.3853±0.006 g/L), and the lowest value of EPS was obtained in the control medium (0.2267±0.037g/L), respectively. It was reported that the mushroom and medium supplementation species could influence EPS production. Wu et al. (2008) studied the EPS production in A. auricula in a submerged culture. They optimized the culture condition in the shaking flask. They found the highest EPS production at the concentration of 3.9±0.04 g/L. Similarly, Zhang et al. (2011) studied the enhancement of EPS from P.tuber-region by submerged fermentation and reported maximum EPS production at the concentration of 0.73g/L after 7 days of fermentation. It has also been reported that polysaccharides are produced in the submerged culture of two edible P. ostreatus mushrooms. They extracted the internal and EPS and reported the average concentration of EPS and internal polysaccharides between 0.1-2g/L and 0.07-1.5g/L. Lim et al. (2004) studied the biomass production and EPS from Lignosus rhinoceros. They reported that the maximum polysaccharide concentration watch was 1.2 g/L. Liu et al. (2018) studied EPS hyperproduction by submerged Ganoderma culture. They found that the size of the inoculums was an essential factor for producing EPS and mycelial biomass. They reported that the mycelial biomass increases with the increase in inoculum size. They obtained biomass at 15.34 g/L and EPS at 0.76 g/L. Thus, fortified mushroom mycelium will be helpful in fortified food materials, and it has potency and efficacy in treating diseases like diabetes and cardiovascular diseases, etc.

Conclusion

The fortifying mushrooms with minerals have become a potential functional food, nutritional value, and food supplement. Fortifying mushrooms with Zn helps treat and prevent diseases such as cancer, cardiovascular diseases, immunological diseases, etc. The different concentrations studied the fortification of a mineral. The series of mineral concentrations in the submerged culture enhanced the production of EPS. The importance of this study is that a good concentration of EPS can be obtained from the submerged culture of C.indica. Another advantage of submerged cultivation is that the culture process is low-cost and easy for biomass production in fewer days than the in vivo culture. Mycelia produced through an optimal submerged culture with the potato dextrose medium is a good source of nutritional value and is also subject to an addition to bio-fortified food products. The growth conditions can be enhanced on the fermentation level to produce higher mycelial biomass. Thus, biofortified mushrooms have a good quality of alternative nutritious substrates and fight against malnutrition, a significant global problem. However, the technology for developing mineral enrichment techniques is needed for the large-scale commercial production of biomass.

Acknowledgement

The authors are thankful to the Head of the Department of the School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, for providing all the necessary facilities to execute research.

Tables

Table 1. Production of mycelia biomass at different pH and different concentrations of mineral supplementation

Data were represented as mean ± standard deviation; means within a column that are followed by the same letter do not differ (p< 0.05), and below is the ANOVA for the respective table.

Table 2. Production of EPS at different pH and different concentrations of mineral supplementation

Data were represented as mean ± standard deviation; means within a column that are followed by the same letter do not differ (p< 0.05), and below is the ANOVA for the respective table.

Figure 1. Production of mycelia biomass and EPS in g/L, influenced by pH

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