Mycelial growth of Philippine mushroom Lentinus tigrinus in selected cucurbit-based media and its antioxidant activity

Lentinus tigrinus, a basidiomyceteous fungus, is known to be nutritious and exhibits biological activities. The mycelial growth performance of L. tigrinus on selected cucurbit-based solid and liquid media, the ability to scavenge 2,2’diphenyl-11picrylhydrazyl (DPPH) free radicals, and the phenolic content of ethanolic extract of mycelia were investigated. Squash decoction gulaman (local crude agar) recorded the widest mycelial diameter (71.99 mm) regardless of the amount. However, 500 g/L of all cucurbit species registered the widest colony diameter of 73.35 mm. In liquid culture, both squash decoction and luffa decoction showed the highest mycelial biomass (0.09 g dry wt.). The highest mycelial biomass (0.14 g dry wt.) was noted at 500 g/L of all broth media. Extract of mycelia grown in winter melon decoction showed the highest scavenging activity (51.56%) and phenolic content (25.04 mg GAE/g sample). Therefore, decoction of the cucurbit fruits could be used as main ingredient of culture media for efficient production of mycelial biomass and improvement of antioxidant properties of L. tigrinus.


Introduction
Lentinus tigrinus, also known as Kabuteng tigre among Filipinos, is an edible fungus that is usually seen growing on decaying logs, stumps, fence posts, and trunks of dead trees. The mycelia of this mushroom have been successfully domesticated and the optimum culture conditions have been established (Dulay et al., 2012). Liquid cultivation of L. tigrinus mycelia using juices of selected tropical fruits such as coconut water, watermelon, honeydew, and grapes (Dulay et al., 2017a), and decoctions of carbohydraterich indigenous sources including rice bran, corn grit, and potato (Dulay et al., 2015a) have been demonstrated in our laboratory. These culture media significantly influenced the production of mycelia and antioxidant properties of L. tigrinus. Moreover, L. tigrinus contains carbohydrates, proteins, dietary fibers, reducing sugars, fats, and minerals, and its lyophilized water extract demonstrates blood glucoselowering effect in alloxan-induced mice (Dulay et al., 2014). Furthermore, Dulay et al. (2017b) reported that Original Article extracts of L. tigrinus also show antioxidant and antibacterial activities. Reyes et al. (2013) reported the anti-inflammatory property of this mushroom as shown by inhibition of IL-8 gene expression. It has also been reported that this mushroom has mycoaccumulation potential of heavy metals (Soriano et al., 2017). In our search for alternative media for liquid cultivation and biomass production of L. tigrinus, cucurbits were identified in the present work. Cucurbits (Cucurbitaceae) fruits are distinctly characterized by a cylindrically green fruit with thin rind when young and become thicker as they mature, with extremely diverse shape and size but with appropriate ratio of length and diameter of fruit (Bisognin, 2002). In Central Luzon, Philippines, cucurbits such as squash (Cucurbita maxima), luffa (Luffa cylindrica), bottle gourd (Lagenaria siceraria), and winter melon (Benincasa hispida) are commonly cultivated by vegetable farmers. Aside from additional source of income, cucurbit farming also provides nutritious foods for Filipinos, as they consumed cucurbit fruits as fresh salads, pickles, vegetable soups, and as among other native vegetable dishes. As nutritious food, they contain valuable nutrients and bioactive compounds. For instance, squash, irrespective of cultivars, has high beneficial fiber and minerals (Czech et al., 2018), whereas luffa contains compounds like phenolics, triterpenoids, ascorbic acids, oleanolic acid, carotenoids, flavonoids, α-tocopherol, chlorophylls, and ribosomeinactivating proteins (Azeez et al., 2013). These bioactive components are highly effective as immunostimulant, anti-inflammatory, anti-diabetic, antitumor, antiviral and oxytocic (Azeez et al., 2013). Kumar et al. (2012) mentioned that bottle gourd has high amounts of carbohydrates, proteins, fats, fibers, calcium, and magnesium, while Al-Snafi (2013) stated that winter melon is rich in saccharides, proteins, carotenes, vitamins, and minerals. Moreover, some cucurbits have also been reported to exhibit anthelmintic, emetic, and purgative properties (Robinson and Decker-Walters, 1997). It is hypothesized that the above-mentioned nutrients are important for the efficient growth of mycelia and could contribute to the nutritional value and functional activities of mushrooms. Given the rich nutritional status of cucurbits, it is therefore necessary to determine the effect of decoction of selected cucurbits at varying amounts on the mycelial growth of L. tigrinus on both solid and liquid culture conditions. It is also of significant interest to determine the antioxidant properties of the extract of the harvested mycelia grown in liquid culture.

Culture
Mycelia blocks were inoculated on potato dextrose agar plates and incubated for seven days. After incubation, a 10 mm-diameter cork borer was used to prepare the mycelial discs as inoculants in the evaluation of mycelia growth.

Preparation of solid and liquid cucurbit-based media
The cucurbits (squash, luffa, bottle gourd, and winter melon) were purchased from the local market. These were peeled, washed, chopped, and weighed at different amounts (100 g, 250 g, and 500 g). Each amount was boiled into 1 L distilled water and the decoction was filtered using cheesecloth. A 500 mL of decoction was reboiled and 10 g of gulaman (agar) was added. This was dispensed into a dry clean 1Lcapacity flask. On the other hand, in preparing liquid media, 50 ml (from the remaining 500 mL decoction) decoction was dispensed into a glass bottle. Ten bottles were prepared for each decoction. Media were autoclaved for 30 min at 15 psi and 121°C. Flask media were pour-plated into sterilized petri plates in triplicate and allowed to solidify prior to inoculation.

Evaluation of mycelial growth
Both solid and liquid cucurbit-based media were aseptically inoculated with the mycelial disc. After five days of incubation at 30°C, mycelial growth diameter was measured using a Vernier caliper. The optimum amount of cucurbit and the best cucurbit type were determined based on the colony diameter and density of mycelia. On the other hand, inoculated culture bottles were incubated for 15 days in a static condition at 30°C. The mycelia were harvested, air dried, and weighed. The best cucurbit type and amount were identified based on the dry weight of mycelial biomass. The air-dried mycelia from the best concentration of each cucurbit were pulverized prior to extraction.

Determination of antioxidant property
Five grams of the pulverized samples were extracted in 300 mL of 95% ethanol and concentrated in a rotary evaporator until dryness and were prepared for the Asian J Agric & Biol. 2020;8(3):323-329 assay. The ability of the extracts as scavengers of 2, 2'diphenyl-1-1picrylhydrazyl (DPPH) free radicals was determined following the procedure of Kolak et al. (2006) with minor modifications. However, phenolic content was estimated using the Folin-Ciocalteu method of Sunita and Dhananjay (2010).

Statistical analysis
The SAS System Version 9.0 (SAS Institute Inc. Cary, NC, USA) was used to analyze and compare all the data.

Mycelial growth and biomass production in cucurbit-based media
The mycelial diameter of L. tigrinus grown on selected cucurbit-based solid media after five days of incubation is presented in Table 1. It can be seen that from among solid media, squash decoction gulaman recorded the widest mycelial diameter, whereas bottle gourd gulaman had the smallest. In terms of the amount, 500 g/L showed the widest mycelial diameter, while 100 g/L registered the smallest. Tukey's HSD revealed a significant difference among cucurbit types and cucurbit amounts. At 500 g/L of all media produced very thick mycelial density except bottle gourd gulaman (Figure 1). However, the mycelial diameter and mycelial density of L. tigrinus were improving in increasing amount of all cucurbits decoction media. The effect of selected cucurbit-based liquid media on the mycelial production of L. tigrinus was also evaluated. Table 2 shows the mean dry weight of mycelial biomass after 15 days of incubation. Both squash decoction and luffa decoction recorded the highest mycelial dry weight regardless of the amount.  On the other hand, the lowest biomass yield was noted in bottle gourd decoction. Similar to solid media, the mycelial biomass yield increased as the amount of cucurbit increases, and so 500 g/L had the highest biomass yield. The liquid cultures of L. tigrinus in the varying amounts of cucurbits are shown in Figure 2. Asian J Agric & Biol. 2020;8(3):323-329 These results indicate that the mycelial biomass production of L. tigrinus is dependent on the amount and type cucurbit as basal media in the liquid culture.

Antioxidant property of mycelia
The percentage radical scavenging activity of the extracts of mycelia is presented in Table 3. Apparently, the scavenging activity was significantly affected by the different cucurbit types. Extract of mycelia from winter melon decoction recorded the highest scavenging activity. This was followed by the mycelial extract from luffa decoction. On the other hand, mycelial extracts of both squash broth and bottle gourd broth registered almost the same activity. Phenolic content of the mushroom extracts was also quantified. Results of the analysis revealed that extract of mycelia grown in winter melon had the highest phenolic content, but statistically comparable with the phenolic contents of mycelial extracts from luffa decoction and bottle gourd decoction (Table 3).

Discussion
In mushroom production, the growth performance of mycelia is dependent on the type of culture media. Mycelial biomass production in submerged cultivation or liquid culture is of greater interest than the conventional lengthy production process because the former is a fast and efficient alternative of producing biomass and metabolites (Dulay et al., 2015b). In this work, four selected cucurbit-based media at varying amounts were evaluated in both solid and liquid culture conditions. Squash decoction gulaman was found to be the most suitable cucurbit type and 500 g/L was the optimum amount of each cucurbit for the luxuriant growth of L. tigrinus mycelia in both state of cultivation. The superiority of squash decoction as solid and liquid media for the growth of mycelia of L. tigrinus could probably be attributed to its essential chemical components. Squash contains carbohydrates, proteins, reducing sugars, amino acids, tocopherols, carotenoids, and minerals (Sharma and Rao, 2013;Adebayo et al., 2013;Czech et al., 2018;Kim et al., 2012), which are essential for physiological function. Moreover, Karanja et al. (2013) reported that squash has βcarotene (provitamin A) and fatty acids (linoleic acid, oleic acid, and palmitic acid). In our previous work (Dulay et al., 2017c), supplementation of varying amounts of vitamin A in the culture media stimulated the mycelial growths of L. tigrinus, Schizophyllum commune, and Ganoderma lucidum, and the vitamin A-enriched substrate improved the yields and biological efficiencies of L. tigrinus and G. lucidum. Luffa decoction was also identified as a suitable medium for mycelial biomass production in liquid culture. Luffa contains sugars and lignocellulose (Siqueira et al., 2010). Petre et al. (2016) mentioned that lignocellulose improves the growth of the mycelium through stimulation of the enzymatic activity. In addition, luffa has high amounts of essential mineral elements such as sodium, potassium, magnesium, and calcium, and rich in nutrients like sitosterol, β-tocopherol, phytol, stigmasterol, and hydrocarbons (Adewuyia and Oderinde, 2012). The above-mentioned nutrients of squash and luffa are also present in coconut water, which serve as the most suitable indigenous medium for the luxuriant mycelial growths of L. tigrinus and L. strigosus (Dulay et al., 2012;Dulay et al., 2017d). Although most of these nutrients are also components of bottle gourd and winter melon, the inferior performance of mycelia in these liquid media could probably be accounted to the presence of phenolics compounds, which are inhibitory to the normal physiology of the fungal cells, thereby affecting the growth and development of the mycelia. Pizzolitto et al. (2015) reported that some natural phenolic compounds showed inhibitory effects on the growth of fungus, Apergillus parsiticus. Several mushrooms have been reported to exhibit antioxidants properties, which neutralize the free radicals that damage the cells resulting to aging and degenerative diseases such as cancer. These antioxidant properties of mushrooms include carotenoids, ascorbic acids, ergosterol, tocopherols, polysaccharides, and phenolics (Sanchez, 2017). In general, the physico-chemical characteristics of mushrooms are dependent on the properties and compositions of medium or substrate (Oyetayo and Ariyo, 2013). The antioxidant properties of the extracts of mycelia grown in 500 g/L of each cucurbit were also determined in our intention to establish the effect of cucurbit-based media not only on the growth performance but also on the antioxidant activity of L. tigrinus. Surprisingly, the squash decoction that showed the highest yield of mycelial biomass exhibited the lowest scavenging activity and phenolic content. This observation does not conform to our hypothesis that the mycelia grown in the most suitable medium would exhibit maximum antioxidant properties. Interestingly, mycelia grown in winter melon decoction recorded the maximum antioxidant properties of L. tigrinus.
The radical scavenging activity and phenolic content of L. tigrinus was found dependent on the type of cucurbit. The same finding was also reported in our previous works. For instance, the scavenging activity of L. tigrinus is dependent on the type of liquid media, in which rice bran broth registered the highest radical scavenging activity (18.94%) among indigenous liquid media (Dulay et al., 2015a). In addition, L. tigrinus mycelial extract from honeydew juice recorded the highest scavenging activity (79.79%) among selected tropical fruit juices (Dulay et al., 2017a). Moreover, the maximum radical scavenging activity and phenolic content of Pleurotus djamor and G. lucidum were found in the extracts of mycelia grown in corn grit decoction and coconut water, respectively (Bustillos et al., 2018). These findings of previous and current works clearly suggest that mycelia of L. tigrinus and other mushrooms can be manipulated by the culture media to exhibit higher antioxidant properties, which indicates a significant advantage in the development of functional food and nutraceuticals.

Conclusion
Altogether, the present study has shown for the first time that decoction of the fruit of cucurbits could be utilized as alternative basal media for the efficient growth of mycelia of L. tigrinus in both solid and liquid culture conditions and for the enhancement of the antioxidant properties. The cultivation technique demonstrated in the present work represents significant contribution in the Philippine mushroom industry as well as in the nutraceutical and pharmaceutical industries. However, it is necessary to further elucidate other biological properties of the L. tigrinus extract, which are essential in the development of useful products for the benefits of mankind.