Our present study demonstrated that a new strain engineered with genetic variance was able to produce an increased proportion of pigments with nutritional value through protoplast fusion. Protoplast fusion allowed the generation of fusant with the properties of parental strains and illustrated the effectiveness of fusant for commercial applications.
The production of protoplasts was a critical step for fusion and regeneration. The age of the mycelium, the combination of enzymes, and the osmotic stabilizer are all essential elements to obtain protoplasts. In the present study, young mycelium was used to isolate protoplasts. The major reason is that the lytic action of digestive enzymes is more sensitive to young mycelium than to old mycelium [21]. Protoplast fusion, gene transfer, and metabolite productions are examples of physiological and genetic research that can benefit from fungal protoplasts. A recent study optimized several critical variables for the synthesis and regeneration of protoplasts from strain CH1. The highest yield of protoplasts was produced using an enzyme mixture consisting of cellulase, glusulase, and driselase in an osmotic stabilizer [37]. Similarly, another study indicated that in an osmotic stabilizer (0.6 M KCl), Trichothecium roseum protoplasts were extracted using the combining the lytic enzyme. A maximum number of protoplasts was achieved at pH 5.5. Protoplast regeneration and reversal attempts revealed a maximum regeneration (60.8 %) ina complete PDA media [9]. Consistent with the above findings, protoplast fusion and regeneration showed maximum regeneration. In the present study, the protoplast yield of M. ruber and P. ostreatus was 2.31×107 cells /ml and 2.33×107 cells/ml under standardized conditions. At pH 6, higher amounts of protoplasts were released from M. ruber (2.21×106 cells /ml) and P. ostreatus (2.24×107 cells /ml). M. ruber treated with chitinase (0.2 mg/ml) and β-glucuronidase (0.1 mg/ml) yielded 2.36×106 cells /ml of protoplasts and for P. ostreatus treated with macerozyme (0.1 mg/ml), cellulase (10 mg/ml), pectinase (0.2 mg/ml) yielded 2.01×107 cells/ml of protoplasts. 0.6M KCl showed a high yield of protoplasts 2.36×106 cells /ml from M. ruber and 2.46×107 cells /ml of protoplast from P. ostreatus.
Osmotic stabilizers, including inorganic salts, sugars, and sugar alcohols, were used to stabilize protoplasts isolated from mycelium. The concentration, molarity, and the type of stabilizer can impact the yield of the protoplast [26, 35]. Depending on their lifestyle, filamentous fungi obtain their nutrients from the environment. Filamentous fungi rely on external carbon sources because they are heterotrophic, but many can absorb inorganic nitrogen and other nutrients for growth and metabolism. They can make all the necessary amino acids from inorganic nitrogen [29]. Many protoplast isolation studies indicated that 0.6 M KCl acts as the best osmotic stabilizer [15, 18]. On the contrary, some protoplast isolation studies indicated that 0.4 M NaCl was effective in protoplast yield [14, 25]. Notably, the molecular weight of PEG is essential in the fusion process. A recent study indicated that the optimal osmotic stabilizer for the maximum interspecific fusion frequency was 40 % PEG with 10 mM CaCl2,10 mM Tris-HCl (pH 7.5). Intraspecific fusion frequency was 6.2 and 7.2 % for T. harzianum and. viride [31]. Another investigation indicated that T. reesei and T. harzianum 40 % PEG with STC buffer weresuitable for intrafusion. When the protoplasts were exposed to the PEG solution, they were attracted and connected together as pairs, and the plasma membrane collapsed into merged protoplasmic contents, finally culminating in the fused protoplasts becoming single and enormous [34]. The present study used incubation with PEG (50% v/v), 10 mM CaCl2, and 50 mM glycine for the protoplast fusion. The maximum level of fusion frequency was 7.1%.
The culture medium for regeneration is significant for the regeneration of fused protoplasts. PDA supplemented with 0.6 M KCl was used as culture for protoplast regeneration in the present study. The regeneration frequency was 71%. The resulting fusant grows faster than the parent strains. A report indicated that the frequency of regeneration of G. putredinis and T. harzianum was 51 and 61%, respectively [38]. A study reported that a good regeneration medium for T. harzianum protoplasts was Potato Dextrose Yeast Extract Agar medium supplemented with 0.6 M KCl, and the fusant showed a faster growth rate compared to the parents [10, 40]. The regenerated colonies were screened for the resistant character to confirm fusant. Colony resistance to nystatin and fluconazole is confirmed because the regenerated colonies obtained are fusant, which is also confirmed by the mycelial patterns of both the parent and fusant strains. It was also used to identify the colony morphology greatly differs in the fusion products [10, 40]. In M. ruber, hyphae - a thread-like tubular filamentous structure had septate in its cross walls with lipid droplets. In contrast, the fusant doesn't have any septate in its cross walls with lipid droplets. In the present study, the mycelia growth pattern and colony morphology were also used as a marker for fusant identification.
The extracellular and intracellular pigment production of M.ruber and Fusant were presented. The pigment level was increased in the fusant strain than in the parental strain. Fusion of protoplast from M. ruber and P. ostreatus may result in genetic inference at the metabolic level, which was the reason behind the elevation of pigment in fusant colonies. A recent study observed a variation or stimulation of pigment production during protoplast hybridization in other species of fungi [22]. The proximate composition of parental and fusant strains contains 7.28% of moisture, 4.87 % f ash content, 11.01% of cured protein, 7.16% of curde fat, 51.01% of carbohydrates, and 8.43% of crude fiber, respectively.
The freshness, flavor, and storage performance of edible fungus are affected by moisture content. Among the 23 edible fungi, there was a difference in the moisture content, nearly about 6.9∼15.5 g/100 g. Ash content reflects the minerals, and minerals were the sole source of bones, hemoglobin, and acid-base balance of the body [47]. Torres et al., 2000 reported that the ash levels were compared to other nutritional sources. It was possible to observe much greater levels than those found in foods such as milk, eggs, and some beef cuts [41]. Lentinus edodes (Berk.) Sing had the lowest ash content of 1.27 g/100 g [47]. Carbohydrates are essential for structural compositions and energy release. Dietary fiber is known as the "gut scavenger." therefore, a higher intake of edible fungi rich in dietary fiber can help prevent various diseases and meet the requirements of changing the diet structure generally advocated at home and abroad [47]. Five kinds of edible fungi have more than 50% dietary fiber content, including Ganoderma lucidum (Leyss. ex Fr.) Karst. had the highest dietary fiber content, 70.2 g/100 g [47]. The fiber content in the mushrooms is good for essential compounds that exhibit different physiological and nutritional benefits [42]. The crude includes lipids, free fatty acids, mono, di, triglycerides, sterols, sterol esters, and phospholipids. Fatty acid compositions differ from each fungi species and are an essential component of organelles comprising about 30–70% of fungi [36]. Pleurotus species contain low lipids and excellent sources of fatty acids like linoleic acid and oleic acid [36]. The earlier research evidence that Pleurotus species are good candidates for anti-inflammation and hypocholesterolemia in the human diet [36]. Pleurotus mushrooms are exceptionally high in folic acid (B9), also known as folic acid, a nutrient that cannot be produced in the body and must be supplied by the diet [36]. Pleurotus species are good sources of palatable proteins and are rich in minerals (Na, Ca, P, Fe, and K) and vitamins (vitamin C and B complex) [36]. A considerable difference was observed in the micronutrients and minerals present in the fusant than the parental strain in the present study, and it was confirmed that the hybrid has the characteristics of M. ruber and P. ostreatus.
In the present study, we have developed a new strain with genetic variance capable of producing an increased level of pigment with nutritional value through protoplast fusion. Protoplast fusion allows the generation of fusant with the characteristics of parent strains and elucidates the effectiveness of fusant for commercial applications.