Research Article | Open Access
Anindita Sinha and Keka Sarkar
Department of Microbiology, University of Kalyani, Kalyani, Nadia, West Bengal, India.
Article Number: 8567 | © The Author(s). 2023
J Pure Appl Microbiol. 2023;17(3):1458-1470. https://doi.org/10.22207/JPAM.17.3.07
Received: 13 March 2023 | Accepted: 19 June 2023 | Published online: 20 July 2023
Issue online: September 2023
Abstract

This study aims to reconcile industrially suitable strains of higher red pigment producing Monascus purpureus MPA5 and higher Monacolin K producing Monascus ruber MRA7, were tried to fuse for development of increased efficacy of targeted secondary metabolites production. The optimum concentration of protoplast mixture was standardized, before the fusion experiment. The effective fusion was observed with the solution of 30 % PEG 6000, CaCl2.2H2O (0.01 M) and glycine (0.05 M). Colonies of the fusants were segregated using complete medium. The fusants were irradiated under UV- light in LD50 dose. On the basis of non-segregation of colony appearance after repeated subculturing in complete medium, 8 colonies were selected. Finally, one of the stable fusant MF11 grew more rapidly than one of the parental strain M. purpureus MPA5, but not as fast as M. ruber MRA7, were selected and grown on low grade rice as solid substrate for production of metabolites. Production of red pigments, alpha-amylase and acid protease were at intermediate stage between two parental strains but enhancement of Monacolin K was observed. High performance liquid chromatography (HPLC) analysis was revealed that Monacolin K content was increased as much as two and half times than the parents. No fluorescent yellow band was detected in TLC plate, indicating of citrinin negativity of the fusant MF11. It is the first report of enhanced Monacolin K production by a stable fusant of Monascus sp.

Keywords

Protoplast fusion, Monascus purpureus, Monascus ruber, Monacolin K

Introduction

Protoplast fusion is a skilled technique which generates and induces genetic recombination and has been attempted in prokaryotic and eukaryotic cells including filamentous fungi.1 Fusion can be done either between wild type or mutated strains, strains with incompatible heterokaryon, auxotrophic mutant and prototrophic strains.2 Experimentally two protoplasts are tried to fuse to acquire parasexual hybrid protoplast, which contain heteroplasmic cytoplasm and fused nuclei of two parents.3

However, development of hybrid by protoplast fusion could be achieved either by closely related species or even distantly related.4 The differentiation of the whole organism involves by the regeneration of protoplast into a normal cell.5 Only a few reports exist on intergeneric fusion6 but intra-specific and interspecific protoplast fusion are more common and includes Mucor,7 Rhizopus,5 Aspergillus,8 Penicillium9 and Trichoderma.10 Monascus sp is a filamentous fungus which is important for production of pigments that are polyketide origin.11 Monascus contain three major species (M. purpureus, M. pilosus and M. ruber) under the family Monascaceae within the class Ascomycetous.12 The Monascus is massively used for coloring of rice, cheese, soybean and meat in different oriental countries like wise food colorant by using the red pigment.13 Apart from this, in Asian countries the pigment gains a long-established food value though it is still forbidden in Europe and America. However, in recent years there are a number of patents registered in Japan, the United States, France and Germany reporting Monascus as food pigment. In spite of pigments, Monascus fermented red rice is also important for different metabolites like monacolin K and L (act as antihypercholesterolemic agents),g-aminobutyric acid or GABA (act as hypotensive agent), citrinin (a mycotoxin) are found.14

Fermentative production of pigments and monacolins can be obtained using Monascus sp. in both solid state fermentation (SSF) and submerged fermentation (SmF).11 However, solid state fermentation leads to higher yield than submerged.15 An active methylated compound, Monacolin K was recovered from fermented broth of M. ruber. 16 Monacolin K (lovastatin, mevinolin and mevacor), a competitive inhibitor of HMG-CoA reductase, a very important enzyme for rate limiting action in biosynthesis of cholesterol, which is helpful in the catalysis of the reduction of 3-hydroxy-3-methylglutaryl coenzyme-A to mevalonate in biosynthesis of cholesterol.17 Monacolin K was used as common medicine in the treatment of hypercholesterolemia.18 FDA approved Monacolin K (MK) to use as hypocholesterolemic drug and this is the first fungal secondary metabolite to received FDA approval in August 1987.19 In addition to its lipid-lowering activity, some studies have shown that MK prevents thrombosis,20 reduces the incidence of atherosclerosis,21 and stimulates bone formation,22 induction of cancer cell death,23 Parkinson’s dsiseases24 and Alzheimer’s treatment of disease.25 Therefore, RMR can be considered as a functional important compound and applied to the food industry. Though Monascus sp. is able to synthesize different useful secondary metabolites, but able to produce citrinin also, which is a mycotoxin having hepatotoxic and nephrotoxic effects in humans.26 Therefore, citrinin becomes a very critical issue in consideration of red rice as a safe food supplement. Maximum permitted levels of citrinin in functional foods are approved at 0.2 mg/kg in Japan and 2 mg/kg in Taiwan.27 However, some reports have been found where the citrinin concentrations in some commercially available red rice where were detected between 0.28 to 2,458.8 mg/kg.12

Traditionally Monascus was grown on rice to prepare red yeast rice, though different liquid media or solid media were applied to produce the secondary metabolites. The traditional method of fermentation is SSF but most of the industry used submerged or liquid fermentation for industrial fermentative processes.15 Though it is possible in modification on cultural conditions in Monascus sp. positively have an effect on secondary metabolites constituents.28 Variation of environmental factors may conclusively affect the production ability of the test organism, likewise in Monascus sp.

The experimental work emphasizes to standardize the protocol for the fusion technique to develop hyperactive hybrids, in terms of quality or quantity. In this study, attempts have been made to develop phenotypically stable fusant between high pigment producing M. purpureus MPA5 and higher Monacolin K producing strain of M. ruber MRA7 and to select strain with dual traits.

Materials and Methods

Organisms and Media
M. purpureus MTCC 410 and M. ruber MTCC 1880 were used as source organisms. MPA5 and MRA7 are the mutant strains developed from M. purpureus MTCC 410 and M. ruber MTCC 1880, respectively and used as parental strain for protoplast fusion. Cultures were grown and maintained in Malt Yeast (MY) medium (g-1): glucose 10.0, yeast extract 3.0, malt extract 3.0 and peptone 5.0; pH 7.0, agar 2% used for solidification. Minimal medium (MM with50 µg ml-1 amino acids and 0.2% yeast extract was added, Czapek Dox (CD) medium was used to select fusants. For protoplast fusion and regeneration hypertonic (0.6 M KCl) Malt Yeast Agar (MYA) and Czapek Dox Agar (CDA) were used.

Mutagenesis of Parental Strains for Auxotroph Formation
Conidial suspension of M. purpureus MTCC 410 and M. ruber MTCC 1880 were prepared from 7-day-old culture in MYA and passed through filter paper to obtain 10 ml suspension of 1×108 conidia ml-1. This conidial suspension in watch glass was exposed to ultraviolet radiation in a laminar air flow for 5 to 30 minutes, with an increment of 5 minutes. To avoid photo reactivation after irradiation, the samples were kept at dark for 5 hours29. After dark treatment 0.1 ml of the irradiated suspension were plated in MYA and incubated at 30±2°C for 4 days. From the plate of 1% survival, colonies were selected visually and the isolates were transferred to plates containing MYA medium. Purity of the isolates verified by repeated streaking in MYA plate. Selected colonies were tested for their auxotrophic nature. Conidial suspension of the isolates was grown in Czapek Dox broth as minimal medium, incubated at 30±2°C for 4 days. Ungerminated spores (mutant) separated from developed germlings by glass wool filter. This process was repeated thrice to confirm that the remaining spores were not of the wild type. The selected isolates were repeatedly grown in Czapek Dox broth with amino acids(s) (50µg l-1) supplementation and in combination to confirm their auxotrophic nature.

Protoplast Generation
For protoplast generation, 1 ml of spore suspension (~2×108 ml-1) of the selected auxotrophs were inoculated into 100 ml MY broth medium and then incubated at 30±2°C for 48 h in rotary shaker (120 rpm). After incubation both the organisms were centrifuged in 10,000 rpm for 20 min and the suspended pellets were aseptically washed using suitable osmotic stabilizer (0.6 M KCl), at pH 6.0 after standardization.

Following the method of Klinsupa and co-researcher (2016), the germinated spores were suspended in hypertonic solution of KCl, NaCl, MgSO4 and Sucrose (0.6 M) separately containing cell wall lytic enzymes of chitinase (1% – 3%) and cellulase (0.2% -0.5%), incubated at 30±2°C up to 5 h at 50 rpm. Samples from suspension were checked for protoplast formation at every 30 min intervals under microscope to determine the time required for the development of protoplasts. Then protoplast suspension was centrifuged (1000 rpm for 5 min) to remove enzymes and enumerated with a hemocytometer under a phase contrast microscope. The rate of protoplast formation (%) was estimated by the numbers of protoplast divided by the total cells.

Protoplast Fusion and Regeneration
Protoplast fusion was attempted with certain modification. Both the protoplasts were mixed (1:1) and maintained at 0.6 M KCl solution and centrifuged at 1000 rpm for 10 min. The pellets were suspended in the solution containing verify concentration of PEG with 0.01 M CaCl2.2H2O and 0.05 M- glycine in 1:1:1 ratio, pH 6.0 and incubated at 30±2°C up to 15 min and observed under microscope. The fused protoplasts were centrifuged at 1000 rpm for 10 min. The pellet was washed with 0.6 M KCl and centrifuged twice. The pellet suspended in hypertonic Czapek Dox medium and incubated at 30±2°C for 24 h.

After incubation the culture (1 ml) was spread over minimal and complete medium, at hypertonic condition and covered with the same soft agar (0.5%) medium to make a sandwich plate and incubated at 30±2°C for 7 days. The colonies developed in the minimal medium were selected the fusants that could grow without any requirement of amino acids. The protoplast formation rates were considered by the number of colonies which appeared on minimal medium as a percentage in number that grew on complete medium.

The isolated regenerants were grown onto MYA medium at 30±2° C for 7 days. The conidial mass (~1×108 conidia/ml) was exposed to UV for different time interval to find LD50. For isolation of stable fusants, the conidial sample (~1×108 conidia/ml) was treated at LD50 dose and kept in dark for 5 hours. Then the sample was put on MYA medium plate and incubated at 30±2°C for 7 days. The appeared colonies were transferred to MYA slant and checked it for their stability.

Reversion of Protoplasts
Mycelial protoplasts of two isolates of Monascus sp. mutant (isolate MPA5 and isolates MRA7) were cultured in MY broth medium containing 1.2 M sorbitol and incubated at 30±2°C for 24 h. Different dilution at appropriate concentration were prepared, they were spreaded onto MY agar medium with 1.2 M sorbitol as supplementary. 0.5% agar was followed to cover and allowed to incubate at 30±2°C for 7 days. The following formula was used to calculate the reversion of protoplast onto mycelia and cell as percentage:

% protoplast reversion= Number of colonies grown on MY agar medium / Number of initial protoplasts׳100

Measurement of Secondary Metabolites
Extracellular Red Pigment Production and Quantification
Parental cells, their auxotrophs and the isolated fusants were grown on MYA medium at 30±2°C for 7 days. Then spore solution was prepared (0.9% NaCl and 0.2% Tween 80).30 Homogenous spore suspension (0.5 ml) was inoculated into 10 g of sterilized rice with 56% humidity and incubated at 30±2°C for 14 days. The rice was air dried in room temperature after fermentation and pigment was extracted by using 5 ml of ethanol in 95% concentration under shaking (110 rpm) for 60 min. The centrifuged of the extract was done at 10,000 rpm for 15 minutes, the supernatant was decanted and the extracellular red pigment concentration determined by spectrophotometer at 500 nm.12

Quantitative Estimation of Lovastatin (Monacolin K):
Slightly different method was applied for extraction and estimation of monacolin K from fermented solid materials, liquid chromatography procedure followed using ODS columns (150׳4.6 nm ID) were used, and varied elution systems were attempted. Solid fermented products were dried and ground into powder. Powered sample (1 g) was mixed with 10 ml of orthophosphoric acid /water solution (75:25) and kept at 60°C under agitation for 10 minutes. Ethyl acetate (10 ml) was added to the extract solution, followed by 30 minutes treatment in a water bath at 60°C and centrifugation at 5000 rpm for 10 minutes. Vacuum concentrated supernatant (1 ml) supernatant was filtered through 0.45µM syringe membrane before infecting 20µL into column for HPLC. The mobile phase was used as a linear gradient of concentrated acetonitrile (ACN) (eluent A) and 0.1% TFA (eluent B) with the flow rate of 0.5 mL min-1. The chromatogram was detected at 237 nm, and the temperature of column was set at 28°C.

Enzymatic Estimation
For detection of common hydrolyzing enzymes, amylase and acid protease estimation were carried out. After SSF, the fermented rice (10 gm) material was taken in a flask (100 ml), the enzymes extracted with 0.5% NaCl (100 ml) and filtered through Whatman number 1 filter paper. Then filtrate was analyzed for estimation of amylase,31 and acid protease (Ichishima 1970) by starch and casein, respectively, as substrate. Amylase activity was assayed in terms of liberated reducing sugars (glucose equivalents) using dinitro-salicylic acid (DNSA). Acid protease was estimated in terms of released tyrosine equivalent using Folin-Ciocalteau Phenol reagent.

Qualitative Estimation of Citrinin
To detect citrinin, thin layer chromatography (TLC) was performed.32 Fermented substrates was extracted with the presence of ethyl acetate and acidified to pH 5. Then the aqueous layer was evaporated to dryness. The concentrated organic layer was loaded to TLC plate 60 F254 aluminum sheets (Merck, Germany). The mobile phase was used as ethyl acetate: acetone: water as 4:4:1. After completion of TLC run the plates were examined under UV light at 350 nm. A fluorescent pale yellow band was detected for presence of citrinin.

Biomass Estimation
After incubation the biomass of the fungal culture during growth was estimated by a gravimetric analysis method by using a digital balance. After filtration of the culture broth, the mycelia were separated from the broth on pre-weighed filter paper (Whatmann filter No. 1 filter paper). The culture was dried at 80°C and biomass was determine in grams per litre.

Statistical Analysis
Experiments carried out in triplicate and the graphical data were presented as mean ± standard error (SR) as required. The statistical analysis was performed using OriginPro 8.5 statistical software.

RESULTS AND DISCUSSION

Formation of Auxotrophic Mutants from Monascus Purpureus and Monascus Ruber
Spores of M. purpureus and M. ruber, that were UV irradiated for different time periods were plated in MYA medium and incubated for colony development. Primary selection of colonies was done from plates that showed 1% survival rate of the irradiated spores. UV exposure of 25 min and 30 min provided this survival rate for M. purpureus and M. ruber, respectively (Figure 1). Finally, 1×105 colonies were selected. Then the selected isolates were sub-cultured three times in the same medium and checked for stability of their physiological and morphological characters. To facilitate the process of selection during protoplast fusion, auxotrophic strains are preferred. To determine the auxotrophic nature of the isolates were allowed to grow in both minimal and complete media and three auxotrophic colonies of each parental strain were considered on the basis of their linear growth (mm). For identifying the amino acid requirements, auxotrophs were grown in Czapek Dox medium with supplementation of amino acid (s) either singly or in combination. On the basis of growth rate that measured as mycelial mass (g) on MY medium (100 ml) two strains were selected. The isolates MPA5 (arg), mutant of M. purpureus and the isolate MRA7 (thi, met), mutant of M. ruber were selected for further study (Table 1). Various treatments were found in literature for auxotrophic mutant formation like a combination of UV-light and sodium nitrate was used to induce mutation of Trichoderma sp.33 Nitrosoguanidine ((N-methyl-N’-nitro-N-nitrosoguanidine) and UV-light separately used as mutagenic agent for auxotroph formation of P. griseoroseum and P. expansum respectively.34

Table (1):
Mycelial mass and secondary metabolites estimation of M. purpureus, M. ruber and their stable selected mutants after incubation in MY medium at 30 ±2°C for 7 days.

Strains
Auxotrophs
Red pigment assay (U/gdfs)
Mycelial mass (g/100 ml)
M. purpureus
58.12
9.0 ± 0.4
MPA4
Thi,Arg
70.23
5.2 ± 0.3
MPA5
Arg
75.15
8.5 ± 0.4
MPA8
Leu
72.22
6.5 ± 0.2
Strains
Auxotrophs
Monacolin K assay (mg/g)
Mycelial mass (g/100 ml)
M.ruber
0.34
9.5 ± 0.2
MRA3
Thi, Leu
0.71
8.5 ± 0.2
MRA7
Thi, Met
0.84
10.5 ± 0.2
MRA9
Lys
0.63
7.5 ± 0.2

Figure 1. Determination of % survival of spores of M. purpureus and M. ruber during different time exposure (mins) of UV light. Values represent the mean±SR

Protoplast Formation from Mycelia of MPA5 and MRA7
For protoplast formation, 48 hr-old mycelia of MPA5 and MRA7 were incubated with enzymes of chitinase and cellulase (Sigma, USA) in different concentrations and combinations along with osmotic stabilizer (Figure 2). To find out a suitable osmotic stabilizer, the mycelia of MPA5 and MRA7 were treated with 1% chitinase + 0.5% cellulase enzymes in presence of various osmotic stabilizers in 0.6 M concentration and KClwas found most effective (Figure 3 A & B). Thereafter, the enzyme concentrations were varied to identify the optimum condition for protoplast formation. In case of MPA5, 1% chitinase (10 mg/ml) and 0.5% cellulase (5 mg/ml) found to produce maximum number of protoplasts (5.1×105/ml) in 4 hrs (Figure 4A) wherein MRA7 produced maximum number (7.2×105/ml), using 1.2% chitinase and 0.3% cellulase (Figure 4B). When the protoplast were incubated in minimal medium with 0.6 M KCl (regeneration media) the mycelia were found regenerating. The regeneration frequency of MPA5 and MRA7 were found 35.6% and 8.2%, respectively. KCl (0.6 M) was also found most effective osmotic stabilizer for protoplast formation in Aspergillus awamori and A. oryzae35and in Monascus anka.36 Protoplast formation frequencies of M. anka MKA1 were optimized using 3% Usukizyme8 and using 0.3% snailase + 0.3% cellulase37. Protoplast formation was induced with 2% Usukizyme+ 0.4% Novozyme 234 in A.oryzae AOK18 and additionally 0.5% lywallzyme in A. terreus CA99.37 So from the previous report it showed that our result is in accordance with that reports.

Figure 2. Microscopic photograph of protoplast formation mixture of MPA5 and MRA7

Figure 3. Effect of 0.6 M KCl (a), NaCl (b), MgSO4(c) and Sucrose (d) during protoplast formation of  MPA5 (A) andMRA7 (B) at different time interval using 1% chitinase + 0.5% cellulase enzyme treatment. Values represent the mean±SR

Figure 4. Effect of various concentration of chitinase and cellulase on the protoplast formation of MPA5 (A) and MRA7 (B) at different time interval using 0.6 M KCl osmotic stabilizer. Values represent the mean±SR

Inter-specific Protoplast Fusion between MPA5 and MRA7
To study the frequency of formation of prototrophic colonies, the efficacy of fusogen (PEG 4000 and 6000) were tested. Figure 5 showing the representative photograph of protoplast fusion between isolate MPA5 and MRA7. After protoplast fusion, it found (Figure 6) that the prototrophic colonies appeared in all plates in the range of tested PEG concentration but PEG 6000 at 30% induced maximally (1.24×103 CFU/ml). Thus, the colonies developed in hypertonic minimal medium appeared as fusants between MPA5 and MRA7, (Figure 7 A, B) while no colonies appeared in non hypertonic medium. Also the fusants could found divided into white and red portions while they were allowed to grow in complete medium for 10 days (Figure 8A). Stability of the fusants were tested by irradiated with UV exposure in different time intervals and kept for dark treatment. After plating the UV exposed plates, LD50 dose was determined and found that 6×102 colonies were grown in complete media. From them eight colonies were further selected because the hyphae were not segregated even after sub cultured several times in the complete media (Figure 8B). Finally, the stable fusant MF11 grew more rapidly than one of the parental strain M. purpureus MPA5, but not as fast as M. ruber MRA7. PEG-induced protoplasts fusion of fungus was influenced by the presence of cation and different concentration of them. PEG with 0.01 M CaCl2 and 0.05 M glycine increased the number of fused colonies in M. anka36. The fusion frequency of M. purpureus MPA5 and M. ruber MRA7 was ascertained as 0.3% (Table. 2). The protoplast fusion frequencies were observed as Penicillium rouqefortii and Penicillium chrysogenum as 0.1% to 0.7% (Leza et al., 2011), M. anka and A. oryzae as 0.14% 8. In this present study, the protoplast fusion frequency is not less significant than reported once.

Table (2):
Protoplast fusion frequency, reversion and fusion formation estimation between MPA5 and MRA7.

Strain Used Strain Used Reversion frequency onto medium (%) No. of fusant (CFU/ml)
MPA5 2.3 x 105 10.2 1.24 x103
MRA7 5.5 x 105 12.6

aNumbers of mycelial colonies on regeneration medium before protoplast fusion treatment

Figure 5. Microscopic photograph showing the protoplast fusion between MPA5 and MRA7

Figure 6. Effects of PEG concentration on colony formation by counting regenerating colonies after protoplast fusion between MPA5 and MRA7.Values represent the mean±SR

Figure 7. Colony morphology of isolate MPA5 (A) and isolate MRA7 (B) on malt yeast agar

Figure 8. Growth pattern of segregated fusant (A) and non-segregated stable fusant (MF11) (B) after UV exposure on malt yeast agar

Extracellular Red Pigment Production by Fusants
All the fusants were grown in sterilized rice at 30±2°C up to 14 days. After fermentation, the pigment was extracted using ethanol as a solvent (after standardization) with agitation and centrifuged. The supernatant was tested for pigment production. The auxotroph MRA7 reduces the production of pigments than its parental strain. The Figure 9 showed that all the fusants produced the red pigments but much less than the parents except the two fusantMF8 and MF11. The pigment production of fusant MF11 was observed since 72 h of fermentation and reached to its optimum on 11th day (35.5 U/gdfs) whereas the parental strain M. purpureus and its auxotroph MPA5 produced much higher quantity of pigments were measured as 58.1 U/gdfs and 75.2 U/gdfs respectively. Many reports are available in the literature for the pigment production capabilities of M. purpureus like extracellular red pigment production of M. purpureus maximum yield was recorded in production media supplemented with 20g/L glucose and 0.3% monosodium glutamate (MSG).38 The medium with fructose as a carbon source and low concentration of sodium nitrate promoted mycelium growth and production of red pigment in M. purpureus.39 In solid state fermentation jackfruit seed was found as novel substrate for pigment production and adding of nitrogenous compound as an external source proved a betterment of pigment production of M. purpureus which is water soluble in nature.40

Figure 9. Red pigment producing capacity of M. purpureus, its auxotroph MPA5 and all the fusants. Values represent the mean±SR, gdfs=gram per dry fermented substrate

Monacolin K Production by Fusants
Monacolin K content was measured from the solvent extracted supernatant of fermented rice and presented in Figure 10. All the fusants are capable to produce Monacolin K in SSF but the fusant MF11 produced maximally (2.22 mg/g) and found no less than 250% increased from the parents. Several reports were found on lovastatin production of M. purpureus and M. ruber.17­,41,42 In most cases the lovastatin yield is very less generally less than 20 mg/L. The production by the fusant may further increased after optimization.

Figure 10. Monacolin K production rate of M.purpureus, M.ruber, their auxotrophs MPA5, MRA7 and all the fusants. Values represent the mean±SR.

Amylase and Acid Protease Activity of Fusants
The isolates, fusant (MF11) and auxotrophs (MPA5, MRA7) were used for production of amylase and acid protease. From the Figure 11, it is found that production of alpha-amylase and acid protease of the fusant MF11 was 3015.5 U/g and 2200.5 U/g, respectively, much higher than the MPA5 but less than the MRA7. It could be correlated with the growth which was supported by the synthesis of hydrolyzing enzymes. It could be assumed that larger amount of enzymes production would probably assist them to grow fast. Generally, interspecific and intraspecific fusants obtained from mold showed higher enzyme productivity than their parental strains.43,44 Though the enzyme production by the fusant MF11much less than the parent MRA7 but without optimization.

Figure 11. Enzyme profile of MPA5, MRA7 and their fusant MF11 during incubation in broken rice at 30 ± 20C, (a) amylase, (b) acid protease. Values represent the mean±SR

Citrinin Estimation
In TLC chromatogram no citrinin was detected in solid state culture (data not shown). Citrinin is a nephrotoxic and hepatotoxic agent, produced by Monascus sp. is related to serious health hazards.45 But it is produced in very trace amount (nearer 0.1-500 mg kg-1), whereas the content is allowed to consume below 200 mg/ml. TLC method was commonly used to separate citrinin from the Monascus sp.32 During the time of rice fermentation, some risk of citrinin contamination may be defined in the fermentation process. Though this situation could be avoided either by detoxification of the mold or using any citrinin non-producer strain or by optimization of the fermentation process for reducing the amount of citrinin production. In this experiment, in case of fusant no citrinin was detected in comparison to the parents in the TLC plate, perhaps the citrinin pro was production was below of detection level by TLC.

This study suggested a protocol for protoplast fusion between the auxotrophs developed from M. purpureus and M. ruber. The fusants (MF11) is found as stable in nature. Thus, the result show promise for its biotechnological application. The fusants (regenerants) could grow and produce successfully the pigments and Monacolin K in SSF using rice. But the fusant MF11 produces Monacolin K at significantly high amount than the parents but there is scope for further enhancement through production optimization. Apart from Monascus, some filamentous fungi like Penicillum, Hypomyces, Doratomyces, Gymnoascus, Trichodermaetc were found to produce Monacolin K but only A. terreus was used for large scale production.46 MonacolinK containing A. terreus broth is not directly consumable because this organism is non edible and therefore its purification costs much. Wherever, Monascus species are nonpathogenic and used to make rice wine, soybean cheese, red rice etc in oriental countries. So enhanced rate of lovastatin from non toxic stain is scant. The production of hydrolyzing enzymes could be further correlated with the growth support. Till date there is no previous report of hybridization between M. purpureus and M. ruber by protoplast fusion.

CONCLUSION

Extensive efforts are being invested by researchers in harnessing new species of filamentous fungi for the manufacturing of economically important secondary metabolites. Monacolin K is a popular drug that effectively lowers blood cholesterol in human by blocking the enzyme that controls the rate of cholesterol synthesis and Monascus sp have the potential to synthesized monacolin K effectively. The protoplast fusion is one of the most acceptable techniques for strain improvement. The aim of this study is to construct a stable fusant between two mutants of M. purpureus (Arg) and M. ruber (Thi , Met) auxotrophs. Though M. purpureus (Arg) mutant is a higher red pigment producer but low in Monacolin K production, whereas M. ruber (Thi , Met) mutant is a high Monacolin K producer. From this investigation, we have developed a stable fusant (MF11) with enhanced Monacolin K producing capability, higher than both the parental mutants of Monascus sp. Shuffling of whole genome through protoplast generation and their fusion in various mutants of Monascus sp can be employed in industrial strain enhancement strategies.

Declarations

ACKNOWLEDGMENTS
The authors would like to thank Department of Microbiology, University of Kalyani, Kalyani, for providing Laboratory Facilities to carry out the experimental work. Authors also Acknowledge Mr. Saikat Banerjee for his co-operation and editing of the manuscript.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
AS conceptualized the study, and performed experiments and formal analysis. KS supervised the study. AS and KS wrote, reviewed and edited the manuscript. Both authors approved the final manuscript for publication.

FUNDING
None.

DATA AVAILABILITY
All datasets generated or analyzed during this study are included in the manuscript.

ETHICS STATEMENT
Not applicable.

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