An Innovative Prosopis cineraria Pod Aqueous Waste as Natural Inhibitor for Enhancing Unsaturated Lipids Production in Yeast Cell Using Banana Peel

Single cell oil (SCO) produced by yeast is an attractive alternative due to higher yield of lipid in a limited space with opportunity for naturally manipulating its quality. In the present study, the banana peel is used as a source of carbon for biotransformation by Rhodotorula mucilaginosa to lipid. Further, the quality and quantity of the lipid are enhanced using discarded aqueous Prosopis cineraria pod extract as a natural inhibitor. Prosopis cineraria aqueous extract was quantified using HPLC to contain phenylpropenoids such as epicatechin (0.068%), gallic acid (0.29%), quercetin (0.34%), epigallocatechin (0.091%), rutin (0.141%), ellagic acid (0.141%), along with glucose (1.22%), and sucrose (2.36%). The sucrose and glucose were isolated from the aqueous extract, and further characterized through NMR and TGA. Hence, this natural inhibitor is found expedient as compared to the chemical inhibitor (statin) in terms of lipid production with desirable quality. It is achieved by inhibitors blocking of yeast competitive mevalonate pathway to promote higher lipid accumulation in the microbial cells. The anti-cholestrolemic activity of this natural inhibitor might be influenced lipid accumulation by blocking the mevalonate pathway. Thus, the reducing sugars as well as phenylpropenoids were worked in synergy to enhance the accumulation of unsaturated lipid in the microbial cells. Phenylpropenoids may inhibit the key enzyme HMG reductase, which controls the mevalonate pathway for ergosterol formation to induce lipid accumulation. This lipid isolation from yeast cell was improved using green solvent viz. liquid-CO2. This liquid-CO2 extract was enriched with unsaturated lipid (46.96%) including ω-fatty acids such as linoleic (17.61%) and linolenic (5.35%). Thus, the SCO is produced using food waste as the source of carbon as well as an inhibitor, and this lipid is treated as natural to find suitable for nutritional purposes.


Introduction
The per capita consumption of vegetable oil is in increasing trend due to modern food practices. Conventional oilseed production has attained a plateau; hence the alternative non-conventional source of lipids is the need of the hour [1]. Fruits peels are a rich source of starch and protein to display an important role in the lipid production by oleaginous yeast [2]. The cultivation of bananas in India occupies around 830.5 thousand ha area with total production is approximately 31.78 × 10 6 tons in the year 2020 [3]. India contributed 16% of World banana production [4]; hence its peel waste is produced in bulk. This waste could be utilized as a substrate for lipid production, which is again used as a food supplement.
Oleaginous yeast like Rhodotorula mucilaginosa (Y-1) was found to promising for lipid production in our earlier studies [2,5,6]. Further enhancing the lipid yield, our study is focused on its metabolic pathway for lipid synthesis, and in this regard, the mevalonate pathway is very important. If the competitive mevalonate pathway was blocked with the help of inhibitors, then the flux shifted towards triacylglyceride (TAG) biosynthesis. The pigments like ergosterol, β-carotenoids were produced by the mevalonate pathway, and the key enzyme for this is 3-hydroxy-3-methyl-glutaryl (HMG) reductase enzyme. In our previous study, it was reported that the inhibition of this enzyme by chemical inhibitor rosuvastatin was helpful in the down-regulation of HMG reductase enzyme, this resulted to enhance the lipid production in the yeast cell [5]. The cholesterol production in human and ergosterol synthesis in yeast follows the same pathway, so targeting HMG reductase enzyme inhibition for lowering the production of cholesterol and ergosterol. The prolonged use of statin in humans had shown some side effects [7]. On the other hand, plant origin natural inhibitor has no side effect, and it can substitute the chemical inhibitor. In this regard, the desert grown vegetation Prosopis cineraria have been known to possess potent bioactive compounds [8][9][10]. Various parts of this plant were augmented with alkaloids, saponins, flavonoids, polyphenols, and tannins. Thus, it is selected as a natural inhibitor for increasing microbial oil production. This study was focused to inhibit the HMG-CoA reductase by adding natural inhibitor P. cineraria pod extract in the media with banana peel as a carbon source for the production of lipid. The result of the natural inhibitor was compared with the chemical inhibitor (rosuvastatin) in terms of biomass as well as lipid yield. Besides this, the microbial oil was extracted by conventional extraction processes and it was compared with the green extraction process viz. liquid CO 2 .

Chemicals and Real-Life Substrates
All the chemicals such as dextrose, peptone, malt extract, yeast extract, sodium citrate, etc. were purchased from Merck Darmstadt (Germany). Inhibitor rosuvastatin tablets (Cipla, India) of 5 mg potency was procured from Apollo pharmacy, New Delhi. Other HPLC grade solvents were supplied by SRL Ltd. (India).

Preparation of Substrate
Banana peel (at its green stage) was collected from the local market, it was then sun-dried and pulverized with the help of a domestic mixer grinder. This dried grounded powder (average of 0.52 mm to 1 mm particle size) was used as carbon source in media preparation for the growth of yeast. This production media was autoclaved before inoculation step. The dried P. cineraria pod was procured from the Bikaner district situated in the desert of Rajasthan, India, and pulverized it for the extraction purpose. Pods (100 g) were soaked in 250 mL autoclaved HPLC grade water overnight and then filtered. The aqueous filtrate was added to the media, and then it was autoclaved for 6 h.

Conditions for the Culture Development
The laboratory culture of R. mucilaginosa (Y-1) was acquired and maintained on malt extract, dextrose, yeast extract, peptone (MGYP) and agar slants with a ratio of 0.3:2.5:0.3:0.5, respectively at 4 °C, and it was sub-cultured in the regular interval of 1 month. All the media were autoclaved at 121 °C under 15 lbs pressure for 15 min before inoculation. The rosuvastatin has been selected because of its distinct orange/ red-coloured pigments to use for visual monitoring through colour development. Then, it was transferred a loopful of MGYP cultured broth (pH 7), this was done under sterilized conditions and incubated for 2 days in an incubator cum shaker (Model lab Therm, Kuhner, Switzerland) at 28 °C in 120 rpm. Media was prepared using 8% banana peel, 0.05% MgSO 4 , 0.1% K 2 HPO 4 (w/v) for the fermentation. At the time of inoculation, the fermentation broth is having real life substrate as source of carbon and nitrogen. However, it is not readily available in its free form as compared to chemical nitrogen sources. It gets released as oleaginous yeast slowly releases enzyme such as amylase, inulinase etc. This process may take few hours to some days. During this span yeast cells faces the nitrogen deprived or deficit condition which in turn creates stress on the oleaginous cells.
However as per the analysis the initial carbon and nitrogen concentration in media with banana peel as substrate with Prosopis cineraria pod as real-life substrate is approximately 33.12% and 1.58% respectively. Therefore, this C/N ratio is around 20.96 which could be supportive throughout the production phase of the cells to ultimately generates the lipid. Li et al. had reported that the banana peel with C/N ratio of 16.4 [11], which was supported the yeast multiplication. Further, this peel contained inulin as well as starch, which facilitated yeast growth to result enhance lipid accumulation in the yeast cell [12].
However, synthetic and natural statin have some dissimilarity, both have 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)-like moiety which behave as HMG-CoA reductase inhibitor. It lowers the cholesterol level in human and ergosterol synthesis in yeast [13,14]. It in turn inhibits the mevalonate pathway, which further enhances the production of lipid. In previous research Chaturvedi et al. (2021), observed that by the use of an inhibitor, rosuvastatin, the yeast may reduce the production of the pigment and enhances the lipid production [5,6].

ICP-OES Analysis of Banana Peel
The dried banana peel substrate was analyzed for their compositional analysis. The ICP-OES was used for the elemental analysis of an acid digestible sample using PerkinElmer, Optima 530 V, USA machine. The standard mixture of Ca, Mg, Al, Mn, K, B, Sr, Cu and P were used for the quantitative estimation.

FT-IR Spectra of Banana Peel
The PerkinElmer FT-IR spectrum Gx was used for the qualitative estimation of a different class of compounds. For that, 10 mg of banana peel was thoroughly mixed with 150 mg dried KBr for the preparation of pellets. Spectra were recorded in between 600 to 4000 cm −1 in the range of 2 cm −1 .

Quantification of Carbohydrates and Phenylpropanoids
Prosopis cineraria pod aqueous extract was freeze-dried, and the extract was used for reducing sugar analysis. The dried P. cineraria pod aqueous extract was hydrolyzed as per the standard NREL protocol [15]. The sample was neutralized using NaHCO 3 and kept in refrigerator for quantification. The reducing sugars were quantified using RP-HPLC-RI system of Waters (Milford MA, USA) make. The system was equipped with 5 mL capacity binary pumps (Waters model 515), a rheodyne (model 7125) manual injector, and Refractive Index (RI) detector. The chromatograms were obtained on Empower Pro software (Waters, USA). HPX-87H Bio-Rad Aminex column (300 mm × 3.9 mm) of water (USA) make at 80 °C was used for the analysis of carbohydrates. An isocratic solvent system comprising of degassed acetonitrile: water (70:30) of 20 min run time was used for the quantification. The flow rate was 0.5 mL/min throughout the run with a column temperature (60 °C), and the sample injection volume of 20 μL. All samples were accurately weighed and dissolved in water with thorough sonication, and filtration before injecting into the system. For the analysis of isolated sugars, 2 mg of each glucose and sucrose were separately diluted in 1 mL of water.
In addition, the dried P. cineraria pod aqueous extract was used for quantification of phenylpropenoids using RP-HPLC-PDA system of the above Waters machine. The system was equipped with PDA detector, Sunfire C 18 RP Column (4.6 mm × 250 mm, 5 μm particle size; Waters, USA) at 30 °C for quantification. Each standard (1 mg) such as epicatechin, gallic acid, quercetin, epigallocatechin, rutin and ellagic acid was dissolved in 1 mL of methanol to make the standard solutions at a concentration of 1 mg/mL. Sample analysis was carried out at 30 °C using a gradient solvent system. The mobile phase for gradient elution comprised of solvent A (Methanol, 100%) and B (0.1% TFA in water), following profile was found best suitable for peak resolution and separation of the individual compound in the sample as 0-25 min (25% A to 35% A), 25-50 min (35% A to 50% A), 50-60 min (50% A to 80% A), and 60-70 min (80% A to 100% A) at a flow rate of 0.8 mL/min with λ max 210-280 nm were used for the quantification.

Confirmation of Compounds Through HRMS Analysis
High resolution mass was recorded using 6545 QTOF LC/ MS of Agilent technologies system. The peaks were identified through high-resolution mass spectra along with their isotopic mass analysis.

Quantitative Evaluation of Yeast Biomass
Yeast biomass was collected with the help of Millipore (0.45 µm) membrane filtration system after completion of the incubation period. The accumulated biomass over the membrane was dried at 80 °C, and the weight was taken by gravimetrically for calculation of the yeast cell weight.

Cell-Morphology Visualization of R. mucilaginosa
The morphological difference of R. mucilaginosa was closely observed with the help of SEM images (Model EVO 50, ZEISS EVO series, Germany) as per the reported protocol [16]. The samples were accumulated at the log phase of the yeast cell growth. On the gold-plated metallic stub, the cell biomass sample was kept with the help of two-sided adhesive carbon tape for imaging.

Extraction of Lipid Through Different Extraction Techniques
The grinded and dried biomass was used for the isolation of lipid through different processes.

Soxhlet Extraction
In the soxhlet extraction or hot extraction process, 8 g of dried biomass was taken in a cellulose thimble, which was extracted in hexane or ethyl acetate for 7 h. Then, filtered the solution and evaporated the solvent in a rotary evaporator under vacuo.

Cold Percolation Method for Lipid Extraction
In the cold percolation method, the sample (8 g) was treated with hexane (50 mL) and kept for 4 h. The hexane solution was filtered out and the marc was extracted (50 mL × 2) times successively in similar conditions. The hexane solutions were pulled together and evaporated the solvent in a rotary evaporator under a vacuum. The weights of the collected samples were recorded.

Extraction of Lipids Through Liquid CO 2 Extraction
The liquid CO 2 extraction was carried out in a high-pressure stainless-steel vessel. The experimental setup and methodology have appeared in our earlier publication [17]. In brief, the modified glass apparatus was placed inside this pressure vessel for keeping the biomass in the upper soxhlet type unit to follow a beaker just below it. The pressure was maintained in the vessel to 70 bar at 15 °C, and it was placed in a water bath maintained at 35 °C. First, the CO 2 was condensed and collected in the soxhlet for filling-up to the siphon level. Then, the CO 2 along with dissolved lipid was siphoned to the lower beaker. Again, the liquid CO 2 was evaporated from the lower beaker due to heat transform from the hot water (35 °C) bath. This cycle of evaporation and condensation was continued for 4 h, and each cycle was completed around 6 min to ensure the complete extraction of lipid. Then, the CO 2 was slowly released from the apparatus to collect the lipids from the beaker.

Physico-chemical Analysis of Lipids
Acid value, saponification value and iodine number are the physico-chemical properties of the lipid were determined as per the standard titration methods [1].

Gas Chromatography (GC) and GC-Mass Spectrometry (MS) Analysis
The extracted lipid was converted into fatty acid methyl ester (FAME), and qualitative analysis was carried out by Perki-nElmer auto system XL Gas chromatography along with a flame ionization detector (FID). The fatty oil (100-200 mg) was taken in an RB. Approximately 5 mL of reagent mixture of methanol: toluene: sulphuric acid (20:10:1) was added and reflux this mixture for 10 min. Add water and hexane to this, taking out the hexane layer and dried it over Na 2 SO 4 [18]. Then, it was concentrated and used for GC-FID and GC/MS analyses.
T h e c a p i l l a r y c o l u m n o f E l i t e wa x o f 30 m × 0.25 mm × 0.25 µm film thickness was used for the analysis. Hydrogen was used as carrier gas with a column head pressure of 7.5 psi. Sample (0.1 µL) was injected in the port with a split ratio of 1:40. The injector and FID temperature were maintained at 240 °C with a column linear temperature program of 40 °C to 120 °C (rate of 3 °C/ min), 120 to 140 °C (2 °C/min) and 140 to 230 °C (5 °C/ min). PerkinElmer SQ8C MS interfaced with a Turbomass Quadrupole mass spectrometer Clarus 680 GC was used for the GC/MS analysis with similar conditions as mentioned above. Helium was used as a carrier gas with a constant pressure of 1.5 psi. The injector and transfer line temperature were kept at 240 °C, electron ionization (EI) 70 eV, and mass scan range 40-450 amu.

Nuclear Magnetic Resonance Spectroscopy (NMR) Analysis
The NMR analysis was performed with the help of Brucker NMR500 MHz machine. For the analyses of samples, 8 mg of lipid was dissolved in CDCl 3 for the recording of proton NMR. In this, the internal standard was TMS, and 200 scans were performed for recording the spectra. The chemical shifts (δ) of protons were interpreted for getting information about the nature of protons.

Thermo Gravimetric Analysis (TGA) of the Lipid
The TGA of lipid was run using the equipment of Mettler-Toledo, TGA/DSC 1 make. For this, 5 mg sample was taken in a silica crucible. Devolatization study was conducted in between 50 to 550 °C at the rate of 10 °C per min. Nitrogen was used as a purge gas with a flow rate of 60 mL/min. In a similar methodology, the standard soybean oil devolatilization behavior was studied for comparison purposes. The spectra were obtained according to the volatility of the samples, and their first derivatives were computed using software for the clear interpretation of devolatilization behavior.

Isolation of Carbohydrates from P. cineraria Pod
Pods powder (300 g) was extracted with 1 L of hexane at 50 °C for defatting. Then, the solution was decanted to follow a quick wash with 500 mL of fresh hexane. The combined extract was evaporated at 40 °C under 160 mbar pressure using Buchi (Switzerland make) rotatory evaporator. The weight of the dried extract was recorded and kept at 4 °C until further analysis. The defatted pod powder was again extracted with 1 L of methanol-water solvent system (4:1) at 50 °C for 4 h. Then, the solution was decanted and repeated the same extraction procedure for two more times. The combined extract was evaporated at 65 °C under 80 mbar pressure. Dried extract was weighed and kept at 4 °C until further analysis. The extract 1 3 was dissolved in a minimum amount of methanol and kept at room temperature to furnish the colorless crystals (Scheme 1). Crystals were centrifuged out at 10,000 rpm and thoroughly washed using methanol and subjected to different spectroscopic analysis. The sugars are quantified as described in "Conditions for the Culture Development" section.

Statistical Significance
The data are recorded as an average of three repeated experiments. The statistical significance was validated by 16.0 software of SPSS data editor (Microsoft). The correlation was established in between the dependent and independent variables using bivariate Pearson, two tailed mode for p < 0.01 and p ˂ 0.05 with 99% and 95% of assurance level, respectively.

Results
The data were taken in triplicate and the average values were represented. The appropriate days for the collection of samples were decided based on the growth curve of yeast culture.

Characterization of Banana Peel and P. cineraria Aqueous Extract
The banana peel was contained glucose and sucrose (> 30%), along with protein (10.81%). The banana peel is contained good amounts of micronutrients (mg/100 g) such as K  Table 1). Most of the micronutrients were increased, when the media is treated with P. cineraria aqueous extract. On the contrary, there is no significant change of nutrients, when media is treated with chemical inhibitor (Table 1). On the other hand, P. cineraria aqueous extract (100 mg/mL) is contained glucose (1.22%), sucrose (2.36%) with proteins (3.77%) ( Table 2); while, the pod is contained 8.44% of protein. The phenylpropenoids contained in the aqueous extract are presented in Table 3 and Figures S4-S5.

Effect of Natural Inhibitor on Yeast Cell Morphology
The SEM pictures of cells clearly signified the changes in cell membrane integrity. In the presence of inhibitor, cells shrank and distorted with wrinkled cell appearance.

Scheme 1 Extraction process for phytocompounds of natural inhibitor P. cineraria pod waste
In the treatment of inhibitor, the cell size was diminished (1.014 µm breath and 1.825 µm length of the cell) at 200 mM rosuvastatin as compared to control (2.129 µm breath and 2.807 µm length). Addition of inducer has influenced the changes in cell structure (Fig. 1). Due to the presence of natural inhibitor, the structure of yeast cells was enlarged with normal morphology; hence there was no shrinkage or crumbling of the cell surface as compared to the cells treated with the chemical inhibitor.

Effect of Inhibitor on Yeast Biomass and Lipid Yield
The dry yeast biomass and lipid production are measured after 3 days (72 h) of fermentation broth in the presence of chemical and natural inhibitors ( Table 4). The highest amounts of biomass (26.99 g/L) were obtained using natural inhibitor followed by the chemical inhibitor (24.04 g/L). Whereas, the total lipid and extracellular lipid content were highest in chemical inhibitor (30.9, 22.5%) followed by natural inhibitor (25.18, 21.12%), respectively. The different solvents were tried for the extraction of lipid from yeast cells ( Table 4). The Soxhlet extraction using hexane was provided the total lipid 25.68% and extra-cellular lipid of 22.14%. Therefore, this solvent was tried in cold percolation method, the yield of total lipid and extracellular lipid was 24.72 and 21.92%, respectively. On the other hand, liquid CO 2 was given a higher yield of total lipid (35.54%) and extra-cellular lipid (30.17%). In paired samples test using SPSS software, the paired difference between control and natural inhibitor with ethyl acetate extraction, natural inhibitor with hexane extraction, natural inhibitor with soxhlet extraction, and natural inhibitor with liquid CO 2 extraction were greater than zero at 95% confidence level. Correlation analysis is also performed for fatty acid content, 0.05 level correlation in between myristic and palmitic acid, and 0.01 level of correlation is reported for myristic and behenic acid content (Tables S1-S3).

Fatty Oil Estimation
In dried fresh-peel (F), spent-peel (P) (residue obtained after P. cineraria aqueous treatment) and spent-peel (S) (residue obtained after rosuvastatin treatment) are used for the recording of FT-IR spectra (Fig. 2). The intensity of -OH at 3434 cm −1 was more prominent in spent-peel (S). The stretching vibrations of -CH gave a sharp peak at 2923 cm −1 .
The diketones stretching vibrations of carbohydrate group and bending vibration of the absorbed water at 1626 cm −1 were recorded. Amine functional group was recorded only in the unspent dried banana peel biomass at 778.2 cm −1 . Whereas, rosuvastatin treated biomass sample has an acid chloride group peak (C-Cl) at 625.8 cm −1 . On the other hand, the stretching aromatic ring (C=C) functional group was recorded at 1531.3 cm −1 in the P. cineraria treated sample. Another peak due to bending vibration of alkanes (CH 2 , CH 3 ) was seen in between 1383.8 and 1443.6 cm −1 . The presence of polysaccharides (pyranose ring C-O-C stretching vibrations) has appeared in the range of 1033.4 to 1069.5 cm −1 , which was due to the reducing sugars.  The saturated and unsaturated lipid composition is estimated using 1 H-NMR ( Figure S1). There are ten fatty acids identified in different extracts through GC-FID and GC/MS ( Table 6). The saturated fatty acids (SFA: 57.31%), monounsaturated fatty acids (MUFA: 18.92%) and polyunsaturated fatty acids (PUFA: 16.75%) were recorded in control sample. Similarly, the chemical inhibitor sample was contained SFA (63.44%), MUFA (20.57%), and PUFA (9.36%), respectively. Besides, the natural inhibitor sample extracted in different solvents such as ethyl acetate, hexane and liquid CO 2 were contained SFA (55.07, 50.63, 45.81%), MUFA (21.89, 22.61, 23.8%) and PUFA (19.89, 19.86, 22.66%), respectively (Fig. 3a). In TGA evaluation, the yeast oil is volatilized at low temperature and then followed by soybean oil (Fig. 3b).

Quantitative Analysis of Polyphenols-Flavonoids and Sugars in P. cineraria Pod Extract
From the HPLC-RI-PDA analysis of the aqueous extract of P. cineraria pod, sucrose (2.3%) and glucose (1.2%) were the primary components in the extract, and fewer polyphenols such as gallic acid (0.29%), quercetin (0.34%), rutin (0.14%), and ellagic acid (0.14%) are also present in the aqueous extract (Tables 2 and 3).

Isolation of Reducing Sugars from P. cineraria
The precipitate was contained sucrose (84.86%). Further, the glucose and sucrose were isolated and spectroscopically characterized. The glucose and sucrose in the aqueous  Figure S2). The devolatilization behavior of isolated glucose and sucrose are fully matched with the pure standards as depicted through TGA and DTG ( Figure S3). The discoloration of initial media after treatment with P. cineraria aqueous solution is presented in Fig. 4.

Importance of Substrate and Inhibitor
The banana peel was contained a high amount of organic matter (91.5%) and minerals. It contained 30% of starch and 10.81% of protein. It is a good source of many essential nutrients as presented in Table 2. Banana peel has appropriate amounts of nutrients for the regular growth of oleaginous yeast. Thus, it is used throughout the experiment as a carbon source in the production media. The single cell oil (SCO) produced from cell factories of yeast is followed lipid production pathway, and an alternative competing mevalonate pathway produces ergosterol pigment formation. To inhibit the pathway for ergosterol formation, the activity of a key enzyme HMG CoA reductase needs manipulation. Similarly, the mevalonate pathway is inside human and animals are responsible for cholesterol formation. So, to increase the flux towards fatty acid production, the competing mevalonate pathway inhibition through HMG CoA reductase (inhibitor) should be done. For this chemical inhibitor rosuvastatin has been used in humans [19], and animals for a long, but its side effects were also being reported. In our previous work on oleaginous yeast, the chemical inhibitor rosuvastatin has also been used to increase lipid production [5].
Due to the side effect of rosuvastatin and for the safe production of lipid, the plant origin P. cinararia aqueous waste extract as a natural inhibitor has been tried in the present work. P. cinararia pod contains 3.77% of protein, carbohydrate, phenylpropenoids and other bioactive compounds. Due to the nutraceutical value of the pod and its aqueous washing part is discarded in food preparation. It is assumed that by inhibiting the enzyme HMG CoA reductase, the aqueous extract of P. cineraria may be effective to enhance the lipid accumulation in the yeast. The addition of the P. cineraria aqueous extract in 10, 20, 30, 40 and 50 mL (Fig. 5), and it is observed to inhibit the competitive pathway in 100 mL media. The optimum result was noticed in 30 mL, and it was used throughout the experiment. As reported, the human as well as animal hypercholesteremic alignments were treated using the extracts of P. cineraria [20,21]. Due to hypercholesteremic activity of P. cineraria aqueous extract, our group explores this as a natural inhibitor for enhancing the biomass production with a higher accumulation of lipid in cells. This attempt of using a natural inhibitor for HMG CoA reductase enzyme inhibition in oleaginous yeast is studied first time. The total dis-colouration is seen by using this waste P. cineraria pod aqueous extract (Fig. 4). In the present analysis, it found that the aqueous extract was contained glucose (1.22%), sucrose (2.36%), protein (3.77%) along with epicatechin (0.068%), gallic acid (0.29%), quercetin (0.34%), epigallocatechin (0.091%), rutin (0.141%), and ellagic acid (0.141%) might be facilitated for enhancing higher lipid accumulation by the yeast cell (Fig. 5). The natural inhibitor is produced higher biomass (27.99%) as compared to the chemical inhibitor (24.04%) ( Table 4). But the chemical inhibitor was produced an enhanced percentage of total lipid and extra-cellular lipid (30.9, 23.5%) as compared to the natural inhibitor (25.68, 22.64%), respectively. As interpreted, the rosuvastatin is given more stress to the R. mucilaginosa, so the overall biomass production is not improved and only accumulated higher fatty acid to overcome the stress as observed in SEM pictures. On the other hand, the natural inhibitor was provided a better environment for a significant increase of biomass as well as lipid. Hence, the overall enhancement of the total lipid is 21.3% in natural inhibitors, whereas total lipid is 18.2% in the chemical inhibitor sample ( Table 5).

Importance of Green Extraction Methods
The extraction of lipid from oleaginous yeast is a difficult task due to its lipid being trapped inside the cell, which is bound by the hard cell wall. This lipid is tried to extract using different organic solvents such as CH 3 Cl, CH 2 Cl 2 , ethyl acetate, and hexane. It was found that the ethyl acetate and hexane gave significant yield. Due to the prohibited nature of CHCl 3 and CH 2 Cl 2 in industrial application, these solvents were not considered for optimization study. Ethyl acetate and hexane gave comparable amounts of lipids (Table 4). Lipids are generally non-polar in nature; hence hexane gave a slightly higher yield as compared to the semipolar ethyl acetate through the percolation method. For complete isolation of lipids, the soxhlet extraction in hexane has been tried and gave maximum yield in 8 h, and then after there was no significant increase of lipid yield. Thus, the percolation as well as soxhlet extraction was not suitable for the complete isolation of lipids due to difficulty in penetration of solvent inside the hard cell wall. The liquid CO 2 extraction has completely isolated the lipids (35.54%) due to their superior solvation properties. Liquid CO 2 has gas-like properties such as high diffusion, low surface tension with medium density and non-polar nature makes it an excellent solvent for dissolving the lipids inside the cell, hence facilitating complete extraction of lipids. These properties are helped to enhance the yield of lipids by about 30%, and 1 3 having greener along with GRAS properties encourages use in food products. The advantages of liquid-CO 2 is no need to remove the extracting solvents, improving the yield and most suitable to extract the thermo-sensitive compounds [17,22]. By the liquid-CO 2 extraction, the smaller chain fatty acids were easily extracted through this method in compared to the solvent extraction. Lack of longer chain fatty acid like behenic and lignoceric fatty acids in liquid-CO 2 extracted oil makes the overall lipid suitable for using in food.

Quality of Fatty Acids for Nutrition
The fatty oil quality is determined by GC-FID and GC/ MS analysis as presented in Table 6. The results of 1 H-NMR are in agreement with the GC-FID and GC/MS. Since, 1 H-NMR is directly analyzed the lipid, hence there is no need for sample preparation step to process without derivatization [23]. The higher percentages of unsaturated fatty acids (UFA) were considered as a preferable combination for food [22]. This higher UFA not only enhances the glucose metabolism and equilibrium of insulin but also lessens cardiac issues [24]. Liquid-CO 2 extraction was giving an enhanced yield of lipids (35.54%) in 4 h due to its superior salvation properties. The novelty for using mevalonate pathway rate limiting enzyme HMG CoA reductase Total lipids %

Volume of P. cineraria aqueous extract (ml)
Total lipids Log. (Total lipids ) inhibition with the use of natural inhibitor P. cineraria, hence flux shifted towards lipid production (Fig. 4). The similar discoloration was observed when rosuvastatin a chemical inhibitor was used for inhibition of HMG CoA reductase enzyme in our previous work [6]. It observed that the biomass (banana peel) was better utilized in the natural inhibited process as compared to the chemical inhibitor.
The chloride was detected in chemical inhibited residue along with un-utilized rosuvastatin, which impact higher stress on the yeast cell-wall. The clear difference is noticed in the comparative FTIR spectra of fresh, spent and statin treated biomass (Fig. 2). The chemical inhibitor is made the cell-wall rigid with a higher accumulation of SFA, this combination is possibly provided better protection to yeast cell against osmosis. On the other hand, the natural inhibitor is provided environment for healthy growth of yeast, higher biomass with improved percentage of UFA. The lipid is also selectively isolated (MUFA and PUFA) from the yeast with improved yield using liquid-CO 2 solvent (Table 4). The acid value (1.2 mg/g KOH), saponification value (172 mg/g KOH), and iodine value (82 g/100 g) were fallen in the range of the vegetable oil [1]. Liquid-CO 2 is helped to screen out the high molecular SFA like behenic acid (1.56%) and lignoceric acid (1.77%); these fatty acids at higher percentage are not recommended for food applications due to their adverse impact in cardiac related issues. In our previous work, it was reported the chemical inhibitor like NaCl and ionic liquid in isolation or combination were shifted the flux towards the accumulation of a higher percentage of SFA in a yeast cell. This SFA may be provided better protection from cell membrane osmosis [6]. In reverse, the natural inhibitor encouraged yeast for the production of UFA, which may be influenced by the phytomolecules of P. cineraria aqueous extract.
The liquid-CO 2 extracted oil was contained an improved percentage of low molecular fatty acids such as myristic (7.29%) and palmitic (25.86%) along with MUFA (24.0%) and PUFA (22.96%), so it volatilized at 375 °C. On the other hand, soybean oil was devolatilized at 425 °C. Overall, the liquid-CO 2 extracted yeast oil is suitable for edible purposes. This is a novel finding to manipulate the production of UFA through SCO and liquid-CO 2 extraction made the complete process interesting.

Mechanism of the Mode of Action
Novelty for using mevalonate pathway is the rate limiting enzyme HMG CoA reductase inhibition by using natural inhibitor P. cineraria to display the complete discolouration in production medium (Fig. 4). The similar discolouration was observed when rosuvastatin was used for inhibition of HMG CoA reductase enzyme. The phytocompounds epicatechin, gallic acid, quercetin, epigallocatechin, rutin, ellagic acid, glucose and sucrose in the aqueous P. cineraria extract as detected through NMR, FTIR, and HPLC techniques were responsible for HMG CoA reductase inhibition as well as reducing sugars helped in the initial nourishment of the yeast. The flavonoids are identified in the aqueous extract of P. cineraria pods (Table 3), could be responsible for the pathway inhibition. Generally, overnight soaked pods of P. cineraria aqueous extract are usually discarded as waste, and banana peel is also a waste to use as a source of carbon and nitrogen for enhancing the lipid production in oleaginous yeast. In other words, 'Wealth from waste' though greener approach to produce food grade lipids to meet the present edible oil demand.

Conclusion
The yeast SCO was produced using banana peel and further enhanced the yield through natural inhibitor P. cineraria waste aqueous extract. The glucose, sucrose and mineral nutrients in both the wastes are responsible for the easy growing of oleaginous yeast. The phyto-molecules might be responsible for enhancing growth as well as accumulation of higher fatty oil. The significant growth of yeast was not observed when applying a chemical inhibitor. Hence, a higher lipid yield (21%) was given by the natural inhibitor sample. This observation was noticed in the SEM pictures Table 6 Fatty acid profile of chemical as well as natural inhibitor lipids extracted using different solvents. The value in bold is referred to natural inhibitor samples contain enhanced percentage of UFA and palmitic acid CIn chemical inhibitor extracted using hexane, NInEa natural inhibitor extracted using ethyl acetate, NInHx natural inhibitor extracted using hexane, NInSc natural inhibitor extracted using liquid CO 2 that the chemical inhibitor was influenced to shrink the yeast cell. Unlike chemical inhibitors (rosuvastatin), the natural inhibitor has shifted the flux to accumulate the ω-3-fatty acid (linolenic acid), which is a precursor for the synthesis of vitamin-E. Different conventional organic solvents have been tried for the extraction of fatty oil, but hexane was found to be most suitable and already practiced for the isolation of fatty oil from oilseeds on an industrial scale. The isolation of fatty oil from chemical inhibitor cells was difficult as compared to natural inhibitor one. The percolation method is energy-efficient as compared to the soxhlet method, and the former can be used in the scale-up process. In addition, liquid CO 2 has been given the highest yield (⁓ 30%) with superior quality due to its faster diffusivity, low surface tension and medium density helped for leaching out lipid from hard cell-wall yeast glands. Thus, liquid-CO 2 was helped for selective isolation of improved percentage of MUFA (24.0%), and PUFA (22.96%) with fewer percentages of high SFA (45.21%) like behenic acid (1.22%) and lignoceric acid (1.77%). Liquid-CO 2 extract was enriched with low molecular fatty acids and UFA (46.96%), hence is an ideal composition for the nutritional purpose.