4.1 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 4.41% of protein. It is a good source of many essential nutrients as presented in Table 1b. 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 ofa 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 [13], 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 [4].
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 5.44% of protein, carbohydrate, phenyl propenoids 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, 50, 30, 40 and 50 mL (Fig. 4b), and it was 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, inside human and animal hypercholesteremic alignments are being treated through P. cineraria [14, 15]. Due to hypercholesteremic activity of P. cineraria aqueous extract, our group explores this as a natural inhibitor for enhancing biomass production with a higher accumulation of lipid. This attempt of using a natural inhibitor for HMG CoA reductase enzyme inhibition in oleaginous yeast is studied first time to the best of our knowledge. The total dis-colouration is seen by using this waste P. cineraria pod aqueous extract (Fig. 4a). 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. 4b). The natural inhibitor was produced higher biomass (27.99%) as compared to the chemical inhibitor (24.04%) (Table 2a). 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 2b).
4.2 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 CH3Cl, CH2Cl2, ethyl acetate, and hexane. It was found that the ethyl acetate and hexane gave significant yield. Due to the prohibited nature of CHCl3 and CH2Cl2in industrial application, these solvents were not considered for optimization study. Ethyl acetate and hexane gave comparable amounts of lipids (Table 2b). Lipids are generally non-polar in nature; hence hexane gave a slightly higher yield as compared to the semi-polar 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 CO2 extraction has completely isolated the lipids (35.54%) due to their superior solvation properties. Liquid CO2 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 having greener along with GRAS properties encourages use in food products. These findings were also been supported by our previous work that, there is no need of removal of extracting solvents, improving the yield and most suitable to extract the thermo-sensitive compounds, higher selectivity, shorter extraction time and no need to involve toxic organic solvents, low cost, non-flammable, non-toxic [12, 16]. By the liquid-CO2extraction, the smaller chain fatty acids were easily been extracted through this method as a comparison with solvent extraction. Lack of longer chain fatty acid like behenic and lignoceric fatty acids in the procured oil makes the overall lipid suitable more for the food grade quality.
4.3 Quality of fatty acids for nutrition
The fatty oil quality is determined by GC-FID and GC/MS analysis as presented in Table 3. The results of 1H-NMR are in agreement with the GC-FID and GC/MS. Since,1H-NMR based process analyses the neat microbial oil so no need to convert the fatty acids into their methyl ester form [17]. The higher percentages of unsaturated fatty acids (MUFA and PUFA) were considered as a preferable combination [16]. This higher UFA not only enhances the glucose metabolism and equilibrium of insulin but also lessens cardiac issues [18]. Liquid-CO2extraction was giving an enhanced yield of lipids (35.54%) in 4 h due to superior salvation properties. The novelty for using mevalonate pathway rate limiting enzyme HMG CoA reductase inhibition with the use of natural inhibitor P. cineraria by showing it for completed is coloration of the production media (Fig. 4a). The similar discoloration was observed when rosuvastatin a chemical inhibitor was used for inhibition of HMG CoA reductase enzyme in previous work [5]. It observed that the biomass (banana peel) was better utilized in the chemical inhibited process as compared to the chemical inhibitor. The chloride was detected in chemical inhibited residue along with rosuvastatin residues, which impact higher stress on yeast cell-wall (Fig. 2). Thus, the cell-wall is rigid with a higher accumulation of SFA, this combination might be provided better protection to yeast. On the other hand, the natural inhibited residue contained more unsaturated groups, higher yeast biomass yield and higher accumulation of UFA. The lipid is also profitably isolated from the yeast biomass (Table 2a). The acid value (1.2 mg g− 1 KOH), saponification value (172 mg g− 1 KOH), and iodine value (82 g/100g) were fallen in the range of the vegetable oil.
This solvent 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 [5]. 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-CO2 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 375oC. On the other hand, soybean oil was devolatilized at 425oC. Overall, the liquid-CO2 extracted yeast oil is suitable for edible purposes. This is a novel finding to manipulate the production of UFA through SCO and liquid-CO2extraction made the process interesting.
4.4 Mechanism of the mode of action
Novelty for using mevalonate pathway is the rate limiting enzyme HMG CoA reductase inhibition with the use of natural inhibitor P. cineraria by showing the complete discolouration in production medium through waste management approach (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 sugars helped in the initial nourishment of the yeast. The flavonoids are identified in the aqueous extract of P. cineraria pods (Table 1c), could be responsible for the pathway inhibition.
Generally, overnight soaked pods of P. cineraria aqueous extract are usually discarded as waste and the same as in the case with banana peel waste but it can be utilized not only to enhance the lipid production in oleaginous yeast but also for the benefit of human health suffering from hypercholesteremic diseases. In other words, ‘best out of waste’ can be made is appropriate for this approach along with a greener approach for the environmental benefit.