INTRODUCTION:

Citrus is one of the most important organic plants in all major countries of the globe. With approximately 102.64 million tons of Citrus, which are considerably credited to its differentiated use and developed world interest, the world's leading organic product is most probable. Citrus natural products are mainly used by companies that prepare juice while peels are frequently washed out. Because the citrus yield is less than 50 percent of the weight of the organic product, many peels are formed every year by squanders¹for instance.

Peel squander is extremely short-lived and occasional and a problem for preparing companies and organisations observing contamination. The provision of precious products from waste products and citrus sprays are not specific instances is dependably taken more into account. Suitable methods should be used to convert items included to consider². Recovery of side effects from organic product squanders can enhance the overall economic elements of the management units. Furthermore, it can considerably decrease the problem of ecological contamination. They are rich in additional products and contain countless phytochemicals, which can also be used efficiently as medicines or as food supplements. Whenever it shows antibacterial motion, citrus peels can also be used in the same food industry which generates large strip squanders as a feed additive. The increasing amount of episodes of sustenance-borne disease caused by certain pathogens gradual concerns were raised by nutrition processors, sanitation researchers and administrative organizations.^3,4,5. Citrus is a wealthy source of flavanones and various polymethoxylated flavones, which are very rare in various plants⁶. The antimicrobial capacity of basic oils such as citrus oil appears to be a particularly fascinating area of use for sustenance and restoration applications⁷. It has also been used as an adversary of the diabetic⁸, antimicrobial⁹, antifungal¹⁰, hypotensive¹¹, cell-reinforcement¹², carminative, cracky, antifungal, larvae, antiviral, uricosuric, yeast harmful, anti-hepatotoxic and antimutagic¹³. Carica papaya Linn is a plant with imminent restorative hugeness, which is commonly referred to as paw-paw. A large number of users worldwide are made of consumable papaya. The papaya crude product is used as a purging and abortion agent. The leaves are also used in the therapy of pyrexia, diabetes, gonorrhea, syphilis, worsening, and wound dressing alongside the natural product¹⁴. The aim of this research is to focus on peel and pulp combinations as carbon and to produce microbial dyestuffs.

Materials:

The Penicillium purpurogenum NCIM 713 - was purchased from NCI Pune, and Potato Dextrose Agar (PDA) slants retained the inventory culture.

The peels of Citrus.limon and pulp of Carica.papaya were collected in fresh container.

PDB (2%), MgSO₄(1%), MnSO₄(1%), K₂HPO₄ (1%) and KH₂PO₄ (1%) and Urea(0.5%) with pH 5.5

Methodology:

The peels were initially washed with tap water, followed by distilled water to remove soil and other contaminants. Then, equal amounts of pulp of papaya and peels of lemon were weighed and ground into a paste and used as the carbon source.

Fermentation:

In the respective 250mL Erlenmeyer flask, 10ml broth was inoculated with a 100mL of PDB-composed manufacturing medium (2%), MgSO4 (1%), MnSO4 (1%), K2HPO4 (1%) and KH2PO4 (1%) and Urea (0.5%) with pH 5 and 5.5. On a rotary shaker (200rpm) at 25^oC the inoculated flake had been incubated for 7 to 10 days.

Pigment extraction:

After incubation, the broth obtained was taken and heated on a heating mantle at 70 degrees Celsius for 2 hours. After heating, the broth was filtered, separating the biomass and the filtrate. The pH of the filtrate was checked. The solution obtained was evaporated and concentrated at 70 degrees Celsius. The water molecules are slowly removed on evaporation leaving the solid concentrate. The concentrate was cooled immediately. The crude extract obtained was subjected to crystallization to form crystals of the pigment. The pigment obtained was purified and weighed.

UV-Visible Spectroscopy (UV-Vis):

The spectrophotometer (SPECORD 210-222K333 UV-Vis) at 500 nm of wavelength (peak absorbance of the extracted and dried colored pigment powder) was determined¹⁵.

Fourier Transform InfraRed (FT-IR) spectroscopy:

The Fourier spectrum of FT-IR was registered with the Bruker FT-IR and a spectral range of 4000 to 500 cm-1¹⁶. A Shimadzu FT-IR 8000 spectrophotometer scanned the dried powder of the colored pigment in the range 4000–400 cm−1 using the KBr approach at the temperature rate of 27°C.

Nuclear Magnetic Resonance Spectroscopy:

A dimethylsulfoxide(DMSO d6) with purified pigment has been dissolved with a sample injected into a spectrometer of nuclear magnet resonance (NMR) (Bruker 400 MHz)¹⁷.

Gas Chromatography-Mass Spectrometry:

A qualitative test procedure for the existence of phytochemical compounds, accompanied by a GC-MS assessment to identify novel compounds, was tested in the present research with the microbial extract of fruit & pulp. In the microbial extract, the mass spectrum of the compounds was combined with the NIST (National Institute of Standards and Technology) and Wiley library.

RESULTS AND DISCUSSION:

The pH 5.5, temperature 37°C and the production time of 8 days are the conditions under which the pigment is produced. The highest peel and pulp mixture output of pigments was 2.8%, that is to say 28.5g/L, and peak UV absorption was 275nm (0.944). The pigment produced was characterized by various techniques, such as UV Visible Spectroscopic, FTIR, 1h NMR and GC-MS.

Absorption	Absorption	Specific type of bond
Peak value	range
3346.5	3500-3300	1^O amines (doublet), 2^O amines N-H stretch
3267.41	3500-3200	Alcohols, phenols (H-bonded), O-H stretch
2937.59	3000-2830	Alkanes C─H stretch
1664.57	1670-1640	AmidesC=O stretch (Amide II band)
1629.85	1640-1550	Amides, 1^O and 2^O amines N-H stretch
1450.67	1450-1375	Alkane –CH₃ bend
1404.89	1450-1375	Alkane -CH₃ bend
1135.43	1300-1000	Alcohols, esters, ethers, -COOH,
		Anhydrides C-O stretch
943.19	1000-650	Alkenes C-H out of plane bend
617.22	800-600	Chloride C-Cl stretch

Figure no 1 :- UV Spectra and FTIR Spectra of Citrus limon and Carica papaya

Figure no 2:- H-NMR-Spectra of Citrus limon + Carica papaya

Figure no 3:- GC – MS Spectrum of Citrus limon & Carica papaya

Table no 1:- Major phytochemical compounds identified in microbial extract of Citrus limon and Carica papaya

S. No	RT(min)	Name of the phytochemical compound	Mol. Formula	Mol. Wt.	Peak region %	Pharmacological purpose
1	4.239	Silanediol dimethyl	C₂H₈O₂Si	92	2.92	organosilanediol.
2	4.842	3-furaldehyde	C₅H₈O2	96	1.39	role as a metabolite.
3	12.886	4-hepten-3-one	C₁₀H₁₈O	154	1.06	Flavouring agent
4	14.170	3-undecen-5-yne, (Z)	C₁₁H₁₈	150	1.24	No activity reported
5	15.745	3-cyclohexon-ol, 1-methyl	C₇H₁₂O	112	1.03	No activity reported
6	17.218	3,4,5-Trimethylpyrano[2,3-c]pyrazol-6(1H)-one	C₉H₁₀N₂O₂	178	12.88	Heterocyclic compound
7	19.458	N-didehydrohexacarboxyl-2,4,5-trimethylpiperazine	C₁₃H₂₂N₂O	222	1.94	Antimicrobial activity and cytotoxic activity
8	19.584	N-didehydrohexacarboxyl-2,4,5-trimethylpiperazine	C₁₃H₂₂N₂O	222	12.84	Antimicrobial activity and cytotoxic activity
9	19.679	6,10-Dimethoxy-3,3-dimethyl-1- (methylsulfanyl) -2-aza-spiro [4.5] deca-1,6,9-trien-8-one	C₁₄H₁₉NO₃S	281	10.43	Spirocyclohexadienones
10	20.471	9-Octadecenoic acid	C₁₈H₃₄O₂	284	1.54	Surface-active agent, FDA Food Additive
11	20.643	2H-1-benzopyran-2-one, 8-methoxy-	C₁₀H₈O₃	176	1.04	Coumarin, Secondary metabolite
12	22.849	Lilial	C₁₄H₂₀O	204	2.03	Odor agents
13	25.529	Di-n-octyl phthalate	C₂₄H₃₈O₄	390	1.16	Food Additive, Plasticizers
14	25.633	2-[2-(4-chloro-phenyl)-vinyl]-1-(2-phenoxy-ethyl)-1h-benzoimidazole	C₂₃H₁₉ClN₂O	374	1.03	No activity reported
15	25.719	Cyclohexane, eicosyl	C₂₆H₅₂	364	1.39	A surrogate mineral hydrocarbon
16	26.698	3',4',5,6,7,8-Hexamethoxyflavone	C₂₁H₂₂O₈	402	10.25	Polyketides, Flavonoids

Nowadays, the study and activity of organic compounds in plants has increased. GC–MS-is a useful instrument to identify bio-active compounds reliably [6]. In this study, GC-MS analysis has identified 150 compounds from the combination microbial extract of Citrus. limon and Carica.papya. Silanediol dimethyl, 3-furaldehyde, 4-hepten-3-one, 3-undecen-5-yne, 3-cyclohexon-ol, 3,4,5-Trimethylpyrano[2,3-c]pyrazol-6(1H)-one, N-didehydrohexacarboxyl-2,4,5-trimethylpiperazine, 6,10-Dimethoxy-3,3-dimethyl-1- (methylsulfanyl) -2-aza-spiro [4.5] deca-1,6,9-trien-8-one, 9-octadecenoic acid, 2H-1-benzopyran-2-one, Di-n-octyl phthalate, 2-[2-(4-chloro-phenyl)-vinyl]-1-(2-phenoxy-ethyl)-1h-benzoimidazole, lilial, Cyclohexane, and 3',4',5,6,7,8-Hexamethoxyflavone were the 16 most abundant in the mixed extract.

CONCLUSION:

The most important result of this research was the production of mustard yellow pigment using P. purpurpurogenum under different environmental conditions. P. purpurogenum could be seen to react by generating elevated levels of lemon peel pigment with papaya pulp. The findings of optimization and characterization show that the isolated pigment with various phytochemicals has been made with antibacterial flavoring agents. The GCMS structural elucidation demonstrates the pigment's primary component structure.

ACKNOWLEDGEMENTS:

The authors are thankful to Miss. Tejal Sheth, from Laxmi Analytical Laboratories, Mumbai and Nanotechnology Research Centre, SRM University, Kattankulathur, Tamilnadu for successful completion of the work.

REFERENCES:

1. Manthey. A and K. Grohmann, (2001): Phenols in citrus peel by products: concentrations of hydroxycinnamates and polymethoxylated flavones in citrus peel molasses, J. Agric. Food Chem. 49 pp. 3268.

2. Nand, K. (1998): Recent advances in the treatment of liquid and solid wastes of food processing industries for biogas production and pollution abatenront. Proc. 4th International. Food Convention, Mysore, pp.35.

3. Wilson CL, Droby GG, (2000): Microbial Food Contamination, CRC Press, Boca Raton, FL, 1-304 (149-171)

4. Friedman M, Henika RP, Mandrell ER. (2002): Bactericidal activities of plant essential oils and some of their isolated constituents against Campilobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica. Journal of Food Protection. 65, pp.1545-1560

5. Marina Soković, Petar D. Marin, Dejan Brkić, Leo J. L. D. van Griensven. (2007): Chemical Composition and Antibacterial Activity of Essential Oils of Ten Aromatic Plants against Human Pathogenic Bacteria Global Science Books Food, 1(1)

6. Ahmad, M.M., Salim-ur-Rehman, Z. Iqbal, F.M. Anjum and J.I. Sultan. (2006): Genetic variability to essential oil composition in four citrus fruit species. Pak. J. Bot., 38(2): pp.319-324.

7. Caccioni, D. R., Guizzardi, M., Biondi, D. M., Renda, A. and Ruberto, G. (1998): Relationship between volatile components of citrus fruit essential oils and antimicrobial action on Penicillium digitatum and Penicillium italicum, International Journal of Food Microbiology, 43, pp. 73–79.

8. Hamendra, S. P. & Anand, K. (2007): Antidiabetic potential of Citrus sinensis and Punica granatum peel extracts in alloxan treated male mice, Bio Factors, 31, pp.17–24.

9. Stange Jr., R. R., Midland, S. L., Eckert, J. W. & Sims, J. J., (1993): An Antifungal Compound Produced by Grapefruit and Valencia Orange After Wounding of the Wounding of the Peel, Journal of Natural Products, 56, pp.1637–1654.

10. Kumamoto, H., Matsubara, Y., Iizuka, Y., Okamoto, K. and Yokoi, K. (1986): Structure and Hypotensive Effect of Flavonoid Glycosides in Orange (Citrus sinensis OSBECK) Peelings, Agricultural and Biological Chemistry, 50, pp. 781–783

11. Proteggente, A. R., Saija, A., De Pasquale, A. & Rice-evans, C. A. (2003): The Compositional Characterisation and Antioxidant Activity of Fresh Juices from Sicilian Sweet Orange (Citrus sinensis L. Osbeck) Varieties, Free Radical Research, 37 pp. 681–687.

12. Kanaze, F. I., Termentzi, A., Gabrieli, C., Niopas, I., Georgarakis, M. and Kokkalou, E. (2008) : The phytochemical analysis and antioxidant activity assessment of orange peel (Citrus sinensis) cultivated in Greece-Crete indicates a new commercial source of hesperidin, Biomedical Chromatography, 23 pp.239-249

13. Han, S. T. (1998): Medicinal Plants in the South Pacific, World Health Organization, (WHO), Regional Publications West Pacific Series, No. 19, Manila

14. Natarajan S., Vidhya R.M. Potential medicinal properties of Carica papaya Linn.-A mini review. Int J Pharm Pharm Sci 2014, 6, 1-4.

15. Lian, X., Wang, C., Guo, K., 2007. Identification of new red pigments produced by Monascus ruber. Dyes Pigm., 73, 121–125.

16. Fessenden RJ., Fessenden JS., Feist P., Organic Laboratory Techniques.Brooks/Cole Thomson Leaning, Inc. Canada. p. 133–138.

17. Cledir S., Marcelo E., Zofia K., Nelson L., Fourier transform infrared as a powerful technique for the identification and characterization of filamentous fungi and yeasts, Res Microbiol 2010; 161: 168–175.

Received on 27.11.2019 Modified on 27.03.2020

Research J. Pharm. and Tech. 2021; 14(5):2417-2425.

DOI: 10.52711/0974-360X.2021.00426