INTRODUCTION:

Essential fatty acids, or EFAs, are fatty acids that humans and other animals must ingest because the body requires them for good health but cannot synthesize them^[1]. Only two fatty acids are known to be essential for humans: alpha-linolenic acid (an omega-3 fatty acid, ω-3) and linoleic acid (an omega-6 fatty acid, ω-6)^[2]. Some other fatty acids are sometimes classified as "conditionally essential", meaning that they can become essential under some developmental or disease conditions; examples include docosahexaenoic acid (an omega-3 fatty acid) and gamma-linolenic acid (an omega-6 fatty acid). In the body, essential fatty acids serve multiple functions. In each of these, the balance between dietary ω-3 and ω-6 strongly affects function. Omega−3 fatty acids, also called ω−3 fatty acids or n−3 fatty acids^[3] are polyunsaturated fatty acids (PUFAs)^[4,5].

The fatty acids have two ends, the carboxylic acid (-COOH) end, which is considered the beginning of the chain, thus "alpha", and the methyl (-CH₃) end, which is considered the "tail" of the chain, thus "omega". One way in which a fatty acid is named is determined by the location of the first double bond, counted from the tail, that is, the omega (ω-) or the n- end. Thus, in omega-3 fatty acids the first double bond is between the third and fourth carbon atoms from the tail end. However, the standard (IUPAC) chemical nomenclature system starts from the carboxyl end.

The three types of omega−3 fatty acids involved in human physiology are α-linolenic acid (ALA), found in plant oils, and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), both commonly found in marine oils⁵.Marine algae and phytoplankton are primary sources of omega−3 fatty acids^[6]. Common sources of plant oils containing ALA include walnut, edible seeds, clary sage seed oil, algal oil, flaxseed oil, sacha inchi oil, echium oil and hemp oil, while sources of animal omega−3 fatty acids EPA and DHA include fish, fish oils, eggs from chickens fed EPA and DHA, squid oils, and krill oil. Dietary supplementation with omega−3 fatty acids does not appear to affect the risk of death, cancer or heart disease^[7,1]. Benefits of omega-3 fatty acids may be summarized as improve heart health, battle mental disorders and decline, reduce inflammation and associated with lowered cancer risks.

The recent report of Arzu et al., showed that the Fusarium sp., are capable of producing single cell oil containing various PUFA’s. They also observed that the Fusarium sp., can accumulate up to 78% of total fatty acids as unsaturated fats. The nutritional potential of biomass and metabolites from filamentous fungi had focused on the metabolites produced by filamentous fungi that have been showing potential for use by food industry such as sources of antioxidant agents, flavors, color pigments, vitamins and essential fatty acid^[8].

Different microorganisms like bacteria, yeasts, filamentous fungi and micro algae are able to synthesize polyunsaturated fatty acids. More frequently the test is made with filamentous fungi as the results say that they have capacity to produce DHA and EPA polyunsaturated fatty acids. When compared with vegetable oils, oils from microorganism have advantages such as shorter life cycle, abundant cheap raw material and production in large scale. Using metabolic engineering, the PUFA production from native microorganism is increased.

MATERIALS AND METHODS:

Screening and isolation of Fusarium sp.:

The soil samples were screened from BIHER, Chennai for the omega-3 producers.1.0gm of soil sample was suspended in 9 ml sterile saline water, serial 10-fold dilutions of that suspension were made and then 0.1ml of sample from 10̵ ² to 10̵ ⁸ was spread evenly on the surface of PDA (Potato dextrose agar) medium^[9,10]. The plate was then incubated at RT. After 4 days of incubation, the selective fungal white colonies were transferred to fresh potato dextrose agar and incubated at room temperature (28°C±1) for sub culturing. Then the absolute cultures were identified, maintained on the PDA plate or slants at 4°C. Fusarium sp., was identified through LPCB stain and used for the production. Pure culture of the above mentioned organism was prepared by quadrant streaking.

Production medium for omega-3 fatty acid:

Seed culture was prepared in 50ml medium containing (g/L) Glucose- 20; Yeast extract-10; the pH was adjusted to 6.0 and autoclaved at 121°C for 20 mins. Pure single cell colony was inoculated in the seed medium and the flasks were incubated for 48 hrs at 28°C. The fermentation medium contained (g/L) Starch- 20g, yeast extract - 5g, KNO₃ - 10g, KH₂PO₄- 1g, MgSO₄- 0.5g^[11]. The final pH was adjusted to 6.5. The medium was sterilized at 121°C for 15min. 10% seed culture was inoculated in the production medium and then incubated for 5 days at RT on a rotary shaker at 120 rpm.

Dry biomass determination and lipid extraction:

Biomass production was determined by harvesting the cells through centrifugation and the cells were dried at 55-60ºC overnight. 1g of Dry biomass was thimble macerated with 0.1N HCl for 20 min. 40ml of chloroform and methanol in the ratio of 2:1 was added to the macerated biomass. Mixture was agitated and kept in orbital shaker for 20min.This mixture was filtered with What man paper. To this filterate 0.9% NaCl solution was added. The solution was then transferred in a separating funnel for separation of chloroform and methanol. The chloroform layer was collected separately and heated at 70ºc for 15 mins.

Rapid preparation of fatty acid methyl esters from lipids:

Dried chloroform layer with lipid was evaporated to dryness. The dried lipid was dissolved in 2ml freshly prepared mixture of methyl chloride and methanol at a ratio of 1:20.

Cooled the sample at room temperature. 1ml of extracting solvent (diethyl ether) added and vortexed for about 20sec. Purification of solution was achieved by washing (using 1ml of water), causing the formation of two immiscible phases, which are then allowed to separate. The upper extracted solvent phase was recovered^[10], dried over anhydrous sodium sulphate and analyzed using TLC.

Thin layer chromatography (TLC) examination of methyl ester:

Thin layer chromatography was an excellent tool for micro preparative separation of mixture. The methyl ester mixtures were charged on thin layer of silica gel and developed by ascending technique using the petroleum ether and diethyl ether in the ratio of 60:40 (v/v). After developing, the plates were dried at room temperature and placed in iodine chamber. The fatty acids methyl ester gave yellow color spot with this iodine vapor. The colored spots were marked and the R_f values of the spots were calculated. Thin layer Chromatography with standard methyl ester was carried out and R_f values were compared.

Chemical titration method:

Weighed 0.1g of oil or fat in glass vial and dissolved in at least 50ml of solvent mixture (if necessary by gentle heating). Solvent mixture is the mixture of 95% ethanol and diethyl ether in the ratio of 1:1(v/v). Titrated, with shaking, with the KOH solution (0.1M KOH in ethanol accurately standardized with 0.1M HCl) in a burette graduated in 0.1ml to the end point of the indicator (5 drops), till the pink color persisting for at least 10s.

The acid value is calculated by the formula:

56.1*N*V/M

Where, V is the ml of KOH solution used, N is the normality, M is the mass in g of the sample.

RESULTS AND DISCUSSION:

Screening and isolation of Fusarium sp.:

1g of soil sample was taken from BIHER, Selaiyur, Chennai and was screened for fungal omega 3 fatty acid producer. 9 different strains of filamentous fungi were obtained (Figure-1) and 3 isolates were found to be Fusarium based on LPCB staining. These strains were screened for the omega 3 production and FS1 was found to be the maximum producer (Figure-2). The LPCB stained Fusarium was shown in (Figure-3).

Figure-1 PDA plate .

with various .

fungal isolates

Figure-2 Pure culture of Fusariam sp.

Figure-3 LPCB staining – Fusariam

Figure-4 Seed culture

Figure-5 Production medium

Extraction of omega-3 fatty acid:

The omega –3 fatty acid was obtained from Fusarium sp., by mechanical disruption of its biomass. The biomass was thimble macerated using 0.1N Hcl. The fungal dry powder was blended with chloroform and methanol. The mixture was filtered and sodium chloride solution was added. The methyl ester fatty acid was prepared, the upper layer was extracted and used for the estimation and analysis.

Analysis of omega 3 fatty acid:

The methyl ester was charged on silica gel, developed by ascending technique using petroleum ether and diethyl ether (Figure-6). The plates were placed in iodine chamber. The fatty acid methyl ester gave yellow color spot with the iodine vapor as shown in Figure-7.

The R_f values are calculated. The R_f value was found to be 0.437 which was close to the standard R_f value of methyl stearate.

Figure-6 TLC setup

Iodine band

Estimation of omega-3 fatty acid:

The acid value is a common parameter in the specification of fats and oils which is defined as weight of KOH in milligram needed to neutralize the organic acid present in 1g of fat and it is a measure of free fatty acid in a sample of oil or fat. The amount of free fatty acid was determined volumetrically by titrating with potassium hydroxide.

CONCLUSION:

Omega-3 fatty acids are one of the “good” types of fat; they may help to lower the risk of heart disease. This study focuses on the production of this valuable lipid from the soil fungi Fusarium. The results confirmed that this organism can be exploited for the production of omega-3. However, large scale study and purification is required for commercialization.

REFERENCES:

1. Scorletti E, Byrne C.D. Omega−3 fatty acids, hepatic lipid metabolism, and nonalcoholic fatty liver disease. Annual Review of Nutrition. 2013; 33 (1): 231–48.

2. Arzu A, Kpinar-Bayizit, TulayOzcan, Lutfiye Yilmaz-Ersan, Fikri Basoglu. Single cell oil (SCO) production by fusarium species using cheese whey as a substrate. 2014. Mljekarstvo 64.

3. MacLean C.H, Newberry S.J, Mojica W.A, Khanna P. Effects of omega−3 fatty acids on cancer risk: a systematic review. The Journal of the American Medical Association. 2006; 295 (4):403–15

4. Rizos E.C, Ntzani E.E, Bika E, Kostapanos M.S. Association between omega-3 fatty acid supplementation and risk of major cardiovascular disease events: a systematic review and meta-analysis. The Journal of the American Medical Association. 2012; 308 (10): 1024–33.

5. Grey A and Bolland M. Clinical trial evidence and use of fish oil supplements. JAMA Internal Medicine. 2014; 174 (3): 460–62.

6. Billman G.E. The effects of omega-3 polyunsaturated fatty acids on cardiac rhythm: a critical reassessment. Pharmacology & Therapeutics. 2013;140 (1): 53–80

7. Robert S, Maurice E, Shils. Modern Nutrition in Health and Disease. 1980; 6, 134–138.

8. Whitney Ellie; Rolfes S.R. Understanding Nutrition. 2008; 11,154.

9. Takahash T, Yano T, Zhu J. Hwang G.W, Naganuma A. Overexpression of FAP7, MIG3, TMA19, or YLR392c confers resistance to arsenite on Saccharomyces cerevisiae. J Toxicol Sci. 2010; 35(6):945-6.

10. Kamalambigeswari, Sangilimuthu Alagar, Narender Sivvaswamy. Strain Improvement through Mutation to Enhance Pectinase Yield from Aspergillus niger and Molecular Characterization of Polygalactouronase Gene. Journal of Pharmaceutical sciences and research 2018; pp.989-994.

11. Bhasin S, Modi H.A. Optimization of fermentation medium for the production of glucose isomerase using Streptomyces sp. SB-PI. Biotechnology Research International. 2012: 1-6.

Received on 15.04.2019 Modified on 03.05.2019

Research J. Pharm. and Tech 2019; 12(9):4295-4298.

DOI: 10.5958/0974-360X.2019.00738.8

S.NO	Fat sample used (g)	Volume of fat sample in (ml)	Initial reading(ml)	Final reading(ml)	Average reading
1.	3.95	2	0	0.4	0.4
2.	0	2	0	0.2	0.2