اثر منابع کربنی و نیتروژنی بر روی پروفایل اسیدهای چرب قارچ مورتیرلا وینشیا

Introduction

Lipid biotechnology is currently emerging as a significant field due to numerous applications of fatty acid and lipid derivatives in various applications such as food and pharmaceutical industries. The n-3 and n-6 (for example linolenic acid and linoleic acid, respectively) chains of polyunsaturated fatty acids (PUFAs) are introduced as potential food additives or pharmaceuticals for their biological activities. The PUFAs can be applied for the treatment of cardiovascular illnesses, cancers and as a participant in the inflammatory reactions (1-9).

Oleaginous microorganisms, such as microalgae, yeasts, fungi and bacteria can produce high levels of lipids and do not need arable lands. The fatty acid profiles are dependent on oleaginous microorganism's types and the growth conditions. Environmental conditions such as temperature, pH, substrate, C/N ratio and oxygen pressure have an effect on the productivity of accumulating lipids. Accumulation of lipids in yeasts and fungi chiefly is deliberate because these microorganisms often produce polyunsaturated fatty acids(10-15).

There are various reports in the literature for lipid production in Mortierella. For example, it has been reported that total lipid in Mortierella alpina was 36%. In another research by Kimura et al, the total lipid level in Mortierella isabellina was 33%. In both of the trials, glucose was selected as a carbon source. In another study, the concentration of linoleic acid was reported 9.8% in media by Mortierella alliacea and percentage of linolenic acid was 3.4% (14- 16). However, the outstanding emphasis of this study is improvement and production of lipids and fatty acids in Mortierella vinacea PTCC 5262, using suitable substrates at the optimum conditions.

Materials and methods

Chemicals: All solvents, chemicals and carbohydrates were purchased from Merck Company. Mineral salts were prepared from Sigma Company.

Culture conditions: Mortierella vinacea PTCC 5262 was purchased from Iranian Research Organization for Science and Technology (IROST) and it was applied for fatty acid production. Mortierella vinacea was maintained on potato dextrose agar (PDA) slants with repeated sub-culturing for its viability. The culture was incubated in 250 ml Erlenmeyer flask for 72 hours at 28°C on a controlled rotary shaker at 180 rpm. The production medium (basal medium, BM) contained (g/l): glucose 40, yeast extract 1.5, NH₄NO₃ 0.286, KH₂PO₄ 0.75, MgSO₄·7H₂O 0.4 and CaCl₂ 0.4 (pH = 5.6). The flask containing 50 ml culture medium was inoculated with suspension containing 1×10⁸ spores/ml. The total number of spores was determined by counting under microscope using haemocytometer (Neubauer counting chamber). The fungal spore count was adjusted to 1 × 10⁸ spores/ml by making appropriate dilutions (17).

Determination of biomass: Biomass was obtained from culture broth through Whatman No. 1 filter paper. Then it was washed twice with distilled water and it was dried at 60°C for sufficient time to reach to a fixed weight (usually after 24h). The dry cell weight was determined gravimetrically (18, 19).

Lipid extraction: The produced lipids were extracted from mycelia according to a modified procedure by Bligh and Dyer. Briefly, cells were accumulated through Whatman No.1 filter paper, then the fungi was washed twice with distilled water, then 1g of cells were added to 10 ml of 4 M HCl, and they were incubated at 60°C for 2h. The acid hydrolyzed mass was shaken with 20 ml of chloroform/methanol mixture (1:1) at room temperature for 2h to 3h, followed by centrifugation at 2000 rpm for 5 min at room temperature using Sigma 3K30 centrifuge in order to separate the aqueous upper phase from organic lower phase. The upper phase was removed by a Pasteur pipette and the lower phase containing lipids was gathered by a sampler and then it was evaporated under a reduced pressure for 10 min (19).

Fatty acid analysis

Preparation of fatty acid methyl esters (FAMEs): Free fatty acids were esterified and then o-acyl lipids were transesterified by heating them with a large excess of anhydrous methanol in presence of an acidic catalyst. Fatty acids were methyl esterified according to the procedure by Christie. The sample of lipid (50 mg) was dissolved in a test tube fitted with 1 ml toluene by a condenser. Then 2ml 1% sulfuric acid in methanol was added and this solution was maintained at 50°C for 24h. The methylated fatty acids were separated from the aqueous layer by adding 5ml of 5% NaCl and then it was dissolved in 2×5ml n-hexane. The n-hexane layer was separated by Pasteur pipettes, which was washed with 4 ml of water containing 2% potassium bicarbonate. Then it was dried over anhydrous sodium sulfate. The solution is filtered and the solvent was removed under reduced pressure in a rotary film evaporator or in a stream of nitrogen (20).

Fatty acids content determination by gas chromatography (GC): GC was performed on Agilent 19091J-413 Series Gas Chromatograph which is equipped with a FID and the capillary column HP5 (30 m ´ 0.25 mm i. d., 0.25 µm film thickness; USA). Injector and detector temperatures were maintained at 260°C and 300°C, respectively. The oven was programmed at 100°C for 2 min, and then it was increased to 160°C at 3 min. Therefore, it was maintained for 2 min at 215°C, it was increased more to 217°C at 2 min, then it was maintained at 218°C for 2 min and finally it was increased to 260°C at 2 min. The carrier gas, nitrogen, was used at a flow rate of 1.5 ml/min. The injection volume was 1 µL, with a split ratio of 50:1.

Determination of glucose concentration: The glucose concentration was estimated with the dinitrosalicylic acid assay in the medium according to a method described by Xiong et al. (21).

Effect of different carbon sources on lipid and fatty acid content: Glucose in the basal medium was replaced by sucrose, fructose and lactose and the medium was incubated at 28°C for 72h. 40 g/l of the carbon source was added to the basal medium.

Effect of different nitrogen sources on lipid and fatty acid content: Nitrogen source in the basal medium was replaced by 1.5% of peptone and triptone at 28°C for 72h. Lactose (40 g/l) was consumed as a carbon source in both of them.

Results

Growth characteristics and lipid production of M. vinacea:Lipid production of the fungal cells was increased gradually and then it was declined at 96h. Maximum yield of lipid was observed in the beginning of the stationary phase (4.76% in 72h). The initial glucose concentration was 40 g/l which was consumed during the cell growth and lipid accumulation and it was depleted in 96h. Value of the initial pH in all tests was 5.5-5.8 (Fig. 1).

Fig. 1- Kinetics monitoring of biomass (g/l), cellular lipid in total biomass (%) and glucose concentration (g/l), in Mortierella vinacea PTCC 5262 on the medium. Medium conditions: initialglucose concentration 40 g/l, initial yeast extract concentration 1.5 g/l, initial pH = 5.6,T=28°C,Incubation Time 96 h. Data are presented as mean values from triplicate experiments.

Effect of different carbon sources on lipid and fatty acid content of M. vinacea: The effect of several carbon sources on biomass and lipid production was investigated (Table 1). The best cell growth was obtained by fructose followed by sucrose, glucose and lactose. Although, higher quantities of lipid in absolute values (L_max, g/l), biomass (x, g/l) and total biomass yield on the consumed carbon source (Y_X/S, g/g) were produced on fructose more than on lactose (0.84, 6.4 and 0.16 against 0.32, 1.2 and 0.03g/l respectively) during growth. Cellular lipid in total biomass (Y_L/x, %) was 13.12 % in the fructose. But, the highest level of Y_L/X was found in the media containing lactose and yeast extract (26.66 %).

The lipid yield on the consumed carbon source (Y_L/S, g/g) was equal in the presence of glucose, sucrose, fructose in the media (also the media contained lactose and peptone). In all of the tested carbon sources, palmitic acid (16:0) was produced, but stearic acid (18:0) was only found in the presence of glucose and fructose. When the media contained lactose, sucrose, fructose and glucose, oleic acid (18:1) contents of 9.18, 9.82, 18.41 and 44.11% were obtained respectively. However, the maximum oleic acid yield was detected by glucose. Linoleic acid was produced only in the presence of lactose and its level was 25.7%. Therefore, lactose was selected as a carbon source in the following experiments.

Table 1- Growth and fatty acid composition of Mortierella vinacea PTCC 5262 in nitrogen limited media with carbon based sources as substrates.

Strain PTCC 5262 was cultivated in a medium containing 40 g/l carbon sources and 1.5 g/l nitrogen sources, pH 5.6, with shaking at 28°C for 72h. Representation of biomass (X, g/l), total biomass yield on the consumed carbon source (Y_X/S, g/g), cellular lipid in total biomass (Y_L/X, %) and lipid yield on the consumed carbon source (Y_L/S, g/g) when the maximum quantity of cellular lipids (L_max, g/l) was achieved. C16:0, palmitic acid; C18:0, stearic acid; C18:1(n-9), oleic acid; C18:2(n-6), linoleic acid; C18:3(n-6). Minor fatty acid components are not shown. Data are presented as mean values from triplicate experiments conducted by using different inocula.

Strain PTCC 5262 was cultivated in a medium containing 40 g/l carbon sources and 1.5 g/l nitrogen sources, pH 5.6, with shaking at 28°C for 72h. Representation of biomass (X, g/L), total biomass yield on the consumed carbon source (Y_X/S, g/g), cellular lipid in total biomass (Y_L/X, %) and lipid yield on the consumed carbon source (Y_L/S, g/g) when the maximum quantity of cellular lipids (L_max, g/L) was achieved. C16:0, palmitic acid; C18:0, stearic acid; C18:1(n-9), oleic acid; C18:2(n-6), linoleic acid; C18:3(n-6). Minor fatty acid components are not shown. Data are presented as mean values from triplicate experiments conducted by using different inocula.

Effect of different nitrogen sources on lipid and fatty acid content of M. vinacea: Utilization of some nitrogen sources by M. vinacea was investigated (Table 1). The best cell growth among other nitrogen sources (yeast extract, triptone and peptone) was peptone. In all of the tested nitrogen sources, palmitic acid was produced, but stearic acid was not detected in all of them. Linoleic acid and oleic acid were produced in the media containing yeast extract and peptone, whereas there werenot any linoleic acid and oleic acid in the presence of triptone (Fig. 2). M. vinacea produced the highest level of linoleic acid (52.76%) in the medium that contained peptone. Linolenic acid was only obtained in the presence of triptone. Hence, peptone and triptone were chosen as nitrogen sources for M. vinacea.

Fig.2- Fatty acid composition (%) of the Mortierella vinacea PTCC 5262 lipid produced in different nitrogen sources (1.5 g/l) in the medium. Lactose (40 g/l) used as a carbon source in all of them (Incubation Time, 72h). Data are presented as mean values from triplicate experiments conducted by using different inocula.