阅读说明：本技术 一种提高裂殖壶菌发酵生产油脂中奇数碳脂肪酸产量的方法 (Method for increasing yield of odd-carbon fatty acid in oil produced by schizochytrium limacinum fermentation ) 是由 蓝东明 雷长梅 肖正 王永华 莫舒欣 于 2021-03-26 设计创作，主要内容包括：一种提高裂殖壶菌发酵生产油脂中奇数碳脂肪酸产量的方法,将裂殖壶菌(Schizochytrium sp.)27173-E接种在发酵培养基中,进行低温有氧发酵,控制培养基中的总碳源和氮源质量比为5:1～20:1,温度在15℃～35℃,发酵时间为60～120h。裂殖壶菌27173-E发酵生产OCFA的最优化条件是：总碳源和氮源质量比为10:1,有机氮与无机氮比为8:1,发酵温度为23℃,发酵时间84h。应用此方法,得到的发酵液中细胞最大干重达到78.87g/L,OCFA占总脂肪酸的31.82％,OCFA产量为7.48g/L。本发明极大提高了OCFA的产量,进一步扩大了裂殖壶菌的应用潜力。(A method for improving the yield of odd-carbon fatty acids in oil produced by fermentation of Schizochytrium limacinum is characterized in that Schizochytrium limacinum (Schizochytrium sp.)27173-E is inoculated in a fermentation culture medium for low-temperature aerobic fermentation, the mass ratio of a total carbon source to a nitrogen source in the culture medium is controlled to be 5: 1-20: 1, the temperature is 15-35 ℃, and the fermentation time is 60-120 h. The optimized conditions for producing OCFA by fermenting schizochytrium 27173-E are as follows: the mass ratio of the total carbon source to the nitrogen source is 10:1, the ratio of organic nitrogen to inorganic nitrogen is 8:1, the fermentation temperature is 23 ℃, and the fermentation time is 84 h. By using the method, the maximum dry weight of cells in the obtained fermentation liquor reaches 78.87g/L, the content of OCFA in the fermentation liquor is 31.82 percent of the total fatty acid, and the yield of OCFA in the fermentation liquor is 7.48 g/L. The invention greatly improves the yield of OCFA and further expands the application potential of schizochytrium.)

1. A method for improving the yield of odd-carbon fatty acids in oil produced by fermentation of Schizochytrium limacinum is characterized in that Schizochytrium limacinum (Schizochytrium sp.) is inoculated in a fermentation culture medium for low-temperature aerobic fermentation, the mass ratio of a total carbon source to a nitrogen source in the culture medium is controlled to be 5: 1-20: 1, the temperature is 15-35 ℃, and the fermentation time is 60-120 hours.

2. The method of claim 1, wherein the mass ratio of the total carbon source to the nitrogen source in the medium is 10: 1.

3. The method according to claim 1, wherein the mass ratio of the organic nitrogen N1 to the inorganic nitrogen N2 in the culture medium is 1: 1-9: 1.

4. The method of claim 1, wherein the culture medium contains organic nitrogen N1 and inorganic nitrogen N2 in a mass ratio of 8: 1.

5. The method according to any one of claims 1 to 4, wherein the fermentation temperature is 23 ± 2 ℃ and the fermentation time is 84 ± 12 h.

6. The method according to any one of claims 1 to 4, wherein the culture conditions are: the pH value is 7-8, the aeration is 3vvm, and the rotating speed is 600 rpm.

7. The method according to any one of claims 1 to 4, wherein the fermentation medium comprises, per 1L of the medium, 20 to 80g of glucose, 0.5 to 1.5g of potassium chloride, 5 to 10g of yeast powder, 4 to 8g of magnesium sulfate heptahydrate, 3 to 5g of potassium dihydrogen phosphate, 20 to 40g of anhydrous sodium sulfate, 5 to 40g of sodium glutamate, 1 to 6g of ammonium sulfate, 2 to 3mL of a metal ion solution, and 0.0005 to 0.001g of an antifoaming agent.

8. The method of claim 7, wherein the metal ion solution formulation (g/L): CoCl₂·6H₂O 0.2-0.5；CuCl₂·2H₂O 0.5-1.0；Na₂·EDTA 2.0-10.0；H₃BO₃ 2.0-5.0；MnCl₂·H₂O 1.0-5.0；NiCl·6H₂O 0.5-1.0；ZnSO₄·7H₂O 2.0-5.0；NaMoO₄·H₂O 1.0-3.0。

9. The method according to claim 1 or 2, wherein the Schizochytrium sp is Schizochytrium 27173-E.

Technical Field

The invention belongs to the field of microbial fermentation, and particularly relates to a method for increasing the yield of odd-carbon fatty acid in oil produced by schizochytrium limacinum fermentation.

Background

Odd-carbon fatty acids (OCFAs) function like unsaturated fatty acids and contribute to the improvement of cell membrane fluidity. It is rich in some bacteria, but not more than 1% in animal and lower plant. After the odd-carbon fatty acid is metabolized, sugar residues are generated, so that the nutrition effect is better; in addition, triglycerides containing odd-carbon fatty acids can reduce the frequency of secondary complications of sepsis and intensive care therapy, or reduce inflammation or shorten the course of disease (CN 200980145702.6). In recent years, studies have found that odd-chain fatty acids, which are present in small amounts in humans, have important physiological functions, and studies have shown that the odd-chain fatty acids in humans are negatively associated with diabetes, cardiovascular diseases, obesity, and the like.

The carbon chain length of most fatty acids in animal and vegetable oil is even number, and the carbon chain length of few fatty acids is odd number, such as pentadecanoic acid and heptadecanoic acid. Odd-carbon fatty acids are mainly derived from ruminal bacteria of ruminants, are mainly present in animal milk fat, and C15:0 and C17:0 account for about 1.2% and 0.54% of the total fatty acids, respectively. Odd-numbered fatty acids can be produced by two pathways, alpha oxidation of even-numbered fatty acids, and fatty acid synthesis. Alpha oxidation generally occurs when there is a modification of a group at the beta position of a fatty acid, which normally does not accumulate in large amounts in the cell. At present, fatty acid products with odd carbon chains are not produced, the main reason is that the sources of the fatty acids with odd carbon chains are few, the two existing improvement ways are mainly adopted, on one hand, by adding propionate, Zhang and the like, 1% -1.5% of isopropanol is added into a fermentation culture medium of rhodococcus erythropolis, and finally the proportion of OCFA in the total fatty acids is increased to 50%. On the other hand, the yield was increased by adding a precursor for OCFA synthesis (CN 201280054084.6). This patent improves the production of OCFA by enhancing propionyl-CoA synthesis, but the total amount of OCFA is only 0.325 g/L.

Microorganisms are a good source for producing odd-chain fatty acids, and schizochytrium also known as schizochytrium limacinum, a marine fungus belonging to the family thraustochytriaceae. Schizochytrium limacinum can accumulate a large amount of active substances, such as DHA, DPA, carotene, squalene, etc. Glucose or glycerol is used as a carbon source for fermentation, the dry weight of cells can reach 150g/L, and oil accounts for more than 70% of the dry weight of the cells. The schizochytrium limacinum has strong lipid accumulation capacity and relatively pure fatty acid composition, and is an excellent lipid synthesis host. Schizochytrium limacinum can be used for producing fatty acids with odd carbon number, and Chang et al introduce that Schizochytrium limacinum suspected strain of thraustochytriaceae can accumulate 15.4% of OCFA. In EP0823475A, the ratio of C15 to 0 and C17 to 0 in odd-carbon fatty acid of Schizochytrium SR21 is 10.1% and 1.8% respectively. Chang K et al fermented schizochytrium ATCC20888 in batches, and the highest amounts of C15: 09.16% and C17:0 were detected at 72h of fermentation at 2.91%. (Chang K, Mansource M P, Dunstan G A, et al. odd-chain polyunsaturated fatty acids in thraustochytrids [ J ]. Phytochemistry,2011,72(11-12): 1460-.

There are many reports of fermentation and lipid production of schizochytrium limacinum, but microbial fermentation for producing OCFA requires propionate induction, and has low biomass, low lipid yield and low content of OCFA. The genetic modification cost is high, most of the genetic modification cost is in a theoretical research stage, and the highest value of OCFA obtained through genetic engineering reported in the existing literature is 3.32 g/L. The invention can overcome the defects of the prior art and provide a method for improving the content of odd-numbered chain fatty acid in schizochytrium limacinum grease.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a method for improving the yield of odd-numbered fatty acid in oil produced by schizochytrium limacinum through fermentation, aiming at improving the percentage content of odd-numbered fatty acid in the oil product of the schizochytrium limacinum and improving the production efficiency.

The method comprises the steps of inoculating Schizochytrium sp in a fermentation culture medium, and carrying out low-temperature aerobic fermentation, wherein the mass ratio of a total carbon source to a total nitrogen source in the culture medium is controlled to be 5: 1-20: 1, the temperature is 15-35 ℃, and the fermentation time is 60-120 h.

Preferably, the mass ratio of the total carbon source and the nitrogen source in the culture medium is 10: 1.

Preferably, the mass ratio of the organic nitrogen N1 to the inorganic nitrogen N2 in the culture medium is 1: 1-9: 1.

Preferably, the mass ratio of the organic nitrogen N1 to the inorganic nitrogen N2 in the culture medium is 8: 1.

Preferably, the fermentation temperature is 23 +/-2 ℃, and the fermentation time is 84 +/-12 h.

Preferably, the culture conditions are: the pH value is 7-8, the aeration is 3vvm, and the rotating speed is 600 rpm.

Preferably, the fermentation medium comprises 20-80g of glucose, 0.5-1.5g of potassium chloride, 5-10g of yeast powder, 4-8g of magnesium sulfate heptahydrate, 3-5g of potassium dihydrogen phosphate, 20-40g of anhydrous sodium sulfate, 5-40g of sodium glutamate, 1-6g of ammonium sulfate, 2-3mL of metal ion solution and 0.0005-0.001g of defoaming agent in 1L of medium.

Preferably, the metal ion solution formulation (g/L): CoCl₂·6H₂O 0.2-0.5；CuCl₂·2H₂O0.5-1.0；Na₂·EDTA 2.0-10.0；H₃BO₃2.0-5.0；MnCl₂·H₂O 1.0-5.0；NiCl·6H₂O 0.5-1.0；ZnSO₄·7H₂O 2.0-5.0；NaMoO₄·H₂O 1.0-3.0。

Preferably, the Schizochytrium sp is Schizochytrium sp 27173-E.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for improving the yield of odd-carbon fatty acid in oil produced by schizochytrium limacinum fermentation, wherein the mass ratio of total carbon to nitrogen in the components of a culture medium is controlled to be 10:1, the mass ratio of organic nitrogen to inorganic nitrogen is adjusted to be 8:1, and the temperature condition is 23 ℃. Finally, the fermentation is amplified in 5L, the biomass reaches 78.87g/L at the fermentation end point, the oil yield reaches 24.95g/L at the fermentation end point within 96h, the ratio of the OCFA to the total fatty acid reaches 32.19% within 84h, and the yield of the OCFA reaches 7.48g/L at the fermentation end point within 84 h. Compared with the maximum yield of 3.32g/L obtained by the schizochytrium through genetic engineering at present, the yield is improved by 125 percent.

Drawings

FIG. 1 is a graph of cell growth over time.

FIG. 2 is a graph of grease accumulation over time.

FIG. 3 is a graph of odd-carbon fatty acid accumulation over time.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.

1) The strain is as follows: schizochytrium sp 27173-E was purchased from American type culture Collection, ATCC accession number PTA-9695, and is disclosed in patent application publication No. CN102428185, US 8207363.

2) Culture medium:

slant medium (1L): 80g of glucose, 1g of potassium chloride, 10g of yeast powder, 4.5g of magnesium sulfate heptahydrate, 3.5g of monopotassium phosphate, 37g of anhydrous sodium sulfate, 40g of sodium glutamate, 20g of agar and distilled water with the constant volume of 1000 mL;

seed medium (1L): 80g of glucose, 1g of potassium chloride, 10g of yeast powder, 4.5g of magnesium sulfate heptahydrate, 3.5g of monopotassium phosphate, 37g of anhydrous sodium sulfate, 40g of sodium glutamate, 2mL of metal ion solution and distilled water with constant volume of 1000 mL;

fermentation medium (1L): 80g of glucose, 1g of potassium chloride, 5g of yeast powder, 4.5g of magnesium sulfate heptahydrate, 3.5g of potassium dihydrogen phosphate, 37g of anhydrous sodium sulfate, 40g of sodium glutamate, 1.2g of ammonium sulfate, 2mL of metal ion solution, 0.0005g of defoaming agent and distilled water with the constant volume of 1000 mL.

The antifoaming agent is Dow Corning 1520 antifoaming agent, purchased from Shenzhen, Jeann district, West county, Ju Shu, No. 25 at exit of subway B.

Formula (g/L) of metal ion solution: CoCl₂·6H₂O 0.2；CuCl₂·2H₂O 1.0；Na₂·EDTA 5.0；H₃BO₃ 3.0；MnCl₂·H₂O 2.0；NiCl·6H₂O 1.0；ZnSO₄·7H₂O 4.0；NaMoO₄·H₂O 2.0。

3) Cell density: ultraviolet spectrophotometer, wavelength of 600 nm. Samples are taken to be diluted properly, the measuring range is 0.2-0.8, and the dilution times are multiplied by the measured value during calculation. This was repeated three times.

4) DCW determination: taking 10mL of fermentation liquor, centrifuging for 5min at 5000g, pouring out supernatant, washing the thallus for 2 times by deionized water, and obtaining the wet thallus of schizochytrium limacinum. And (3) drying the wet thalli in an oven at 80 ℃, and weighing the dry thalli to constant weight after drying. This was repeated three times.

5) Determination of total lipids: 10mL of fermentation liquid is taken, 5000g of the fermentation liquid is centrifuged for 5min, and the fermentation liquid is washed for 2 times by deionized water. Adding 5mL of hydrochloric acid into the wet thallus, swirling for 2min, placing in a water bath kettle at 80 ℃ for water bath heating for 1h, and extracting with n-hexane for 3 times until the supernatant is transparent. And (3) completely dissolving the oil sample in the n-hexane solution, recovering the solvent by rotary evaporation, drying the solvent, and weighing the grease. Performing methyl esterification with 3mL of 2% sodium hydroxide methanol at 60 deg.C for 30min to obtain fatty acid methyl ester, and detecting with gas chromatography-mass spectrometer. The gas chromatographic column is CP-Sil88, the used carrier gas is helium, the adopted sample injection mode is split-flow sample injection, and the temperature rise program of the chromatographic column is as follows: the initial temperature is 140 ℃, the temperature is kept for 5min, then the temperature is increased to 220 ℃ at the speed of 10 ℃/min, and the temperature is kept for 17 min. And calculating the contents of the total lipid and each fatty acid by adopting a peak area normalization method according to the internal standard.

Example 1

And (3) investigating the influence of the total carbon nitrogen ratio on the contents of schizochytrium limacinum grease and various fatty acids. Shaking a flask with a 500mL baffle, filling the flask with a liquid volume of 100mL, rotating at a speed of 180rpm, respectively taking glucose as a carbon source and sodium glutamate as a main nitrogen source in a schizochytrium culture medium, setting the mass ratio of total carbon to nitrogen to be 20:1, 10:1 and 5:1 respectively, and collecting thalli for analyzing each component when the thalli are fermented for 120 hours. The parameter change and the result of the fermentation process are shown in the table 1, the thalli grow rapidly in the first 48 hours of the fermentation, the thalli density and the dry weight increase rapidly, and after 2 days of the fermentation, the dry weight and the cell density of all groups of thalli reach the maximum values. Wherein the mass ratio of total carbon to nitrogen is 10: under the condition of 1, the cell density and the cell dry weight are obviously better than those of a nitrogen source excess group (5:1) and a carbon source excess group (20:1), and the maximum value of 45.55 and 86.2g/L is reached in the 2d of fermentation respectively. At the end of fermentation, under the condition that the mass ratio of C to N is 10:1, the dry weight of cells reaches 60.10g/L, and the oil yield is 7.44 g/L. The proportion of odd-carbon fatty acid in the total fatty acid is 9.27%, and the yield of the OCFA grease is 0.684 g/L. After the maximum yield of the OCFA is fermented for 3 days, the oil yield is 7.92g/L, the proportion of odd-carbon fatty acids to total fatty acids is 9.89%, and the oil yield of the OCFA is 0.783 g/L. The yield of OCFA was increased by 305.7% and 36.9% respectively, relative to the maximum yield of 0.193g/L (day 1) and 0.572g/L (day 4) in the nitrogen source excess group (C: N: 5:1) and the carbon source excess group (C: N: 20: 1).

Example 2

And (3) investigating the influence of different organic nitrogen sources and organic nitrogen source ratios on the production of OCFA by fermentation of schizochytrium limacinum. 6 groups of experiments are set, and the mass ratio of the organic nitrogen N1 to the inorganic nitrogen N2 is set to be 1: 1; 2: 1; 4: 1; 8:1 and all-organic nitrogen source and all-inorganic nitrogen source groups, shaking flask fermentation, liquid loading capacity of 100mL, fermentation for 5 days, each group being repeated for two times, based on the conclusion of example 1, the total carbon-nitrogen mass ratio is set to 10:1, other conditions are the same as example 1, and the results are shown in Table 2. The organic nitrogen source is beneficial to the growth of thalli, and the growth speed of the thalli is increased along with the increase of the proportion of the organic nitrogen source. And the inorganic nitrogen source is favorable for accumulating odd-carbon fatty acid, wherein the proportion of the odd-carbon fatty acid to the total fatty acid reaches a maximum value of 15.75% on the 3 rd day of fermentation of the full inorganic nitrogen source group, the dry weight and the density of the cells reach maximum values of 42.98g/L and 68.55 at the end point of the fermentation under the condition that the ratio of N1 to N2 is 8:1, and the oil yield accounts for 17.59% of the dry weight at the end point of the fermentation. Higher than the total organic nitrogen source group (15.91%), which is beneficial to the purification of grease and saves cost. The proportion of odd-carbon fatty acid in the total fatty acid is 10.29% at most, the yield is 0.54g/L at most, and the yield is only 13.64% and 0.679g/L of the total organic nitrogen source.

Although the OCFA yield of the total organic nitrogen source group is higher than that of the group with the carbon-nitrogen mass ratio of 8:1, the total organic nitrogen has high cost, and the results of the example 2 show that the addition of the inorganic nitrogen source can improve the oil content, improve the utilization rate of the carbon source and the nitrogen source, save the production cost and reduce the energy waste.

Example 3

The influence of temperature on the production of OCFA by schizochytrium is examined, the experiment sets three temperatures which are 23 ℃, 28 ℃ and 33 ℃, the liquid loading capacity of a 5L fermentation tank is 3L, the pH value of hydrochloric acid and sodium hydroxide is regulated to be 7, the rotating speed is 600rpm, the ventilation is 3vvm, and other conditions are consistent with those of a shake flask. Fermentation was carried out for 120h, samples were taken every 12h for fermentation parameter evaluation, and the results are shown in Table 3. As can be seen from Table 3, in the 23 ℃ fermentation group, the growth of the cells was fast, and at the end of the fermentation, the oil yield and the oil content in terms of dry weight percentage both reached maximum values, 18.78g/L and 24.31% respectively. After 3 days of fermentation, the cell density and the dry cell weight reached larger values of 127.8 g/L and 78.25g/L, respectively. Meanwhile, the proportion of odd-carbon fatty acid in the total fatty acid reaches 28.51 percent of the maximum value, and the yield is 5.288 g/L. Compared with a 28 ℃ fermentation group (2.747g/L) and a 33 ℃ fermentation group (1.463g/L), the maximum odd-carbon fatty acid yield of the fermentation is respectively improved by 92.5 percent and 261.5 percent. The largest change is C15:0, the proportion of C15:0 in total fatty acid is changed to the largest extent from 9.12% to 20.24% in fermentation of 24h to 48h, and meanwhile, the yield of C15:0 fatty acid reaches the highest value of 4.887g/L after 3 days of fermentation. The maximum value of the temperature of the steel plate is increased by 110.4 percent and 349.8 percent respectively relative to the maximum value under the conditions of 28 ℃ (2.323g/L) and 33 ℃ (1.087 g/L). Therefore, the 23 ℃ fermentation is beneficial to the schizochytrium to accumulate a large amount of odd-carbon fatty acids, particularly C15: 0.

TABLE 2 comparison of fermentation results for different organic and inorganic nitrogen ratios

Example 4

After temperature and culture medium components are optimized, the influence of high-density fermentation of schizochytrium limacinum in a 5L fermentation tank in fed-batch mode to produce odd-number carbon fatty acid (OCFA) is considered. This experiment was carried out in combination with the results of examples 1 to 3, wherein 3L of liquid was filled in a 5L fermenter, and the other conditions were the same as those of example 3, and the fat was extracted and the fatty acid composition was measured after 5 days of fermentation, and the results are shown in FIGS. 1 to 3. As shown in the figure, the cell density and dry weight change trends consistently throughout the fermentation period, and the cell density and dry weight increase rapidly and slowly and finally stabilize with the increase of the fermentation time. The maximum cell density and cell dry weight were 120 and 80g/L, respectively. The sodium glutamate is completely consumed in 36 hours, the glucose consumption rate reaches the maximum in the first 48 hours, and the glucose concentration is maintained at about 40g/L by feeding and supplementing materials in the later period. The oil yield reaches the maximum value of 24.95g/L after 96 hours of fermentation, and the dry weight percentage is 33 percent at most. From the aspect of fatty acid composition, the proportion of odd-number fatty acid to total fatty acid is gradually accumulated along with the time, and the change is maximum between 24 and 36 h. The highest proportion of the total odd-carbon fatty acids reaches 32.19 percent in 84 hours of fermentation, wherein C15:0 fatty acids account for 23.2 percent. The yield reaches 7.48g/L at most. According to the results shown in fig. 2, the biomass (dry weight) is increased by nearly one time compared with the culture before optimization by adopting the optimized temperature and culture medium components for fermentation, the proportion of odd-carbon fatty acids in the total fatty acids reaches 32.19%, the yield of the fat containing odd-carbon fatty acids reaches 7.48g/L, which is higher than the highest value of 3.32g/L obtained by genetic engineering reported in the prior art and is increased by 125%.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.