CN116179619A

CN116179619A - Method for improving DHA content and yield of schizochytrium limacinum by using synergistic induction of 2 synthetic plant growth regulators

Info

Publication number: CN116179619A
Application number: CN202211614895.9A
Authority: CN
Inventors: 唐裕芳; 杨雨欣; 曾彦霖; 李玉芹; 周蓉
Original assignee: Xiangtan University
Current assignee: Xiangtan University
Priority date: 2022-12-15
Filing date: 2022-12-15
Publication date: 2023-05-30

Abstract

The invention discloses a method for improving DHA content and yield of schizochytrium limacinum by using 2 synthetic plant growth regulators in a synergistic induction way. The method specifically comprises the following steps: 1) Coating activated schizochytrium limacinum with a flat plate, culturing schizochytrium limacinum with a seed culture medium until the logarithmic phase, and collecting algae liquid as seed liquid for fermentation; 2) Taking DHA content as an index, optimizing the DA-6 addition concentration in a fermentation medium; 3) And (3) continuously taking DHA content as an index, optimizing the addition concentration of 2,4-D in fermentation culture on the basis of the optimized DA-6 concentration, and constructing a stable culture system for synergistically inducing the DHA of the schizochytrium limacinum by 2 synthetic plant growth regulators and improving the DHA content and the yield. According to the invention, 2 synthetic plant growth regulators DA-6 and 2,4-D are simultaneously added, so that the DHA content and yield of the schizochytrium limacinum are synergistically induced to be improved, and a new way and basis are provided for industrial production of DHA.

Description

Method for improving DHA content and yield of schizochytrium limacinum by using synergistic induction of 2 synthetic plant growth regulators

Technical Field

The invention relates to the technical field of algae biology, in particular to a method for synergistically improving DHA content and yield of schizochytrium limacinum by using 2 synthetic plant growth regulators.

Background

DHA is used as a nutrient substance with a plurality of special physiological functions, plays an important role in cardiovascular health, brain development, vision health care and the like of human bodies, and has wide application prospect in the medicine and food industries. The traditional source of DHA is deep sea fish oil, along with the increasing exhaustion of marine resources, and the extraction of DHA from the deep sea fish oil has the defects of high cost, heavy fishy smell, high cholesterol and the like, so that the replacement resources for sustainable production of DHA are required to be searched, and the current utilization of a biological fermentation method for synthesizing DHA is environment-friendly, renewable and artificially regulated, so that the method becomes a current research hotspot.

Schizochytrium (Schizochytrium) is a class of marine fungi belonging to the phylum fungi, the phylum oomycetes, the order of the family of the hydromyces, the family of the thraustochytriaceae. The schizochytrium limacinum has short growth cycle, does not occupy cultivated land, is not influenced by seasons and climates, has high DHA content of algae oil, is paid attention to, and is considered to be one of potential strains for realizing sustainable commercial production of DHA. There are many strategies to promote DHA accumulation in schizochytrium, abiotic stress (low temperature, nutrient deficiency, high salt, etc.) is a common strategy to promote DHA content increase, but abiotic hypochondriac forces schizochytrium biomass to be significantly reduced, so that DHA yield increase of schizochytrium is not significant. The plant growth regulator is artificial synthesized plant hormone analogue and has the functions of regulating the growth process and cell activity of plant. The research of the Chinese invention CN 102304479A proves that the phytohormone can promote the DHA content and yield of the schizochytrium limacinum to be increased, however, the DHA content is not high, and the industrial production is not facilitated.

Disclosure of Invention

Aiming at the technical problems that the existing strategy is insufficient in DHA content and yield improving capability in the schizochytrium limacinum and is not beneficial to industrialized production, the invention provides a method for synergistically improving DHA content and yield in the schizochytrium limacinum by using 2 synthetic plant growth regulators, wherein 2 plant growth regulators, namely diethyl aminoethyl hexanoate (DA-6) and 2, 4-dichloro naphthyloxy acetic acid (2, 4-D), are simultaneously added during fermentation culture of the schizochytrium limacinum, so that good synergistic effect is exerted, DHA accumulation in the schizochytrium limacinum is promoted, and DHA content and yield are improved.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

a method for synergistically increasing the DHA content and yield of schizochytrium limacinum with 2 synthetic plant growth regulators, comprising the steps of:

(1) Coating activated schizochytrium limacinum with a flat plate, culturing the schizochytrium limacinum with a seed culture medium until the logarithmic phase, collecting algae liquid as a seed culture liquid, and inoculating the seed culture liquid to a fermentation culture medium;

(2) Fermenting and culturing schizochytrium limacinum in a fermentation medium added with 0-1.0 mg/L DA-6, and determining the optimal addition concentration of DA-6 by taking DHA content as an index;

(3) And (3) continuously taking DHA content as an index, determining the optimal addition concentration of 2,4-D based on the DA-6 concentration determined in the step (2), and constructing a stable culture system for synergistically inducing the DHA accumulation of the schizochytrium limacinum and improving the DHA content and the yield of the schizochytrium limacinum by using 2 synthetic plant growth regulators.

Further, the formula of the seed culture medium adopted by the schizochytrium limacinum is as follows: 25.00-35.00 g/L glucose, 4.00-12.00 g/L yeast powder and 15.0-25.00 g/L seawater crystal; the formula of the fermentation medium is as follows: 50g/L to 100g/L of glucose, 5g/L to 30g/L of yeast powder, 0.1g/L to 1g/L of sodium chloride, 0.5g/L to 5g/L of potassium sulfate, 0.1g/L to 1g/L of monopotassium phosphate, 1g/L to 10g/L of crystalline magnesium sulfate and 0.01g/L to 1g/L of anhydrous calcium chloride.

Further, the plate coating activation schizochytrium limacinum specifically comprises the following steps: the schizochytrium limacinum strain is coated on a flat plate for activation for 48-72 hours, the obtained single colony algae strain is picked up and transferred into a seed liquid culture medium, and the single colony algae strain is cultured in a shaking table with the temperature of 25-30 ℃ and the rotating speed of 150-200 rpm for 24-48 hours until the logarithmic phase to obtain a seed culture liquid for being inoculated into a fermentation culture medium.

Further, the vessel for culture was a baffle flask.

Further, inoculating the seed culture solution into a fermentation culture medium containing 0-1.0 mg/L DA-6 according to an inoculation proportion of 2% -10%, culturing for 48-120 hours in a shaking table with the temperature of 25-30 ℃ and the rotating speed of 150-200 rpm, collecting algae liquid, centrifugally separating to obtain schizochytrium limacinum, freeze-drying the algae mud, measuring the DHA content in the schizochytrium limacinum by a gas chromatography, and calculating the DHA content by an internal standard curve method. The optimal addition concentration of DA-6 was determined at the highest DHA content.

Further, the step (3) specifically comprises: on the basis of determining the concentration of DA-6, adding 0-2.0 mg/L of 2,4-D, inoculating a seed culture solution according to an inoculation proportion of 2% -10%, culturing for 48-120 hours in a shaking table with the temperature of 25-30 ℃ and the rotating speed of 150-200 rpm, collecting algae liquid, centrifugally separating to obtain schizochytrium, freeze-drying the algae mud, measuring the DHA content in the schizochytrium by a gas chromatography, and calculating the DHA content by an internal standard curve method. The optimal synergistic concentration of 2,4-D was determined at the highest DHA content.

Further, after saponification and methyl esterification of the schizochytrium limacinum powder, extracting DHA with saturated sodium chloride and normal hexane, determining the DHA content by a gas chromatography method, and calculating the DHA content by an internal standard curve method.

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

the existing strategies for promoting the DHA content and yield of the schizochytrium limacinum include abiotic stress, increasing oxygen dissolution, adding plant growth regulators and the like. The common abiotic stress strategy can improve DHA accumulation but inhibit the growth of the schizochytrium limacinum, so that the DHA yield is limited, and corresponding equipment is required for increasing the dissolved oxygen amount, so that the algae raising cost is increased. The DHA content and yield of the schizochytrium limacinum can be obviously improved by adding the synthetic plant growth regulator, the dosage of the plant growth regulator is small, the addition of the plant growth regulator to a culture medium is simple and convenient, and the production cost of the synthetic plant growth regulator is low. Through researches and analysis, the inventor unexpectedly found that the 2 plant growth regulators DA-6 and 2,4-D are matched to be used at proper concentrations, so that obvious synergistic effect can be exerted, and DHA content and yield of schizochytrium limacinum are obviously improved by a multiple compared with the single plant growth regulator. Therefore, the method for synergistically improving DHA content and yield of the schizochytrium limacinum by adding 2 synthetic plant growth regulators is a relatively economical strategy, and meets the industrial production requirements better.

Drawings

FIG. 1 shows the effect of different concentrations of DA-6 on DHA content and yield in Schizochytrium.

FIG. 2 shows the effect of different concentrations of 2,4-D on DHA content and yield in Schizochytrium.

FIG. 3 shows the effect of 0.05mg/L DA-6 in combination with different concentrations of 2,4-D on DHA content and yield in Schizochytrium.

FIG. 4 is a comparison of DHA content and yield in schizochytrium limacinum when 0.05mg/L DA-6 was used in combination with 0.5 mg/L2,4-D and 0.05mg/L DA-6 alone, 0.5 mg/L2,4-D and no plant growth regulator was added.

Detailed Description

The invention provides a method for improving DHA content and yield of schizochytrium limacinum by using 2 synthetic plant growth regulators in a synergistic induction way. The present invention will be further described with reference to the following examples, but the present invention is not limited thereto. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

The schizochytrium limacinum strain stored at-80 ℃ is coated on a flat plate for activation culture for 72 hours, single colony in the flat plate is picked up and transferred to a seed liquid culture medium, and then the culture is carried out in a shaking table at the temperature of 28 ℃ and the rotating speed of 165rpm for 48 hours, so as to obtain a seed culture liquid.

Seed culture solutions were inoculated into the fermentation media of Schizochytrium limacinum added with 0.01,0.05,0.1,0.5,1mg/L DA-6 at an inoculation ratio of 5% (and a case where no plant growth regulator was added as a control), and cultured in a shaker at 28℃and a rotation speed of 165rpm for 96 hours. Collecting algae liquid, centrifuging, and freeze drying to obtain schizochytrium limacinum powder.

0.02g of algae powder is added with 1mL of 0.5mol/L KOH-CH ₃ OH,1mg/mL of an internal standard methyl nonadecanoate 300 mu L, saponifying for 15min at 75 ℃ in water bath, and adding 2mL of 14% BF after the reaction ₃ -CH ₃ OH, water at 75 DEG CMethyl esterification reaction is carried out for 15min under the bath condition, saturated NaCl solution and normal hexane are added after cooling to room temperature, the mixture is mixed and vibrated, and then the mixture is centrifuged (4000 rpm,8 min), and the supernatant is taken into a brown sample bottle for gas chromatography analysis.

DHA was measured by GC-7900 gas chromatograph, capillary column HP-88 (100 mm. Times.250 μm. Times.0.20 μm). Selecting high-purity nitrogen as carrier gas, wherein the detector is an FID detector, the temperature is 300 ℃, the initial temperature of a column box is 100 ℃, then the temperature is increased to 200 ℃ at 40 ℃/min, then the temperature is increased to 280 ℃ at 8 ℃/min, and the temperature is kept for 5min; the sample loading was 1. Mu.L. And calculating the DHA content by adopting an internal standard curve method. The optimal addition concentration of DA-6 was determined at the highest DHA content.

The results are shown in FIG. 1: when DA-6 was not added (i.e., the addition concentration of DA-6 was 0 mg/L), the DHA content and yield were 38.83% and 3.66g/L, respectively, and when the addition concentration of DA-6 was 0.05mg/L, the DHA content and yield reached 49.48% and 4.54g/L, respectively, which were increased by 0.27-fold and 0.24-fold, respectively, compared to the blank control without DA-6. When the added concentration of DA-6 was increased to 0.50mg/L, there was no significant difference in DHA content and yield. DA-6 concentration exceeds 0.50mg/L, and DHA content and yield are remarkably reduced when the concentration is increased to 1.00 mg/L. Therefore, 0.05mg/L DA-6 is selected to cooperate with 2,4-D induction to improve DHA yield and output of the schizochytrium limacinum.

Example 2

Seed culture solutions were inoculated into the fermentation media of Schizochytrium limacinum added with 0,0.25,0.50,1.00,1.50,2.00mg/L2,4-D at an inoculation ratio of 5%, and cultured in a shaker at 28℃and 165rpm for 96 hours. And (3) centrifugally separating the collected algae liquid, and freeze-drying the obtained algae mud to obtain the schizochytrium limacinum algae powder.

0.02g of algae powder is added with 1mL of 0.5mol/L KOH-CH ₃ OH,1mg/mL of an internal standard methyl nonadecanoate 300 mu L, saponifying for 15min at 75 ℃ in water bath, and adding 2mL of 14% BF after the reaction ₃ -CH ₃ OH at 75 DEG CMethyl esterification reaction is carried out for 15min under the water bath condition, saturated NaCl solution and normal hexane are added after cooling to room temperature, the mixture is mixed and vibrated, and then the mixture is centrifuged (4000 rpm,8 min), and the supernatant is taken into a brown sample bottle for being used for gas chromatography analysis.

DHA was measured by GC-7900 gas chromatograph, capillary column HP-88 (100 mm. Times.250 μm. Times.0.20 μm). Selecting high-purity nitrogen as carrier gas, wherein the detector is an FID detector, the temperature is 300 ℃, the initial temperature of a column box is 100 ℃, then the temperature is increased to 200 ℃ at 40 ℃/min, then the temperature is increased to 280 ℃ at 8 ℃/min, and the temperature is kept for 5min; the sample loading was 1. Mu.L. And calculating the DHA content by adopting an internal standard curve method.

The results are shown in FIG. 2: when the 2,4-D adding concentration is 0-0.5 mg/L, the DHA content in the schizochytrium limacinum is obviously increased from 38.83% to 45.00%, the DHA yield is obviously increased from 3.66g/L to 4.08g/L by 0.16 times, the DHA yield is improved by 0.11 times, and when the 2,4-D content exceeds 0.50mg/L, although the DHA content is increased, the DHA content is not obviously changed, and the yield is obviously reduced. Therefore, the 2,4-D in the concentration range of 0,0.25,0.50,1.00,1.50 and 2.00mg/L is determined to be used for cooperatively inducing the schizochytrium limacinum to improve the DHA content and the DHA yield in combination with 0.05 mg/LDA-6.

Example 3

Seed culture solution was inoculated into the fermentation medium of Schizochytrium limacinum added with 0.05mg/L DA-6 and 0,0.25,0.50,1.00,1.50,2.00mg/L2,4-D at an inoculation ratio of 5%, and cultured in a shaker at a temperature of 28℃and a rotation speed of 165rpm for 96 hours. Collecting algae liquid, centrifuging to obtain algae mud, and freeze drying to obtain schizochytrium limacinum powder.

0.02g of algae powder is added with 1mL of 0.5mol/L KOH-CH ₃ OH,1mg/mL of an internal standard methyl nonadecanoate 300 mu L, saponifying for 15min at 75 ℃ in water bath, and adding 2mL of 14% BF after the reaction ₃ -CH ₃ OH, performing methyl esterification reaction for 15min under the water bath condition of 75 ℃, cooling to room temperature, adding saturated NaCl solution and n-hexane, mixing, shaking and separatingHeart (4000 rpm,8 min), and the supernatant was taken into a brown sample bottle for gas chromatography.

DHA was measured by GC-7900 gas chromatograph, capillary column HP-88 (100 mm. Times.250 μm. Times.0.20 μm). Selecting high-purity nitrogen as carrier gas, wherein the detector is an FID detector, the temperature is 300 ℃, the initial temperature of a column box is 100 ℃, then the temperature is increased to 200 ℃ at 40 ℃/min, then the temperature is increased to 280 ℃ at 8 ℃/min, and the temperature is kept for 5min; the sample loading was 1. Mu.L. And calculating the DHA content by adopting an internal standard curve method. The optimal addition concentration of 2,4-D, which is used to induce a further increase in DHA content in conjunction with DA-6, is determined at the highest DHA content.

As can be seen from FIG. 3, when the addition amount of DA-6 was 0.05mg/L, the addition concentration of 2,4-D was increased from 0 to 0.5mg/L, the DHA content in the schizochytrium was significantly increased from 49.47% to 58.32%, by 0.17-fold, by 0.50-fold as compared with the DHA content (38.83%) in the schizochytrium when no DA-6 and 2,4-D were added, the DHA yield was significantly increased from 4.54g/L (only 0.05mg/L DA-6 was added) to 5.17g/L, by 0.14-fold, and by 0.42-fold as compared with the DHA yield (3.66 g/L) in the schizochytrium when no DA-6 and 2,4-D were added. When 2,4-D exceeds 0.50mg/L, the yield is significantly reduced although DHA content is not significantly changed. Therefore, 0.05mg/L DA-6 is selected to cooperate with 0.50 mg/L2,4-D to induce the schizochytrium limacinum to improve the DHA content and the yield.

As can be seen from FIG. 4, 0.05mg/L DA-6 and 0.5 mg/L2,4-D synergistically induced the accumulation of schizochytrium limacinum DHA, the DHA content and yield reaching 58.32% and 5.17g/L, respectively, being significantly higher than those of the experimental group with 0.05mg/L DA-6 or 0.5 mg/L2,4-D alone and the control group without plant growth regulator (49.48% and 4.54g/L,45.00% and 4.08g/L,38.82% and 3.66 g/L).

Claims

1. A method for synergistically inducing and improving DHA content and yield of schizochytrium limacinum by using 2 synthetic plant growth regulators, which is characterized by comprising the following steps:

(2) Fermenting and culturing schizochytrium in a fermentation medium, adding 0-1.0 mg/L DA-6, and determining the optimal adding concentration of the DA-6 by taking DHA content as an index;

2. The method for synergistically inducing increased DHA content and yield of schizochytrium limacinum with 2 synthetic plant growth regulators according to claim 1, wherein: the formula of the seed culture medium is 25.00-35.00 g/L glucose, 4.00-12.00 g/L yeast powder and 15.0-25.00 g/L seawater crystal; the formula of the fermentation medium is 50g/L to 100g/L of glucose, 5g/L to 30g/L of yeast powder, 0.1g/L to 1g/L of sodium chloride, 0.5g/L to 5g/L of potassium sulfate, 0.1g/L to 1g/L of monopotassium phosphate, 1g/L to 10g/L of crystalline magnesium sulfate and 0.01g/L to 1g/L of anhydrous calcium chloride.

3. The method for synergistically inducing and increasing the DHA content and yield of schizochytrium limacinum with 2 synthetic plant growth regulators according to claim 1, wherein the step (1) is specifically: the schizochytrium limacinum strain is coated on a flat plate for activation for 48-72 hours, and the obtained single colony algae strain is picked and transferred into a seed culture medium for culture for 24-48 hours until the logarithmic phase to obtain a seed culture solution.

4. The method for synergistically inducing increased DHA content and yield of schizochytrium limacinum with 2 synthetic plant growth regulators according to claim 1, wherein: the container for culture is a baffle triangular flask.

5. The method for synergistically inducing increased DHA content and yield of schizochytrium limacinum with 2 synthetic plant growth regulators according to claim 1, wherein: the inoculation proportion of the seed culture solution to the fermentation culture medium is 2% -10%.

6. The method for synergistically inducing increased DHA content and yield of schizochytrium limacinum with 2 synthetic plant growth regulators according to claim 1, wherein: the fermentation culture condition of the schizochytrium limacinum is that the temperature is 25-30 ℃ and the rotating speed is 150-200 rpm.

7. The method for synergistically inducing and increasing the DHA content and yield of schizochytrium limacinum with 2 synthetic plant growth regulators according to claim 1, wherein the step (2) is specifically: adding 0-1.0 mg/L DA-6 into a fermentation culture medium, culturing in a shaking table for 48-120 hours, collecting algae liquid, centrifugally separating to obtain schizochytrium limacinum, freeze-drying the algae mud, measuring the DHA content in the schizochytrium limacinum, and determining the optimal DA-6 adding concentration according to the highest DHA content.

8. The method for synergistically inducing and increasing the DHA content and yield of schizochytrium limacinum with 2 synthetic plant growth regulators according to claim 1, wherein the step (3) is specifically: adding the DA-6 with the optimal concentration determined in the step (2) into a fermentation medium, adding 2,4-D with the concentration range of 0-2.0 mg/L into the fermentation medium, culturing the fermentation medium for 48-120 hours in a shaking table, collecting algae liquid, centrifugally separating the algae liquid to obtain the schizochytrium limacinum, determining the DHA content in the schizochytrium limacinum after freeze drying the algae mud, and determining the optimal adding concentration of the 2,4-D with the highest DHA content.

9. A method for synergistically inducing increased amounts of schizochytrium limacinum DHA with 2 synthetic plant growth regulators according to claim 7 or 8, wherein: after saponification and methyl esterification of the schizochytrium limacinum powder, extracting DHA with saturated sodium chloride and normal hexane, determining the DHA content by gas chromatography, and calculating the DHA content by adopting an internal standard curve method.