阅读说明：本技术 一种高产脂肪酸的制备方法及破囊壶菌培养基 (Preparation method of high-yield fatty acid and thraustochytrid culture medium ) 是由 汪光义 温帅 叶会科 谢云轩 何耀东 于 2019-11-28 设计创作，主要内容包括：本发明公开一种高产脂肪酸的破囊壶菌培养基,成分及比例如下所示：Glycerol 35-65g/l,Peptone 2-8g/l,磷酸二氢钾0.2-1g/l,海盐25-35g/l。还公开高产脂肪酸的制备方法,主要步骤包括：液体培养基MPqd的制备；破囊壶菌液体培养基MP的制备；破囊壶菌固体培养基MP的制备；活化；破囊壶菌种子液的制备；破囊壶菌发酵培养基的制备；最后破囊壶菌生物量的测定和脂肪酸的测定。本发明培养基配方简单,能源利用高,绿色无污染,降低成本,提高脂肪酸的产量,利于工业化生产。(The invention discloses a thraustochytrid culture medium for high yield of fatty acid, which comprises the following components in percentage by weight: 35-65g/l of glycol, 2-8g/l of Peptone, 0.2-1g/l of monopotassium phosphate and 25-35g/l of sea salt. Also discloses a preparation method of the high-yield fatty acid, which mainly comprises the following steps: preparing a liquid culture medium MPqd; preparing a thraustochytrid liquid culture medium MP; preparing a thraustochytrid solid culture medium MP; activating; preparing a thraustochytrid seed solution; preparing a thraustochytrid fermentation culture medium; and finally, measuring the biomass of the thraustochytrid and measuring the fatty acid. The culture medium has the advantages of simple formula, high energy utilization, greenness, no pollution, cost reduction, fatty acid yield improvement and contribution to industrial production.)

1. the culture medium for the thraustochytrid bacteria with high fatty acid yield is characterized by comprising the following components in percentage by weight: glycerol35-65g/l, Peptone 2-8g/l, potassium dihydrogen phosphate 0.2-1g/l, sea salt 25-35 g/l.

2. A method for preparing high-yield fatty acid by using the culture medium of claim 1, which is characterized by comprising the following main steps:

1) preparing a thraustochytrid high-yield fatty acid liquid culture medium MPqd:

adding ultrapure water 1L under natural pH condition, performing ultrasonic treatment for 5-10min, stirring with glass rod, packaging in 100ml sterile conical flask, adding liquid culture medium 50ml per flask, sterilizing in high pressure steam sterilization pot at 115 deg.C for 21 min, and cooling to obtain required culture solution;

2) preparing a thraustochytrid liquid culture medium MP;

3) preparing a thraustochytrid solid culture medium MP;

4) activation of thraustochytrids:

under the aseptic condition, selecting a proper amount of single colony of the thraustochytrid with an inoculating loop into an MP solid culture medium, then scribing lines in three areas in the culture medium, and culturing for 1-3 days in an incubator at the temperature of 26 +/-2 ℃;

5) preparing a thraustochytrid seed solution:

taking out the purified solid culture medium of the thraustochytrid from the incubator, picking a proper amount of single colony of the thraustochytrid in the prepared MP liquid culture medium by using an inoculating loop in a sterile operation platform, and culturing for 1-2 days at the temperature of 26 +/-2 ℃ and 180rpm in a shaking table to obtain the required seed liquid;

6) preparing a thraustochytrid fermentation medium:

sucking 2.5-5ml of the above-mentioned thraustochytrid seed liquor by using a liquor-transferring gun in an ultraclean working platform, placing the above-mentioned seed liquor into MPqd thraustochytrid liquid culture medium in the step 1), and culturing for 4 days in a shaking table at 180rpm and 26 +/-2 deg.C so as to obtain the required thraustochytrid fermentation liquor.

3. The method for preparing high fatty acid yield according to claim 2, wherein the preparation of the thraustochytrid liquid culture medium MP comprises the following steps: adding 1L of ultrapure water into 10-20g/L of Glucose, 1-2g/L of Yeast extract powder, 1.5-2.5g/L of Peptone, 0.1-0.3g/L of monopotassium phosphate and 25-35g/L of sea salt at natural pH, stirring uniformly, subpackaging in 100ml of sterile conical flasks, adding 50ml of liquid culture medium into each flask, sterilizing at 115 ℃ for 21 minutes, and cooling to obtain the required liquid culture medium.

4. The method for preparing high-yield fatty acid according to claim 2, wherein the preparation steps of the thraustochytrid solid culture medium MP are as follows: 10-20g/L of Glucose, 1-2g/L of Yeast extract powder, 1.5-2.5g/L of Peptone, 0.1-0.3g/L of monopotassium phosphate, 25-35g/L of sea salt, 15-20g of agar powder, natural pH, 1L of ultrapure water, performing ultrasonic treatment for 5min, stirring uniformly, subpackaging in 100ml of sterile conical flasks, adding 50ml of liquid culture medium in each flask, sterilizing at 115 ℃ for 21 min, pouring the sterilized culture medium into a sterile culture dish, and cooling to obtain the required solid culture medium.

5. The method for preparing fatty acids with high yield according to claim 2, wherein the step of measuring the biomass of thraustochytrid comprises: sucking 10-30ml of thraustochytrid bacteria liquid into a 50ml centrifuge tube, centrifuging at 4 deg.C and 8000rpm for 10-20min, pouring out the upper layer liquid, washing the lower layer bacteria with 10-30ml of sterile distilled water for 3 times, freeze-drying in a freeze dryer for 36h, and weighing to calculate biomass.

6. The method for preparing fatty acids with high yield according to claim 2, wherein the step of measuring the fatty acids of thraustochytrid comprises: taking freeze-dried thallus (ensuring no moisture), adding 2ml of 4% sulfuric acid methanol solution, adding 100 mu 1g/l nonadecanoic acid, shaking for 30s-60s, carrying out water bath at 80 ℃ in a water bath for 1h, cooling to room temperature, and adding 1ml of ddH into a centrifuge tube₂And O, 1ml of n-hexane, shaking for 1-2min to fully dissolve the n-hexane at 28 ℃, centrifuging at 6000rpm for 5-10min, layering the solution to obtain an upper layer liquid, namely the n-hexane solution containing fatty acid methyl ester, filtering the n-hexane solution containing the fatty acid methyl ester by using a 0.45-micrometer organic filter head, measuring the content of each fatty acid component in the filtrate by using an image chromatograph, and calculating the content of each fatty acid component according to the standard liquid amount.

Technical Field

The invention relates to the field of microbial fatty acid preparation, in particular to a preparation method of high-yield fatty acid and a thraustochytrid culture medium.

Background

Thraustochytrium is a unicellular heterotrophic protist in the ocean and was first discovered in 1936. It has wide distribution range and has been reported to be separated in temperate zone, tropical zone, subtropical zone and polar glacier.

At present, 11 genera mainly include Aurantiochytrium, Thraustochytrium, Botryochytrium, Parietichytrium, Aplanochytrium, Labyrinthuloides, Oblingichytrium, Sicyoiidochytrium, Japonochytrium, Schizochytrium and Ulkenia. Most thraustochytrids can live in rotten plant residues and higher plants, and a few can live in invertebrates such as abalones and clams.

Thraustochytrids are widely recognized because of the high fatty acid content in their cells. The fatty acids contained in the cells mainly comprise saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids. The content of fatty acids in the cells of the thraustochytrid can be between 30% and 50% of the dry weight and the cholesterol content is low. The fatty acid component mainly comprises omega-3 unsaturated fatty acid-DHA (Docosahexaenoic acid, C22:6) and saturated fatty acid-palmitic acid (C16:0), and the fatty acid component accounts for more than 80% of the total fatty acid and has low content of other fatty acid components.

DHA plays an irreplaceable role in the visual development, the brain development and the like of infants, and has obvious effects on preventing diseases such as hypertension, arteriosclerosis, arthritis, cancer and the like for adults. However, DHA is not produced directly in humans, and can only be obtained from the outside. The traditional DHA source is deep sea fish oil, however, due to the problems of fishy smell, potential pollution and the like, the DHA acquisition is limited, and the DHA production by using the thraustochytrid fungi can not only overcome the defects, but also is not limited by time and place, so that the DHA production by using the thraustochytrid fungi is more and more concerned by students.

Because fossil fuels are gradually exhausted on the earth at present and a great amount of pollution is generated in the process of utilizing the fossil fuels by human beings, the search of continuously replaceable green energy is an important way for realizing the sustainable development of economy and society and the ecological environment protection.

Compared with the traditional petrochemical petroleum, the biodiesel has the characteristics of safety, no pollution, full utilization of heat energy and the like. Saturated fatty acid (palmitic acid) and some monounsaturated fatty acid in the thraustochytrid are important raw materials for synthesizing green energy-biodiesel.

With the current market for producing various fatty acids by using microorganisms such as thraustochytrid and the like by human beings opened by people, obtaining high-yield fatty acids by optimizing a culture medium and searching for proper culture conditions is one of important means for making full use of thraustochytrid to contribute to the development and progress of the human society.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a preparation method of high-yield fatty acid and a thraustochytrid culture medium.

The first purpose of the invention is to provide a high fatty acid yield thraustochytrid culture medium, which comprises the following components in percentage by weight: 35-65g/l of glycol, 2-8g/l of Peptone, 0.2-1g/l of monopotassium phosphate and 25-35g/l of sea salt.

The second purpose provided by the invention is to provide a preparation method of high-yield fatty acid, which adopts the culture medium: the culture medium of the thraustochytrid comprises the following components in percentage by weight: 35-65g/l of glycol, 2-8g/l of Peptone, 0.2-1g/l of monopotassium phosphate, 25-35g/l of sea salt,

the method mainly comprises the following steps:

1) the preparation method of the thraustochytrid high-yield fatty acid liquid culture medium MPqd comprises adding ultrapure water 1L under natural pH condition, performing ultrasonic treatment for 5-10min, stirring with glass rod, packaging in 100ml sterile conical flask, adding liquid culture medium 50ml per flask, sterilizing in a high pressure steam sterilization pot at 115 deg.C for 21 min, and cooling to obtain the desired culture solution.

2) Preparation of commonly used liquid medium MP for Thraustochytrium: 10-20g/L of Glucose, 1-2g/L of Yeast extractpowder, 1.5-2.5g/L of Peptone, 0.1-0.3g/L of monopotassium phosphate and 25-35g/L of sea salt are added with ultrapure water for 1L, subjected to ultrasonic treatment for 5min, stirred uniformly and then subpackaged in 100ml sterile conical flasks, 50ml of liquid culture medium is added into each flask, the mixture is sterilized at 115 ℃ for 21 min, and the required liquid culture medium is obtained after cooling.

3) Preparing a thraustochytrid solid culture medium MP: 10-20g/L of Glucose, 1-2g/L of Yeast extractpowder, 1.5-2.5g/L of Peptone, 0.1-0.3g/L of monopotassium phosphate, 25-35g/L of sea salt, 15-20g of agar powder, natural pH, 1L of ultrapure water, ultrasonic treatment for 5min, uniformly stirring, subpackaging in 100ml of sterile conical flasks, adding 50ml of liquid culture medium in each flask, sterilizing at 115 ℃ for 21 min, pouring the sterilized culture medium into a sterile culture dish, and cooling to obtain the required solid culture medium.

4) Activation of thraustochytrids: under the aseptic condition, an appropriate amount of single colonies of the thraustochytrid are picked by an inoculating loop and put into an MP solid culture medium, then three zones in the culture medium are streaked, and the single colonies are cultured in an incubator for 1 to 3 days at the temperature of 26 +/-2 ℃.

5) Preparing a thraustochytrid seed solution: taking out the purified solid culture medium of the thraustochytrid from the incubator, picking a proper amount of single colony of the thraustochytrid in the prepared MW liquid culture medium by using an inoculating loop in a sterile operation platform, and culturing for 1-2 days at the temperature of 26 +/-2 ℃ and 180rpm in a shaking table to obtain the required seed liquid.

6) Preparing a thraustochytrid fermentation medium: absorbing 2.5-5ml MVqd thraustochytrid liquid culture medium in the above-mentioned thraustochytrid seed liquor (1) by using liquid-transferring gun in ultraclean bench, and culturing for 4 days at 26 + -2 deg.C and 180rpm in shaking table to obtain the required thraustochytrid fermentation liquor.

7) Determination of Thraustochytrium Biomass

Sucking 10-30ml of thraustochytrid bacteria liquid into a 50ml centrifuge tube, centrifuging at 4 deg.C and 8000rpm for 10-20min, pouring out the upper layer liquid, washing the lower layer bacteria with 10-30ml of sterile distilled water for 3 times, freeze-drying in a freeze dryer for 36h, and weighing to calculate biomass.

8) Determination of Thraustochytrium fatty acids

The lyophilized cells were collected (to ensure moisture free), and 2ml of 4% methanol sulfate solution was added to the cells, followed by 100. mu.1 g/l of the solutionShaking nonadecanoic acid for 30s-60s, placing in water bath at 80 deg.C for 1h, cooling to room temperature, adding 1ml ddH into centrifuge tube₂And O, 1ml of n-hexane, shaking for 1-2min to fully dissolve the n-hexane at 28 ℃, centrifuging at 6000rpm for 5-10min, layering the solution to obtain an upper layer liquid, namely the n-hexane solution containing fatty acid methyl ester, filtering the n-hexane solution containing the fatty acid methyl ester by using a 0.45-micrometer organic filter head, measuring the content of each fatty acid component in the filtrate by using an image chromatograph, and calculating the content of each fatty acid component according to the standard liquid amount.

The invention has the beneficial effects that: the culture medium has simple formula, high energy utilization, no pollution, reduced cost, increased fatty acid yield, and suitability for industrial production.

1. The traditional solid glucose is changed into liquid glycerol, so that the utilization rate of raw materials is improved, the cost is saved, and the yield of fatty acid is increased.

2. The nitrogen source is replaced by a single nitrogen source, namely peptone, so that the formula of the culture medium is simplified, the time is saved, the cost is reduced, and the improvement of the energy utilization rate is facilitated.

Drawings

FIG. 1 shows the total fatty acid, palmitic acid and DHA content of Thraustochytrium Schizochytrium PKU # Mn4(CGMCC 80912014.04.09) in a conventional liquid Medium (MP) and a high fatty acid production liquid medium (MPqd).

Fig. 2 shows the total fatty acid, palmitic acid, DHA content of thraustochytriales thraustochytriaceae pku # Mn16(CGMCC 80952014.04.09) using the universal Medium (MP) and the high fatty acid liquid production medium (MPqd).

Detailed Description

The invention is further illustrated by the following specific examples and the accompanying drawings. The examples are given for the purpose of better understanding of the present invention by researchers in the relevant fields and are not intended to limit the present invention in any way.

The invention provides a liquid culture medium of a thraustochytrid with high fatty acid yield, which comprises the following components in percentage by weight: 35-65g/l of glycol, 2-8g/l of Peptone, 0.2-1g/l of monopotassium phosphate and 25-35g/l of sea salt.

A culture method adopting the culture medium comprises the following specific steps:

1) the preparation method of the thraustochytrid high-yield fatty acid liquid culture medium MPqd comprises the steps of weighing 35-65g/L of Glycerol, 2-8g/L of Peptone, 0.2-1g/L of monopotassium phosphate and 25-35g/L of sea salt, adding 1L of ultrapure water under the condition of natural pH, carrying out ultrasonic treatment for 5-10min, uniformly stirring by using a glass rod, subpackaging in 100ml of sterile conical bottles, adding 50ml of liquid culture medium into each bottle, sterilizing at 115 ℃ in a high-pressure steam sterilization pot for 21 min, and cooling to obtain the required culture solution.

2) Preparation of commonly used liquid medium MP for Thraustochytrium: 10-20g/L of Glucose, 1-2g/L of Yeast extractpowder, 1.5-2.5g/L of Peptone, 0.1-0.3g/L of monopotassium phosphate and 25-35g/L of sea salt are added with ultrapure water for 1L, subjected to ultrasonic treatment for 5min, stirred uniformly and then subpackaged in 100ml sterile conical flasks, 50ml of liquid culture medium is added into each flask, the mixture is sterilized at 115 ℃ for 21 min, and the required liquid culture medium is obtained after cooling.

3) Preparing a thraustochytrid solid culture medium MP: 10-20g/L of Glucose, 1-2g/L of Yeast extractpowder, 1.5-2.5g/L of Peptone, 0.1-0.3g/L of monopotassium phosphate, 25-35g/L of sea salt, 15-20g of agar powder, natural pH, 1L of ultrapure water, ultrasonic treatment for 5min, uniformly stirring, subpackaging in 100ml of sterile conical flasks, adding 50ml of liquid culture medium in each flask, sterilizing at 115 ℃ for 21 min, pouring the sterilized culture medium into a sterile culture dish, and cooling to obtain the required solid culture medium.

4) Activation of thraustochytrids: under the aseptic condition, an appropriate amount of single colonies of the thraustochytrid are picked by an inoculating loop and put into an MP solid culture medium, then three zones in the culture medium are streaked, and the single colonies are cultured in an incubator for 1 to 3 days at the temperature of 26 +/-2 ℃.

5) Preparing a thraustochytrid seed solution: taking out the purified solid culture medium of the thraustochytrid from the incubator, picking a proper amount of single colony of the thraustochytrid in the prepared MW liquid culture medium by using an inoculating loop in a sterile operation platform, and culturing for 1-2 days at the temperature of 26 +/-2 ℃ and 180rpm in a shaking table to obtain the required seed liquid.

6) Preparing a thraustochytrid fermentation medium: absorbing 2.5-5ml MVqd thraustochytrid liquid culture medium in the above-mentioned thraustochytrid seed liquor (1) by using liquid-transferring gun in ultraclean bench, and culturing for 4 days at 26 + -2 deg.C and 180rpm in shaking table to obtain the required thraustochytrid fermentation liquor.

7) Determination of Thraustochytrium Biomass

Sucking 10-30ml of thraustochytrid bacteria liquid into a 50ml centrifuge tube, centrifuging at 4 deg.C and 8000rpm for 10-20min, pouring out the upper layer liquid, washing the lower layer bacteria with 10-30ml of sterile distilled water for 3 times, freeze-drying in a freeze dryer for 36h, and weighing to calculate biomass.

8) Determination of Thraustochytrium fatty acids

Collecting lyophilized thallus (ensuring no water content), adding 2ml 4% sulphuric acid methanol solution, adding 100 μ l nonadecanoic acid (1g/l), shaking for 30s-60s, water bath at 80 deg.C for 1 hr, cooling to room temperature, adding 1ml ddH into centrifuge tube₂And O, 1ml of n-hexane is shaken for 1-2min to be fully dissolved at 28 ℃, and the n-hexane solution is centrifuged at 6000rpm for 5-10min, after the solution is layered, the upper layer liquid is the n-hexane solution containing fatty acid methyl ester, the n-hexane solution of the fatty acid methyl ester is filtered by an organic filter head with the diameter of 0.45 mu m, then the content of each fatty acid component in the filtrate is measured by an image chromatograph, and the content of total fatty acid, palmitic acid and DHA is calculated according to the standard liquid amount.