CN108424938A - A kind of fermentation medium of thraustochytriale high yield aliphatic acid and its application - Google Patents

Abstract

The invention discloses a kind of fermentation medium of thraustochytriale high yield aliphatic acid and its application, a kind of fermentation medium of thraustochytriale high yield aliphatic acid is made of following methods：Weigh 45 65g of glycerine, 1 2g of peptone, 0.5 1.5g of yeast extract, 0.2 0.3g of potassium dihydrogen phosphate, 25 40g of artificial sea salt in proportion, add ultra-pure water to 1000ml, stir evenly, 115 DEG C sterilize 21 minutes, be cooled to room temperature to get.(1) present invention utilizes glycerine, saves production cost.(2) using the culture medium of the present invention compared with former culture medium, the yield conspicuousness of total fatty acids, saturated fatty acid and DHA improves, and improves raw material utilization efficiency.(3) fermentation condition of fermentation medium of the invention is simple, and easy to operate, production cost is low, is easy to large scale fermentation production, energy saving.

Description

A kind of fermentation medium of Thraustochytrium high-yield fatty acid and its application

technical field

The invention relates to the field of microorganisms, in particular to a thraustochytrium high-yield fatty acid fermentation medium and application thereof.

Background technique

Thraustochytrids are heterotrophic fungi-like marine protists that are widely distributed. Because the cells of Thraustochytrium can accumulate a large amount of fatty acids, which can reach more than 50% of the biomass content, including saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids. Among them, saturated fatty acids and monounsaturated fatty acids are the main raw materials for biodiesel synthesis. As the main component of cell membrane phospholipids, polyunsaturated fatty acids are an indispensable nutrient for human health, especially DHA (docosahexenoic acid). DHA can assist the normal development of brain cells and retina, while DHA also has the effect of preventing cardiovascular disease, hyperlipidemia and anti-inflammation.

In nature, DHA is mainly distributed in marine microorganisms, especially marine organisms living in deep sea and cold regions. At present, the commercial source of DHA is mainly marine fish oil. Sardines, fin fish, etc. contain high fat tissue, and are used to produce fish oil in many countries. The content of DHA in fish oil can reach 20-30%. The price of commercially available food-grade high-concentration DHA (27%-30%) is 730,000-1.1 million yuan/ton. In 2010, the total consumption of DHA in China reached 2,260 tons. China's DHA industry has a very broad space for development. However, the production of DHA from fish oil has many disadvantages, such as fishy smell, complex composition and potential heavy metal pollution. Using microorganisms as an alternative source to produce DHA through microbial fermentation can effectively solve the production problem of fish oil DHA. In the cells of some Thraustochytrium strains, the DHA content can reach more than 25%. Through large-scale fermentation and cultivation of Thraustochytrium to obtain DHA active products, it has many advantages such as less pollution and low cost. Therefore, Thraustochytrium is an effective source of DHA for industrial production and application, and its application prospect has received extensive attention in the world.

Thraustochytrium is a heterotrophic microorganism. The composition of the medium has a great influence on its growth and oil accumulation. Thraustochytrium can utilize various carbon sources such as glucose, fructose, sucrose and mannose. However, the utilization efficiency of different carbon sources varies greatly, among which glucose is the carbon source that most microorganisms can utilize. However, currently, thraustochytrium is fermented and cultured on medium containing glucose, and the yield of fatty acid is not ideal.

Contents of the invention

The purpose of the invention is to overcome the deficiencies of the prior art, and provide a low-cost, high-fatty-acid-yielding fermentation medium of Thraustochytrium that can increase fatty acid production.

The second object of the present invention is to provide an application of a thraustochytrium high-yielding fatty acid fermentation medium.

Technical scheme of the present invention is summarized as follows:

A thraustochytrium high-yield fatty acid fermentation medium, prepared by the following method: weighing 45-65g of glycerin, 1-2g of peptone, 0.5-1.5g of yeast extract, and 0.2-0.3g of potassium dihydrogen phosphate 1. Add 25-40g of artificial sea salt, add ultrapure water to 1000ml, stir evenly, sterilize at 115°C for 21 minutes, cool to room temperature, and get ready.

Application of the above-mentioned fermentation medium for thraustochytrium high-yield fatty acid fermentation culture thraustochytrium high-yield fatty acid.

Advantages of the present invention:

(1) The present invention utilizes glycerin to save production cost.

(2) Compared with the original culture medium (universal culture medium, containing glucose) using the culture medium of the present invention, the output of total fatty acid, saturated fatty acid and DHA is significantly improved, and the raw material utilization efficiency is improved.

(3) The fermentation medium of the present invention has simple fermentation conditions, convenient operation, low production cost, easy large-scale fermentation production, and energy saving.

Description of drawings

Fig. 1 is the total fatty acid of Thraustochytrium sp.PKU#Mn4 (CGMCC 8091) using universal culture medium (M4) and the fermentation medium (abbreviation: MZ) of Thraustochytrium high-yield fatty acid prepared in Example 1 of the present invention , saturated fatty acid and DHA production.

Figure 2 shows the biomass, total fatty acid, saturated fatty acid and DHA production of Thraustochytriidae sp.PKU#Mn16 (JX847368.1CGMCC 8095) using a universal medium (M4) and a high-yield fatty acid fermentation medium (MZ).

Detailed ways

The present invention will be further described below by specific examples.

Thraustochytrium sp.PKU#Mn4 (Latin: Schizochytrium sp.PKU#Mn4, date of deposit: April 9, 2014, depository unit: General Microorganism Center (CGMCC), China Committee for Microbial Culture Collection, number: CGMCC 8091). The address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, 100101.

Thraustochytriidae sp.PKU#Mn16 (Latin: Thraustochytriidaesp.PKU#Mn16, date of deposit: April 9, 2014, depository unit: General Microorganism Center (CGMCC), China Committee for Microbial Culture Collection, number: CGMCC 8095). The address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, 100101.

The artificial sea salt used in the present invention is the clown salt produced by French Red Cross Company. But it does not limit the present invention in any way.

Example 1

A thraustochytrium high-yield fatty acid fermentation medium (abbreviated as MZ) is made by the following method: weigh 60g of glycerol, 1.5g of peptone, 1g of yeast extract, 0.25g of potassium dihydrogen phosphate, and 33g of artificial sea salt. Add ultrapure water to 1000ml, stir evenly, sterilize at 115°C for 21 minutes, cool to room temperature, and obtain.

Example 2

A thraustochytrium high-yield fatty acid fermentation medium is prepared by the following method: Weigh 45g of glycerin, 2g of peptone, 0.5g of yeast extract, 0.3g of potassium dihydrogen phosphate, and 25g of sea salt, and add ultrapure water to 1000ml, stir well, sterilize at 115°C for 21 minutes, cool to room temperature, ready to serve.

Example 3

A thraustochytrium high-yield fatty acid fermentation medium is made by the following method: Weigh 65g of glycerin, 1g of peptone, 1.5g of yeast extract, 0.2g of potassium dihydrogen phosphate, and 40g of sea salt, add ultrapure water to 1000ml, stir well, sterilize at 115°C for 21 minutes, cool to room temperature, ready to serve.

Example 4

Thraustochytrium universal medium (referred to as M4) formula is: glucose 20g/l, yeast extract 1g/l, peptone 1.5g/l, potassium dihydrogen phosphate 0.25g/l, sea salt 33g/l.

Example 5

Thraustochytrium high-yield fatty acid fermentation medium fermentation culture application of Thraustochytrium high-yield fatty acid comprises the following steps:

1. Activation of the strain: Pick a single colony of Thraustochytrium from the solid medium and inoculate it on a new solid medium, draw a line in three zones, and culture it in an incubator at 28°C for 48-72 hours to obtain an activated strain;

2. Preparation of Thraustochytrium seed liquid: pick a ring of activated Thraustochytrium and inoculate it in M4 medium, place it on a shaker at 150rpm and incubate at 28°C for 24 hours to prepare Thraustochytrium seed fermentation liquid;

3. Inoculation and fermentation culture: inoculate the Thraustochytrium seed liquid in MZ according to the inoculum size of 10%, and cultivate it on a 150rpm shaking table for 4 days at a culture temperature of 28° C. to obtain the Thraustochytrium fermented liquid.

4. Determination of biomass: Take 15ml of thraustochytrium fermented liquid in a 50ml centrifuge tube with constant weight, centrifuge at 10000rpm for 10 minutes, discard the supernatant, and wash the lower layer of bacteria twice with 15ml of sterilized ultrapure water respectively. Thraustochytrium thallus cells are obtained, and the thallus cells are freeze-dried in a freeze dryer for 48 hours, weighed, and the biomass of the Thraustochytrium in the fermentation liquid can be obtained.

5. Determination of fatty acid content: Add 2ml of 4% methanolic sulfuric acid solution to the freeze-dried cells, add nonadecanoic acid internal standard solution, vortex and oscillate for 20 seconds to mix well, bathe in 80°C water bath for 1 hour, take it out and let it cool to room temperature, Add 1ml of ultrapure water and 1ml of n-hexane respectively, vortex for 20 seconds to mix, centrifuge at 4000rpm for 2 minutes, take the upper layer of n-hexane and pass it through a 0.45μm nylon membrane to measure the fatty acid content by gas phase phase, and compare it with the fatty acid mixed standard to determine the fatty acid composition .

Example 6

Thraustochytrium Schizochytrium sp.PKU#Mn4 (CGMCC 8091) was fermented on M4 medium and MZ medium (prepared in Example 1). After analyzing the sample, it can be seen that the M4 medium was used for fermentation and cultivation, and after 96 hours, its total fatty acid output was: 0.82g/l, the saturated fatty acid output was: 0.39g/l, and the DHA output was: 0.36g/l; After 96 hours of fermentation, the total fatty acid output was 1.50 g/l, the saturated fatty acid output was 0.63 g/l, and the DHA output was 0.74 g/l. The total fatty acid production significantly increased by 82.93% (P=0.004), the saturated fatty acid production significantly increased by 61.54% (P=0.019), and the DHA production significantly increased by 105.56% (P=0.001). (see picture 1)

Example 7

Thraustochytriidae sp.PKU#Mn16 (CGMCC 8095) was fermented on M4 medium and MZ medium (prepared in Example 2). After analyzing the samples, it can be seen that the M4 medium was used for fermentation and cultivation, and after 96 hours, the total fatty acid output was 0.74g/l, the saturated fatty acid output was 0.41g/l, and the DHA output was 0.27g/l; After 96 hours of fermentation, the total fatty acid output was 2.05g/l, the saturated fatty acid output was 0.85g/l, and the DHA output was 1.01g/l. The total fatty acid production significantly increased by 177.03% (P=0.005), the saturated fatty acid production significantly increased by 107.32% (P=0.008), and the DHA production significantly increased by 274.07% (P=0.003). (See Figure 2)

The MZ medium prepared in Example 3 was used to replace the MZ medium prepared in Example 2 of the present embodiment. Others were the same as in this embodiment. The MZ medium was fermented and cultivated. After 96 hours, its total fatty acid production was: 2.05g/l, Saturated fatty acid yield: 0.85 g/l, DHA yield: 1.01 g/l.

Claims (2)

1. A fermentation medium of Thraustochytrium high-yield fatty acid is characterized in that it is made by the following method: take by weighing glycerol 45-65g, peptone 1-2g, yeast extract 0.5-1.5g, dihydrogen phosphate Potassium 0.2-0.3g, artificial sea salt 25-40g, add ultrapure water to 1000ml, stir evenly, sterilize at 115°C for 21 minutes, cool to room temperature, and obtain. 2. The application of a kind of Thraustochytrium high-yield fatty acid fermentation medium fermentation culture Thraustochytrium high-yield fatty acid of claim 1.