Method for producing DHA through solid culture and liquid fermentation of Schizochytrium

• US 10,006,067 B2
• Filed: 12/30/2013
• Issued: 06/26/2018
• Est. Priority Date: 12/31/2012
• Status: Active Grant

• Current Assignee: Inner Mongolia Kingdomway Pharmaceutical Limited, Xiamen Kingdomway Group Company
• Original Assignee: Inner Mongolia Kingdomway Pharmaceutical Limited, Xiamen Kingdomway Group Company
• Inventors: Chen, Jinqing, Chen, Fangfang, Chen, Junhuang, Lin, Chao, Wu, Meiqiong
• Primary Examiner(s): Hobbs, Lisa J
• Application Number:
  US14/654,897
• Publication Number:
  US 20150361461A1
• Time in Patent Office: 1,639 Days
• CPC Class Codes: C12N 1/125 Unicellular algae isolates
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12R 2001/89 Algae ; Processes using algae

Abstract

The invention relates to a method for fermentation production of DHA, in particular a method for producing DHA through solid culture and liquid fermentation of Schizochytrium. The method comprises steps of activation of a strain, preparation of a suspension of the strain, preparation of a primary seed, preparation of a secondary solid seed, enlarging cultivation of the secondary solid seed in a fermentor, collection of cells after fermentation, and extraction of DHA oil. With the fermentation method, the seed preparation method is improved, the seed enlarging technology is simplified, the period of seed culture and fermentation is shortened, and therefore the investment is reduced and the cost is saved; in addition, the solid culture medium is of eutrophy and rich in a plurality of growth factors, and is more beneficial to the growth of the seed; cells are strong in viability and good in synchronism in solid culture medium.

Claims

1. A method for producing DHA through solid culture and liquid fermentation of Schizochytrium, which comprises the following steps:

(1) activating a strain, wherein a Schizochytrium strain is inoculated into a culture medium, cultured and activated to prepare a suspension of the strain;

(2) preparing a primary seed, wherein the suspension of the strain obtained in step (1) is inoculated into a solid culture medium and cultured to obtain a primary solid seed;

or wherein the suspension of the strain obtained in step (1) is inoculated into a liquid seed culture medium and cultured to obtain a primary liquid seed;

(3) preparing a secondary solid seed, wherein the primary solid seed is prepared as a solution of the primary solid seed, which is inoculated into a solid culture medium and cultured;

or wherein the primary liquid seed solution is inoculated into a solid culture medium and cultured;

whereby a secondary solid seed is obtained;

(4) enlarging culture of the secondary solid seed in a fermentor, wherein the secondary solid seed is prepared as a solution of the secondary solid seed, which is inoculated into a liquid culture medium in a fermentor for enlarging culture; and

(5) collecting cells after fermentation and extraction of DHA;

wherein the solid culture medium in step (2) or step (3) comprises a solid component and a liquid component of nutrient solution,

wherein the solid component is cereal with an amount of 250-400 g/L, wherein the cereal is selected from the group consisting of rice, wheat and corn, wherein the rice is selected from the group consisting of rice, millet and unpolished rice, and wherein the wheat is selected from the group consisting of barley, wheat and oat;

wherein the liquid component of nutrient solution comprises component 1 and component 2, wherein the component 1 comprises 2-5 g/L of glucose, 0.2-0.6 g/L of magnesium sulfate heptahydrate, 1.0-5.0 g/L of dipotassium hydrogen phosphate, 0.1-0.8 g/L of sea salt, 0.2-0.6 g/L of glycine, 1.5-1.8 g/L of threonine, 2-3.5 g/L of methionine, 3-6 g/L of malic acid, water is added to a metered volume, and the pH value is adjusted to 6-7; and

wherein the component 2 comprises 10-15 mg/L of La³⁺, 1-4 mg/L of Ce³⁺, 2-6 mg/L of Sm³⁺, 8-12 mg/L of Nd³⁺, 0.6-1.2 mg/L of Mn²⁺, 0.05-0.1 mg/L of Co²⁺, 0.2-0.6 mg/L of biotin, 0.1-0.3 mg/L of cerulenin, 2-5 mg/L of gibberellin GA, 0.5-1.0 mg/L of VB₁₂, 0.01-0.05 mg/L of folic acid and the balance of water;

wherein the preparation method of the solid culture medium comprises;

mixing uniformly the solid component and component 1 of the liquid component of nutrient solution, steaming or boiling the cereal till no white cores exist at the centers of the cereal and the water content is 50%-65%, whereby a raw material of the solid culture medium is obtained;

mixing uniformly the raw material of the solid culture medium and component 2 of the liquid component of nutrient solution, and loading the resulting mixture with a thickness of 0.2-1.0 cm into a solid fermentation bottle, and sterilizing, whereby the solid culture medium is obtained.

2. The method of claim 1, wherein the Schizochytrium is Schizochytrium sp. selected from the group consisting of Schizochytrium sp. KDW-12, Schizochytrium sp. S31, Schizochytrium aggregatum and Schizochytrium sp. S8.

3. The method of claim 1, wherein step (1) is characterized by one or more of the following items:

a. the culture temperature is 25-28°

C., and the culture is performed till the cell concentration and cell morphology are good; and

b. the cell concentration of the suspension of the strain is 1.0-2.5×

10⁶/mL.

4. The method of claim 1, wherein step (2) is characterized by one or more of the following items:

a. the inoculation amount is 0.5-2%;

b. the culture temperature is 25-28°

C., and the culture is performed till the cell concentration and cell morphology are good; and

c. the liquid culture is subject to shaking at a speed of 180-220 rpm.

5. The method of claim 1, wherein step (3) is characterized by one or more of the following items:

a. the cell concentration of the solution of the primary solid seed is 1.0-2.5×

10⁶/mL;

b. the solution of the primary solid seed is inoculated into the solid culture medium in an inoculation amount of 10%-20% in volume/weight fraction;

c. the primary liquid seed is inoculated into the solid material culture medium in an inoculation amount of 8%-15% in volume/weight fraction; and

d. the culture temperature is 25-28°

C., and the culture is performed till the cell concentration and cell morphology are good.

6. The method of claim 1, wherein step (4) is characterized by one or more of the following items:

a. the solution of the secondary solid seed is inoculated into the fermentor for enlarging culture in an inoculation amount of 10%-20% in volume fraction;

b. at the first 48 h of the fermentation, the temperature is controlled at 25-28°

C. and the dissolved oxygen is kept at 30%-50% by regulating the shaking speed;

c. the temperature is controlled at 18-22°

C. from the 48th hour to the end of the fermentation;

d. the fermentation is terminated when the cell begin to autolyze, the dry cell weight and the yield of DHA are not increased, increased slowly or in a descending trend;

e. the pH of fermentation broth is controlled at 6.0-6.5 with ammonia during the fermentation; and

f. the concentration of glucose is controlled at 4-8 g/L via automatic feeding of glucose solution during the fermentation.

7. The method of claim 1, wherein the culture medium is a slant culture medium in step (1) containing 20-30 g/L of glucose, 10-15 g/L of peptone, 3-5 g/L of yeast powder, 15-20 g/L of sea crystal and 15-25 g/L of agar powder, with a pH value of 6.0-7.0.

8. The method of claim 1, wherein the liquid seed culture medium in step (2) contains 20-25 g/L of glucose, 20-25 g/L of sodium chloride, 0.2-0.5 g/L of potassium dihydrogen phosphate, 3-5 g/L of yeast powder, 8-12 g/L of magnesium sulfate heptahydrate, 0.1-0.8 g/L of sea salt and 0.2-0.6 g/L of glutamic acid, with a pH value of 6.0-7.0.

9. The method of claim 1, wherein the liquid culture medium in the fermentor in step (4) contains 30-60 g/L of glucose, 15-20 g/L of NaCl, 4-8 g/L of MgSO₄.7H₂O, 3-8 g/L of yeast powder, 0.5-1.0 g/L of KH₂PO₄, 0.2-0.5 g/L of (NH₄)₂SO₄, 0.8-1.2 g/L of NaHCO₃, 1.0-2.0 μ

g/L of CoCl₂, 0.5-1.0 μ

g/L of MnSO₄, 0.05-0.1 mg/L of VB₁ and 0.001-0.01 mg/L of VB₁₂, with an initial pH value of 6.0-7.0.