Schizochytrium sp and method for producing docosahexenoic acid grease

A technology of Schizochytrium and seeds, which is applied in the field of fermentation, can solve the problems of high BOD value of fermentation wastewater, increase investment in fixed assets, and high requirements for equipment materials, achieve short fermentation cycle, reduce the amount of seed preparation and its preparation time, and material less demanding effect

Inactive Publication Date: 2014-02-26
HUAZHONG UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Authorized notification number CN101519676B discloses a method for producing DHA oil by fermentation of Schizochytrium fungus. The DHA productivity is 4.86g / L.d, the DHA content is about 50%, and there is still room for further improvement in productivity; The method of cultivating Schizochytrium to produce DHA by fermentation, the DHA output is as high as 26-30g / L, and the productivity is about 6.0g / L.d, but because the fermentation medium components contain a large amount of yeast extract and peptone, the production cost is relatively high, and the whole fermentation The process needs to be maintained in a small pH range, and the pH must be strictly controlled by frequent feeding of acid and alkali substances, so there is a risk of bacterial contamination in the large-scale production process
In addition, the fermentation process requires the dissolved oxygen concentration to be controlled at 30-50%, and the energy consumption is relatively high;
[0007] Application publication number CN102864111A discloses a Schizochytrium strain producing docosahexaenoic acid. The DHA productivity of the strain is 7.63g / L.d, but the sea crystal requirement in its fermentation medium reaches 20g / L. Chloride ions in the fermenter have a strong corrosive effect on the fermenter equipment. Therefore, this method has high requirements on the material of the equipment in the later industrial application process, greatly increasing the investment in fixed assets, and in the fermentation process, the glucose concentration is always maintained at 15g / L, so that the BOD value of fermentation wastewater is high, and the treatment is difficult; the application publication number CN102899254A discloses a method for high-density fermentation of Schizochytrium to produce DHA. The productivity of this strain is as high as 7.2-8.5g / L. Sea crystal is added. Although this formula reduces the corrosion of chloride ions on fermentation equipment, the high concentration of Na 2 SO 4 The addition of (24g / L) makes the fermentation waste liquid contain a high concentration of salt, which is difficult to handle, and various heavy metals, such as manganese, cobalt, nickel, etc., are added to the artificial seawater, which seriously affects the product quality

Method used

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  • Schizochytrium sp and method for producing docosahexenoic acid grease
  • Schizochytrium sp and method for producing docosahexenoic acid grease
  • Schizochytrium sp and method for producing docosahexenoic acid grease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Screening of DHA producing bacteria

[0040] 1) Take a certain amount of water sample and spread it on high-salt YPD solid medium (glucose 20.0g / L, yeast powder 10.0g / L, peptone 20.0g / L, agar 20.0g / L, sea crystal 10.0g / L L), cultivated at 25°C for 48 hours, and obtained multiple single colonies.

[0041] 2) Pick a single colony in the liquid medium, and culture it in a shaker flask for 72 hours under the conditions of a shaker rotation speed of 180 rpm and a culture temperature of 25.0°C, then stain the bacteria with Sudan III for about 1 hour, observe under a microscope, and screen to obtain several strains Strains with high oil production.

[0042] 3) Using the GC-MS method to analyze the oil components of the above strains, one of the strains has a relatively high content of DHA, the specific content is 34.8%.

[0043] 4) The strain was cultured in YPD solid medium at 25°C for 2 days, and a single colony was observed with the naked eye, which was light y...

Embodiment 2

[0045] Therefore, according to the above experimental analysis results, the DHA producing bacterium was identified as Schizochytrium. Embodiment 2. Mutation breeding of Schizochytrium

[0046] 1) Pick a single colony of Schizovibrio and place it in a 250mL Erlenmeyer flask containing 50mL of liquid YPD medium (glucose 20.0g / L, yeast powder 10.0g / L, peptone 20.0g / L), shake at a culture temperature of 25°C Cultivate for 48 h at a bed speed of 180 rpm, centrifuge at 6000 g for 10 min, collect the bacteria, wash once with PBS buffer (100 mmol / L, pH 7.0), add 0.5 mL of PBS buffer (100 mmol / L, pH 7.0) to prepare Bacterial suspension.

[0047] 2) Add 0.5mL of 2.5mg / mL nitrosoguanidine solution, and treat at 30°C for 1 hour; centrifuge the above treatment solution under sterile conditions, wash the bacteria with distilled water to make the bacteria liquid OD 650 After reaching 0.4-0.6, pour it into a petri dish to make the bacterial liquid into a thin layer of liquid with a thicknes...

Embodiment 3

[0051] Embodiment 3. The influence of culture temperature on thalline growth and DHA synthesis

[0052] 1) Add 50mL seed medium (glucose 50.0g / L, yeast powder 5.0g / L, peptone 5.0g / L, MgSO 4 2.0g / L, KH 2 PO 4 2g / L), the inoculum size was 3%, and cultivated for 24h at a culture temperature of 20°C and a shaker speed of 220rpm;

[0053] 2) Add 50mL fermentation medium (glucose 90.0g / L, yeast powder 20.0g / L, MgSO 4 0.5g / L, KH 2 PO 4 1.5g / L, NaNO 3 3.0g / L, Na 2 SO 4 10.0g / L); sterilize at 121°C for 30min, cool down for later use. A total of 15 bottles were prepared, and each 3 bottles was a parallel.

[0054] 3) Insert the cultured seed liquid into each bottle according to the inoculum amount of 10%, and vibrate at 15°C, 18°C, 20°C, 23°C, 25°C, 28°C, and 30°C for 60 hours, and the shaking speed is 220rpm , 6000g centrifuged for 10min to collect the thalline, centrifuged to collect the thalline, after using neutral cellulase and alkaline protease to break the wall, using n...

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Abstract

The invention discloses Schizochytrium sp and method for producing docosahexenoic acid grease. The Schizochytrium sp S056 is preserved in CCTCC in September 29, 2013, and the preservation number is CCTCC M2013459. The Schizochytrium sp S056 with stable and high yield can be obtained by a method combined with mutagenesis breeding and rapid screening, and dissolved oxygen detection supplementary material and temperature control technology is used, and glucose concentration of fermentation medium is regulated and controlled for completely using glucose, and DHA yield of the bacterial strain can reach 23 g / L at the highest, and the productivity can reach 8.2g / L.d at the highest, and the DHA has a high product quality and the DHA content can reach 51%. The bacterial strain can produce DHA, and the fermentation condition is simple and easy to control, and is suitable for growth under the low salinity condition without seawater or seawater crystals with short fermentation period and high production efficiency, and the fermentation waste water is easy to be treated, and the product is suitable for industrial application.

Description

technical field [0001] The invention belongs to the field of fermentation, and relates to a Schizochytrium and a method for producing docosahexaenoic acid (DHA) oil. Background technique [0002] DHA is a polyunsaturated fatty acid with very important physiological functions. It is an important nutrient that the human body cannot synthesize in large quantities but is indispensable. DHA exists in a large amount in the nerve tissue of the human retina and cerebral cortex, which can promote the development of the brain and vision, improve intelligence, memory and vision; in addition, it can also prevent the deposition of cholesterol on the blood vessel wall, prevent or reduce atherosclerosis sclerosis and coronary heart disease. [0003] The traditional production route of DHA mainly comes from the extraction and purification of deep-sea fish oil. However, the resources of deep-sea fish oil are very limited, the production cost is high, the quality is unstable, and the output ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12P7/64C12R1/645
Inventor 余龙江朱圆敏周蓬蓬陈伟谢辰王勇钱凯
Owner HUAZHONG UNIV OF SCI & TECH
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