CN105018539B - A method of culture schizochytrium limacinum high yield DHA - Google Patents

Abstract

The invention discloses a kind of methods for cultivating schizochytrium limacinum high yield DHA, belong to field of microbial fermentation.The method of the present invention includes the following steps: that will reach dry cell weight by medicine bottle culture is that 18~22g/L schizochytrium limacinum seed liquor is inoculated into seeding tank, after culture to the level of transferring, it inoculates in fermentor, using 22~33 DEG C of progress High Density Cultivations of high temperature early period, the mode that later period carries out 18~22 DEG C of low temperature stress carries out Fiber differentiation 110~130 hours, during which maintains glucose sugar, total nitrogen content in fermentation process to keep a certain concentration and pH in 5.5~6.5 sections by stream plus glucose sugar juice, nitrogen source solution and aqueous slkali.Dry cell weight reaches 120~150g/L in the fermentation liquid obtained by the method for the invention, and DHA reaches 40~50g/L, is higher by 1.2~1.5 times than reported DHA productivity, is highly suitable to be applied for the industrialized production of DHA.

Description

A method of culture schizochytrium limacinum high yield DHA

Technical field

The invention belongs to field of microbial fermentation, are related to the production of DHA, and in particular to a kind of culture schizochytrium limacinum high yield The method of DHA.

Background technique

Docosahexaenoic acid (22:6n-3, docosahexaenoic acid, DHA) is a kind of important ω -3 series Polyunsaturated fatty acid (ω -3polyunsaturated fatty acids), can either promote infant's intelligence and eyesight Development, and there is prevention the elderly's cardiovascular disease, anticancer, anti-inflammatory, strengthen immunity and other effects.Traditional extraction how unsaturated The method of fatty acid is obtained from deep sea fish oil, with environmental pollution and the gradually scarcity of marine fishery resources, from deep-sea fish Middle extraction unsaturated fatty acid, which manifests, deposits problems, for example, quality is unstable, exogenous pollution, smell are bad, purifying at This is high, and the EPA and cholesterol contained in fish oil also makes them unsuitable for being added into pregnant baby's food.On the contrary, as microorganism sends out The further investigation of the continuous maturation and microbial fermentation grease technology of ferment technology obtains grease from microorganism and is not only easy Realize large-scale production, but also can overcome traditional fish oil it is generally existing by region and weather conditionality the problem of, answer It is boundless with prospect.

Schizochytrium limacinum (Schizochytrium) is one of the ideal species of current industrialized production DHA, belongs to brokenly capsule A kind of marine microalgae of chytrid section can accumulate significant quantities of fat by high density fermentation culture in vivo, and 70% with triglycerides Form exists, and wherein DHA can account for the 45~55% of total fatty acids.

Schizochytrium limacinum belongs to heterotroph microorganism, and glucose, fructose and glycerol are the good of schizochytrium limacinum fermentation production DHA Carbon source, wherein glucose is most suitable carbon source.Traditional Industrialization production algae oil DHA mainly uses glucose as carbon source, but produces 1 Ton DHA grease take around consumption 5 tons of glucose, it is this conversion be it is inefficient, need to input a large amount of energy in production process in addition Amount, investment in equipment and maintenance is costly, so that the production cost of current algae oil DHA is very high.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the existing technology and deficiency, provides a kind of culture schizochytrium limacinum high yield DHA Method.

The purpose of the invention is achieved by the following technical solution:

A method of culture schizochytrium limacinum high yield DHA includes the following steps:

(1) schizochytrium limacinum strain is seeded in the shaking flask equipped with culture medium, in 25~35 DEG C of shaking tables with 150~ 250rpm revolving speed carries out 20~35h of constant temperature perseverance revolving speed culture.

(2) seed liquor that step (1) obtains is transferred in the shaking flask equipped with culture medium, inoculation volume is that culture medium fills liquid The 5~10% of amount carry out constant temperature perseverance revolving speed 16~20h of culture, measurement gained kind under the conditions of 25~30 DEG C, 150~250rpm The dry cell weight of sub- liquid, if dry cell weight reaches 18~22g/L in obtained seed liquor, directly progress step (4).

(3) if dry cell weight does not reach 18~22g/L in the seed liquor that step (2) obtains, it is further transferred to In shaking flask equipped with culture medium, cultivated according to the method in step (2)；Repeat that the step is one or many to be obtained to culture Seed liquor in dry cell weight reach 18~22g/L.

(4) above-mentioned dry cell weight is reached to the seed liquor of 18~22g/L, dress is inoculated into the inoculum concentration of 5~10% (V/V) Have and carries out fermented and cultured in the seeding tank of culture medium；By ammonium hydroxide or liquid alkaline control fermentation pH maintain 5.5~6.5 it Between, at 25~35 DEG C, ventilatory capacity control exhausts, stops in 0.3~0.5vvm, culture glucose into fermentation liquid for cultivation temperature control Only cultivate；

(5) fermentation liquid in step (4) seeding tank is inoculated into the fermentor equipped with culture medium with 5~10% inoculum concentration Middle carry out fermented and cultured；At 22~33 DEG C, ventilatory capacity control passes through stream plus glucose in 0.3~0.5vvm for cultivation temperature control Concentration of glucose maintains 30~50g/L in solution control fermentation liquid, is controlled in fermentation process by stream plus mixed nitrogen solution The concentration of total nitrogen maintains 15~25g/L, includes yeast extract, Dried Corn Steep Liquor Powder, sodium glutamate, albumen in the mixed nitrogen One of peptone is a variety of；Fermentation process pH, which is controlled, by Feeding ammonia water or liquid alkaline maintains 5.5~6.5；Fermented and cultured is to 70 Low temperature stress culture is carried out after~100h, temperature is reduced to 18~22 DEG C, remaining condition is constant, continues 30~50h of culture.

Above-mentioned steps (1), (2) and the culture medium in (3) formula are as follows: 40~50g/L of glucose, yeast extract 10~ 25g/L, 5.0~10.0g/L of sodium sulphate, 0.6~0.8g/L of potassium chloride, 1.8~2.2g/L of magnesium sulfate, potassium dihydrogen phosphate 1.9~ 2.lg/L, 0.14~0.16g/L of calcium chloride, 0.8~1.2g/L of ammonium sulfate, 1.68~1.72mg/L of tetrahydrate manganese chloride, seven water sulphur Sour 2.8~3.0mg/L of zinc, 1.4~1.6mg/L of cupric sulfate pentahydrate, 0~0.05mg/L of Sodium Molybdate Dihydrate, CoCL2 6H2O 0~ 0.05mg/L。

The formula of above-mentioned steps (4) and the culture medium in (5) are as follows: 75.0~85.0g/L of glucose, Dried Corn Steep Liquor Powder 15~ 20g/L, 2.9~3.lg/L of yeast extract, 5.0~15.0g/L of sodium glutamate, 5.0~10.0g/L of sodium sulphate, potassium chloride 0.6 ~0.8g/L, 1.8~2.2g/L of magnesium sulfate, 1.9~2.lg/L of potassium dihydrogen phosphate, 0.14~0.16g/L of calcium chloride, ammonium sulfate 0.8~1.2g/L, 1.68~1.72mg/L of tetrahydrate manganese chloride, 2.8~3.0mg/L of white vitriol, cupric sulfate pentahydrate 1.4~ 1.6mg/L, 0~0.05mg/L of Sodium Molybdate Dihydrate, 0~0.05mg/L of CoCL2 6H2O.

Schizochytrium limacinum strain described in step (1) is preferably schizochytrium limacinum ATCC20888.It is furthermore preferred that step (1) Are as follows: the schizochytrium limacinum ATCC20888 strain saved with glycerol is inoculated into the 250m shaking flask equipped with 50mL culture medium, in 25~ 25~35h of constant temperature perseverance revolving speed culture is carried out with 150~250rpm revolving speed in 30 DEG C of shaking tables.

The concentration of ammonium hydroxide described in step (4) and (5) is preferably 28~30% (V/V), and the concentration of the liquid alkaline is excellent It is selected as 30~40% (W/V).

The concentration of glucose solution described in step (5) is preferably 700~750g/L, the mixed nitrogen solution Concentration is preferably 200~500g/L.

The volume of seeding tank described in step (4) is preferably 200L, and tank body liquid amount is preferably 150L；In step (5) The volume of the fermentor is preferably 3000L, and tank body liquid amount is preferably 1500L.

The present invention is optimized by culture medium to schizochytrium limacinum fermenting and producing DHA and condition of culture, using traditional Aerobic fermentation method carries out High Density Cultivation using relatively high temperature in the 3000L ferment tank prometaphase, and the fermentation later period carries out low Warm coercing cultivation, which not only reduces fermentation costs, but also reduces the control difficulty of fermentation process, fermentation ends post-fermentation The dry cell weight of liquid reaches 120~150g/L, and DHA reaches 40~50g/L, 1.2 are higher by than reported DHA productivity~ 1.5 times, it is highly suitable to be applied for the industrialized production of DHA.The method of culture schizochytrium limacinum high yield DHA of the present invention has following excellent Gesture:

(1) prometaphase high-temperature cultivation, the low temperature stress Fiber differentiation of short time in later period, with traditional cold fermentation phase are used Than energy consumption at least reduces by 30%, greatly reduces demand of the fermentation process to energy, and the requirement to refrigeration equipment.

(2) used medium formula is compared to traditional zymotic in the present invention, and production level is higher, and cost significantly drops It is low, fermentation risk can be reduced while additional income.

(3) producing the polyunsaturated fatty acids (PuFA) such as DHA by high density aerobic fermentation method culture schizochytrium limacinum can subtract It is few because the market demand it is excessive caused by largely catch bring environment and influence, therefore the invention has the protection of environmental resource Potential meaning, while passing through the products such as Production by Microorganism Fermentation docosahexaenoic acid, the three wastes generated in production process Less, the construction of factory and cost of investment are relatively low.

(4) although the lipid content of presently found High Density Cultivation schizochytrium limacinum fermentation production is usually than traditional fish oil It is low, but the grease of microbial fermentation production contains largely necessary fatty acid, and passes through the fatty acid that microbe fermentation method obtains Form fairly simple, the content of polyunsaturated fatty acid is very high, and enrichment is also than extracting much easier, this height from traditional fish oil Purity is extremely advantageous to subsequent extracted technique, can simplify purification process, can greatly reduce be produced into the industrial production This.

(5) it is not limited by the factor of season and climate change by the technique that microbe fermentation method obtains polyunsaturated fatty acid System, can be produced, and do not limited by raw material and the place of production whole year, and the flexible of polyunsaturated fatty acid production is increased Property.

(6) grease extracted by microbe fermentation method contains more cholesterol unlike fish oil product, increases The purity of product and the applicable surface of product are added, have kept the quality of product more guaranteed.

(7) producing DHA by Schizochytrium in high-density culture through fermentation, strain schizochytrium limacinum are rich in protein, microelement, dimension Raw element, antioxidant etc., can be used as the source of macronutrient and micronutrient in food prescription.

(8) quality of product not only can be improved by what microorganism was fermented, but also can use genetic engineering hand Section simultaneously combines microorganism to produce the metabolic pathway of polyunsaturated fatty acid, and the composition of PuFA is controlled using the method for metabolic regulation, So as to realize the manual control of production.

Specific embodiment

Further detailed description is done to the present invention below with reference to embodiment, embodiments of the present invention are not limited thereto.

Embodiment 1

(1) culture medium is prepared

The preparation method of Shake flask medium is as follows: by component tetrahydrate manganese chloride, white vitriol, the five water sulfuric acid in formula After copper, Sodium Molybdate Dihydrate, CoCL2 6H2O weigh, is sufficiently dissolved with sterile water, be prepared into mother liquor, under acid condition, 121 DEG C of height Press steam sterilizing 20min；After other compositions weigh, dispensed after completely dissolution with sterile water to shaking flask, 121 DEG C of high steams go out Bacterium 20min.

Shake flask medium formula is as follows: glucose 45g/L, yeast extract 10g/L, sodium sulphate 8.0g/L, potassium chloride 0.7g/L, magnesium sulfate 2.0g/L, potassium dihydrogen phosphate 2.0g/L, calcium chloride 0.15g/L, ammonium sulfate 1.0g/L, tetrahydrate manganese chloride 1.7mg/L, white vitriol 3.0mg/L, cupric sulfate pentahydrate 1.5mg/L, Sodium Molybdate Dihydrate 0.05mg/L, CoCL2 6H2O 0.05mg/L。

The preparation method of fermentation tank culture medium is as follows: after weighing proportionally by all components in formula, being filled with sterile water Point dissolution, 121 DEG C of high pressure steam sterilization 20min are spare after cooling.

Fermentation tank culture based formulas is as follows: glucose 80.0g/L, Dried Corn Steep Liquor Powder 15g/L, yeast extract 3.0g/L, Sodium glutamate 10.0g/L, sodium sulphate 8.0g/L, potassium chloride 0.7g/L, magnesium sulfate 2.0g/L, potassium dihydrogen phosphate 2.0g/L, chlorination Calcium 0.15g/L, ammonium sulfate 1.0g/L, tetrahydrate manganese chloride 1.7mg/L, white vitriol 3.0mg/L, cupric sulfate pentahydrate 1.5mg/L, Sodium Molybdate Dihydrate 0.05mg/L, CoCL2 6H2O 0.05mg/L.

(2) it goes bail for and is hidden in the glycerol tube schizochytrium limacinum ATCC20888 strain of ultra low temperature freezer, be seeded to 2 equipped with 50mL In Oscillating bottles of 250mL of Shake flask medium, with the revolving speed of 150rpm in 25 DEG C Oscillating, culture is for 24 hours；Then with 5% inoculation Secondary culture in Oscillating bottles of 250mL be seeded to 300 equipped with 50mL culture medium is measured, is turned in 25 DEG C Oscillating with 150rpm Speed cultivates 19.5h, and measuring the dry cell weight in seed liquor is 18.7g/L, collects seed liquor.

(Oscillating bottles of seed liquors are arrived the 200L level-one kind equipped with 150L fermentation tank culture medium by kind according to 10% kind of amount of pressing by 3) Fermented and cultured is carried out in sub- tank, cultivation temperature is 28 DEG C, ventilatory capacity 0.5vvm, and entire fermentation process is controlled using 30% ammonium hydroxide For the pH of fermentation liquid between 5.5~6.0, fermentation stops hair to when glucose content is lower than 5g/L in fermentation liquid during 35~45h Ferment.

(4) the seed liquor 150L in first class seed pot is all seeded to the hair of the 3000L equipped with 1500L fermentation tank culture medium Fermented and cultured is carried out in fermentation tank, cultivation temperature is 28 DEG C, ventilatory capacity 0.5vvm, and entire fermentation process is controlled using 30% ammonium hydroxide The pH of fermentation liquid is between 5.5~6.0.Start the mixing of stream plus the glucose solution and 230g/L of 750g/L when fermenting to 20h Nitrogen source solution (solution includes 30g/L yeast extract, 100g/L sodium glutamate, 100g/L Dried Corn Steep Liquor Powder), samples every 3h Detect glucose and total nitrogen content in fermentation liquid, by flow rate control concentration of glucose in fermentation liquid maintain 30 always~ 50g/L, total nitrogen content maintain 15~25g/L, when fermented and cultured is to 115h, terminate fermentation.Tank is put by being freeze-dried detection When fermentation liquid in dry cell weight be 121.9g/L；Compound lard content, which is detected, by organic solvent extractionprocess accounts for dry cell weight 61.02%, gas phase analysis detects DHA content in compound lard and accounts for the 51.12% of compound lard dry weight, and DHA's contains in fermentation liquid It measures up to 38.02g/L.The component analysis of gained compound lard is shown in Table 1 after this Batch fermentation:

Table 1

Fatty acid species	Quality percentage composition (%)
		C12:0	0.06
C14:0	0.57
		C16:0	32.16
C16:1	0.06
		C18:0	1.44
C18:1	0.05
		C18:2	0.05
C18:3	0.07
		C20:3	0.36
Squalene	0.45
		C20:5	0.39
C22:0	0.27
		C22:5	12.97
C22:6	51.12

Embodiment 2

Embodiment 2 is different in addition to the method in 3000L fermentation cylinder for fermentation culture compared with Example 1, remaining is all the same, Specifically: the seed liquor 150L in 1 first class seed pot of embodiment is all seeded to equipped with 1500L fermentation tank culture medium Fermented and cultured is carried out in 3000L fermentor, cultivation temperature is 28 DEG C, ventilatory capacity 0.5vvm, and entire fermentation process utilizes 30% Ammonium hydroxide controls the pH of fermentation liquid between 5.5~6.0.Start when fermenting to 20h stream plus 750g/L glucose solution and and (solution is dry comprising 30g/L yeast extract, 100g/L sodium glutamate, 100g/L corn pulp for the mixed nitrogen solution of 230g/L Powder), every glucose and total nitrogen content in 3h sample detection fermentation liquid, concentration of glucose in fermentation liquid is controlled by flow rate and is begun 30~50g/L is maintained eventually, total nitrogen content maintains 15~25g/L.Low temperature stress culture is carried out when fermented and cultured is to 77h, Cultivation temperature is reduced to 20 DEG C in 30min, continues to cultivate 37h, terminates fermentation.It is detected, cell is dry in fermentation liquid when putting tank It weighing to 138.5g/L, compound lard content accounts for the 63.5% of dry cell weight, and DHA content accounts for the 56.85% of compound lard dry weight, The content of DHA is up to 49.99g/L in fermentation liquid.The component analysis of gained compound lard is shown in Table 2 after this Batch fermentation:

Table 2

Fatty acid species	Quality percentage composition (%)
		C12:0	0.02
C14:0	0.58
		C16:0	26.27
C16:1	0.06
		C18:0	1.26
C18:1	0.05
		C18:2	0.08
C18:3	0.05
		C20:3	0.27
Squalene	0.65
		C20:5	0.32
C22:0	0.30
		C22:5	13.24
C22:6	56.85

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Claims (5)

1. a kind of method for cultivating schizochytrium limacinum high yield DHA, it is characterised in that include the following steps:

(1) schizochytrium limacinum ATCC20888 strain is seeded in the shaking flask equipped with culture medium, with 150 in 25~35 DEG C of shaking tables ~250rpm revolving speed carries out 20~35h of constant temperature perseverance revolving speed culture；

(2) seed liquor that step (1) obtains is transferred in the shaking flask equipped with culture medium, inoculation volume is culture medium liquid amount 5~10%, constant temperature perseverance revolving speed 16~20h of culture, measurement gained seed liquor are carried out under the conditions of 25~30 DEG C, 150~250rpm Dry cell weight, if dry cell weight reaches 18~22g/L in obtained seed liquor, directly progress step (4)；

(3) if dry cell weight does not reach 18~22g/L in the seed liquor that step (2) obtains, it is further transferred to and is equipped with In the shaking flask of culture medium, cultivated according to the method in step (2)；Repeat the one or many kinds obtained to culture of the step Dry cell weight reaches 18~22g/L in sub- liquid；

(4) above-mentioned dry cell weight is reached to the seed liquor of 18~22g/L to be inoculated into the inoculum concentration of 5~10% (V/V) equipped with training It supports in the seeding tank of base and carries out fermented and cultured；Fermentation pH is controlled by ammonium hydroxide or liquid alkaline to maintain between 5.5~6.5, At 25~35 DEG C, ventilatory capacity control exhausts, stops in 0.3~0.5vvm, culture glucose into fermentation liquid for cultivation temperature control Culture；

(5) by the fermentation liquid in step (4) seeding tank with 5~10% inoculum concentration be inoculated into the fermentor equipped with culture medium into Row fermented and cultured；At 22~33 DEG C, ventilatory capacity control passes through stream plus glucose solution in 0.3~0.5vvm for cultivation temperature control Concentration of glucose maintains 30~50g/L in control fermentation liquid, controls total nitrogen in fermentation process by stream plus mixed nitrogen solution Concentration maintains 15~25g/L, comprising in yeast extract, Dried Corn Steep Liquor Powder, sodium glutamate, peptone in the mixed nitrogen It is one or more；Fermentation process pH, which is controlled, by Feeding ammonia water or liquid alkaline maintains 5.5~6.5；Fermented and cultured is to 70~100h Low temperature stress culture is carried out afterwards, temperature is reduced to 18~22 DEG C, remaining condition is constant, continues 30~50h of culture；

Above-mentioned steps (1), (2) and the culture medium in (3) formula are as follows: 40~50g/L of glucose, 10~25g/ of yeast extract L, 5.0~10.0g/L of sodium sulphate, 0.6~0.8g/L of potassium chloride, 1.8~2.2g/L of magnesium sulfate, 1.9~2.lg/ of potassium dihydrogen phosphate L, 0.14~0.16g/L of calcium chloride, 0.8~1.2g/L of ammonium sulfate, 1.68~1.72mg/L of tetrahydrate manganese chloride, white vitriol 2.8~3.0mg/L, 1.4~1.6mg/L of cupric sulfate pentahydrate, 0~0.05mg/L of Sodium Molybdate Dihydrate, CoCL2 6H2O 0~ 0.05mg/L；

The formula of above-mentioned steps (4) and the culture medium in (5) are as follows: 75.0~85.0g/L of glucose, 15~20g/ of Dried Corn Steep Liquor Powder L, 2.9~3.lg/L of yeast extract, 5.0~15.0g/L of sodium glutamate, 5.0~10.0g/L of sodium sulphate, potassium chloride 0.6~ 0.8g/L, 1.8~2.2g/L of magnesium sulfate, 1.9~2.lg/L of potassium dihydrogen phosphate, 0.14~0.16g/L of calcium chloride, ammonium sulfate 0.8 ~1.2g/L, 1.68~1.72mg/L of tetrahydrate manganese chloride, 2.8~3.0mg/L of white vitriol, cupric sulfate pentahydrate 1.4~ 1.6mg/L, 0~0.05mg/L of Sodium Molybdate Dihydrate, 0~0.05mg/L of CoCL2 6H2O.

2. the method for culture schizochytrium limacinum high yield DHA according to claim 1, it is characterised in that: step (1) are as follows: will use The schizochytrium limacinum ATCC20888 strain that glycerol saves is inoculated into the 250mL shaking flask equipped with 50mL culture medium, is shaken in 25~30 DEG C 25~35h of constant temperature perseverance revolving speed culture is carried out with 150~250rpm revolving speed in bed.

3. the method for culture schizochytrium limacinum high yield DHA according to claim 1, it is characterised in that: in step (4) and (5) The concentration of the ammonium hydroxide is 28~30% (V/V), and the concentration of the liquid alkaline is 30~40% (W/V).

4. the method for culture schizochytrium limacinum high yield DHA according to claim 1, it is characterised in that: described in step (5) The concentration of glucose solution is 700~750g/L, and the concentration of the mixed nitrogen solution is 200~500g/L.

5. the method for culture schizochytrium limacinum high yield DHA according to claim 1, it is characterised in that:

The volume of seeding tank described in step (4) is 200L, and tank body liquid amount is 150L；

The volume of fermentor described in step (5) is 3000L, and tank body liquid amount is 1500L.