CN101824442A - Fermentation production technique of schizogenesis chytrid - Google Patents

Abstract

The invention provides a fermentation production technique of schizogenesis chytrid, which solves the problems of lower yield and higher production cost in the prior art. The fermentation production technique has the technical scheme that bean pulp is taken as main nitrogen source for fermentation production, and the technique comprises the steps of bean pulp hydrolysis, pressing filter, preparation of fermentation medium, arrangement of a fermentation tank system, collection and drying of the schizogenesis chytrid, and preparing the schizogenesis chytrid dry powder. The new fermentation production technique of the schizogenesis chytrid takes the bean pulp as main nitrogen, thus not only reducing the cost of fermentation production, but also improving the growth rate of the schizogenesis chytrid and promoting fatty acid accumulation of the cells of the schizogenesis chytrid.

Description

A kind of fermentation manufacturing technique of schizochytrium limacinum

Technical field

The invention belongs to the microbial fermentation production field, specifically, relating to a kind of is the fermentation manufacturing technique of the schizochytrium limacinum of major nitrogen source with the dregs of beans.

Background technology

In recent years, (Polyunsaturated fatty acids, PUFAs) vital role to human health more and more is subject to people's attention polyunsaturated fatty acid.Wherein, as the docosahexenoic acid of one of important ω-3PUFAs (22:6, n-3, docosahexaenoicacid, DHA), because of it gains great popularity at human body and the intravital important physiological function of animal.Studies show that DHA is the basal component of cytolemma in some tissue of human body, and baby's vision system and neural growth are played an important role, and also has pharmacological actions such as reducing cholesterol, anticoagulation, preventing cancer.In addition, DHA still is the multiple seawater fish required indispensable fatty acid that grows, and can improve the surviving rate of fry and reduce its albinism.DHA obtains from fish oil traditionally, but the PUFAs that extracts from fish oil exists the output shakiness, yield is low, cost is high and contain other problems such as ω-6PUFAs, and along with the growing tension of fishing resources, traditional DHA resource can't satisfy the growing market requirement.Therefore, the newborn resource of exploitation DHA has become new research focus.

Schizochytrium limacinum (Schizochytrium limacinum) is a kind of thalassiomycetes, belongs to net myxomycota (Labyrinthulomycota), net Myxomycetes (Labyrinthulomycetes), thraustochytriale (Thraustochytriales), thraustochytriale section (Thraustochytriaceae), schizochytrium limacinum genus, schizochytrium limacinum.DH A content is abundant in this bacterium, also contain a spot of clupanodonic acid (Docosapentaenoic acid, DPA), and other unsaturated fatty acid content very low [NaKahara et al.JAOCS, Vol.73, No.11 (1996)].This bacterium is safe in utilization, and it has carried out a series of security detection to white mouse, rabbit humans such as Hammond, does not find any toxic side effect (Hammond, 2001).Therefore, be the latent production-goods source of a kind of good DHA.T.Yokocki etc. adopt several kinds of carbon source and nitrogenous source that schizochytrium limacinum (Schizochytrium limacinum SR21) is optimized cultivation in shaking bottle, the output of the highest DHA that obtains is 4g/L[Yokocki et al, AppiMicrobiol Biotechnol, Vol.49,72-76 (1998)]; People such as Kw Fan are that carbon, nitrogenous source are cultivated schizochytrium limacinum mangrovei with glucose and yeast extract, the output of DHA is 2.79g/L[Kw Fan et al, Journal of industrial Microbiology andBiotechnology, Vo.l 27 (4): 199-202 (2001)].

Existing schizochytrium limacinum fermentation be with yeast product as major nitrogen source, cost is higher.Therefore, seek a kind of high yield, the industrial fermentation new process of production of schizochytrium limacinum then is the new problem that the present invention faced cheaply.

Summary of the invention

The invention provides a kind of fermentation manufacturing technique of schizochytrium limacinum, can solve lower, the production cost problem of higher of output that prior art exists.

For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:

A kind of fermentation manufacturing technique of schizochytrium limacinum, comprises the steps: as the major nitrogen source fermentative production with dregs of beans

1) dregs of beans hydrolysis

Adding mass ratio in hydrolytic decomposition pot is 1: the dregs of beans of 5-10 and water, add concentrated hydrochloric acid regulator solution p H to 1.0-3.0, and be heated to 100 ℃-120 ℃, pressure rises to 0.04-0.1MPa, soaking time 10～40min, pH to 5.0-7.0 is regulated with alkali in the cooling back; In hydrolytic decomposition pot, add the 5-10 seawater doubly of dregs of beans quality again, promptly get dregs of beans hydrolysis material;

2) press filtration

The said hydrolyzed material is squeezed into plate-and-frame filter press, carry out solid-liquid separation, the effective filtration area of plate-and-frame filter press is 80-100m ², adopt 300-350 order filter cloth, filtration temperature 60-80 ℃, filter pressure 0.10-0.30Mpa; Gained filtrate is soybean meal hydrolysate, collects all filtrates and is stored in the fermentative production that is used for next step schizochytrium limacinum in the storage tank;

3) preparation of fermention medium

As follows with dregs of beans: glucose concn 60-90g/L, dregs of beans concentration 20-80g/L, concentration of sodium glutamate 10-50g/L, the volume ratio 1 of seawater and distilled water: 1-2 as the fermentative production prescription of major nitrogen source fermentation schizochytrium limacinum; Dregs of beans concentration is the percentage concentration that the quality of dregs of beans accounts for whole fermentation system;

4) setting of fermentor tank system

Staged fermentor tank system, in each grade fermentor tank system, schizochytrium limacinum seed inoculum size is 5%-10%; Leavening temperature is 24-26 ℃, and pH is 5.5-7.0, stirs and blowing air fermentation time 3-4 days;

5) collection of schizochytrium limacinum and drying

After the fermentation ends, the schizochytrium limacinum fermented liquid separates with whizzer, separates back material solid content at 5%-15%; Material is squeezed into spray-drying tower, and the steam output of spray-drying tower is 200-300kg/h, and inlet temperature is 175-190, and temperature out is 70-90 ℃; The dry powder that spray-dried back obtains is schizochytrium limacinum dry powder, and lucifuge, vacuum cold are preserved fast.

In technical scheme of the present invention, also have following technical characterictic: Heating temperature is 105～115 ℃ in described dregs of beans hydrolysing step, soaking time 15～30min.

In technical scheme of the present invention, also have following technical characterictic: carry out eardrum and operate the filtrate of removing in the filter residue after the press filtration step is finished, working pressure is 0.30-0.45Mpa.

In technical scheme of the present invention, also have following technical characterictic: the screening formulation of described fermention medium: glucose concn is 70-80g/L, and dregs of beans concentration is 30-60g/L, and concentration of sodium glutamate is 10-40g/L; Preferred 1: 1 of the volume ratio of seawater and distilled water.

In technical scheme of the present invention, also have following technical characterictic: described staged fermentor tank system is a 100L-1000L-15000L three grade fermemtation tank body system.

In technical scheme of the present invention, also has following technical characterictic: mend the sugar operation during for 20-30g/L when glucose concn in the fermented liquid, mend at every turn that glucose concn is between the 45-55g/L in the sugared secondary fermentation liquid.

In technical scheme of the present invention, also have following technical characterictic: mixing speed is 100-300rpm in the described fermentor tank system.

In technical scheme of the present invention, also have following technical characterictic: ventilation is than being 0.5-2.0L min in the described fermentor tank system ^-1L ^-1, i.e. VVM, the ratio of bubbling air in the per minute unit volume fermented liquid.

Compared with prior art, advantage of the present invention and positively effect are:

The invention provides a kind of dregs of beans that utilizes and the thalassiomycetes-schizochytrium limacinum (Schizochytrium limacinum) that is rich in DHA is carried out the industrial fermentation new process of production for major nitrogen source, by optimizing dregs of beans processing conditions and zymotechnique, set up the new process for fermenting of a cover schizochytrium limacinum, not only reduced production cost, also improve the output of schizochytrium limacinum, increased DHA content in the cell.

Description of drawings

Fig. 1 is a dregs of beans hydrolysis process schema;

Fig. 2 is the technological process of production figure of schizochytrium limacinum fermentation;

Fig. 3 is the fatty acid analysis collection of illustrative plates that adopts soybean meal hydrolysate fermentative production schizochytrium limacinum.

Embodiment

The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.

Do detailed explanation below in conjunction with accompanying drawing 1,2,3 and concrete experiment at following problem:

Determining of I, dregs of beans hydrolysis process: solid (dregs of beans) and liquid (hydrolyzed solution) ratio, hydrolysis pH value, hydrolysis temperature and hydrolysis time definite;

II, be the schizochytrium limacinum Optimum of culture medium of major nitrogen source with the dregs of beans;

III, mixing speed are to being to the influence of its growth and DHA content with the dregs of beans in the schizochytrium limacinum fermentation of major nitrogen source;

IV, ventilation comparison are the growth of schizochytrium limacinum of major nitrogen source and the influence of DHA content with the dregs of beans.

Embodiment 1

The compound nitrogen source substratum is to the influence of schizochytrium limacinum biomass and DHA content

Concentration is the preparation of the soybean meal hydrolysate (amounting to into the weight of dregs of beans) of 40g/L

Adding 82mL concentration in a, the 40g dregs of beans is the hydrochloric acid of 1mol/L, and adds the distilled water of 231mL.

B, 121 ℃, pyrohydrolysis 30min.

C, adding 1mol/L NaOH regulate pH to 7.0, and be centrifugal, keeps supernatant liquor.

D, adding distil water are settled to 1L with supernatant liquor, and the solution of acquisition is the soybean meal hydrolysate that concentration is 40g/L (amounting to into the weight of dregs of beans).

E, if need the soybean meal hydrolysate of other concentration, only need corresponding adjustment soybean meal amount.

Triangle at 250ml shakes in the bottle, add 25ml natural sea-water and 25ml distilled water respectively, press the concentration shown in the table 1 and add carbon source (glucose) and nitrogenous source (soybean meal hydrolysate and Sodium Glutamate), transferring pH value is 6.0, behind the autoclaving, insert the pre-incubated seed liquor of 5ml, 25 ℃ of following shaking culture are 4 days on the airbath vibrator, and the vibration rotating speed is 200rpm.Centrifugal collection thalline, lyophilize is surveyed its dry weight to constant weight under-50 ℃ of conditions; Get the part thalline, BF3-methyl alcohol method esterification routinely is by the percentage composition of DHA in the gas chromatography determination thalline.The results are shown in Table 1.

The orthogonal test of table 1 schizochytrium limacinum medium optimization

Experiment	Glucose (g/L)	Dregs of beans (g/L)	Sodium Glutamate (g/L)	Biomass (g/L*d)	DHA content (% dry weight)
						??1	??60	??20	??40	??6.83±0.12	??15.4
??2	??60	??40	??30	??6.62±0.31	??16.5
						??3	??60	??60	??20	??7.25±0.29	??11.1

Experiment	Glucose (g/L)	Dregs of beans (g/L)	Sodium Glutamate (g/L)	Biomass (g/L*d)	DHA content (% dry weight)
						??4	??70	??20	??30	??7.05±0.28	??16.1
??5	??70	??40	??20	??6.55±0.23	??15.2
						??6	??70	??60	??40	??5.26±0.56	??14.8
??7	??80	??20	??20	??6.55±0.22	??16.6
						??8	??80	??40	??40	??5.95±0.15	??12.0
??9	??80	??60	??30	??7.55±0.08	??13.9

As can be seen from Table 1, when glucose concn was 60-70g/L, when soybean meal hydrolysate and concentration of sodium glutamate were respectively 40g/L and 20g/L, dry cell weight reached 26-30g/L.The concentration of soybean meal hydrolysate is the Mass Calculation of amounting to into dregs of beans.

Embodiment 2

Different dregs of beans hydrolysis processs are to the influence of schizochytrium limacinum biomass and DHA content

After adopting following manner to produce soybean meal hydrolysate, add the 600L substratum in the 1000L fermentor tank, transferring pH value is 6.0, and adds VITMAIN B1, B12, and H after the sterilization, inserts the bacterial classification seed liquor, cultivates 4 days down for 25 ℃.Centrifugal collection thalline is surveyed its dry weight; Get the part thalline, BF routinely ₃The esterification of-methyl alcohol method is by the percentage composition of DHA in the gas chromatography determination thalline.The results are shown in Table 2.

The hydrolysis scheme

A adds bean cake powder and an amount of water in enamel reaction still, add 30% concentrated hydrochloric acid regulator solution pH to 1.3.Be steam heated to 110 ℃, keep 30min, pH to 6.0 is regulated in the cooling back, and with residue and solution separating, the clear liquid of acquisition is as the nutrient solution of fermentation.

B adds bean cake powder and an amount of water in enamel reaction still, add 40% dense NaOH regulator solution pH to 12, is heated to 60 ℃, insulation 30min, and pH to 6.0 is regulated in the cooling back, and with residue and solution separating, the clear liquid of acquisition is as the nutrient solution of fermentation.

C adds an amount of water in enamel reaction still, be heated to 50 ℃, behind the adjusting pH to 11, adds dregs of beans, and pH is in the 6.5-7.0 scope in control, and with residue and solution separating, the clear liquid of acquisition is as the nutrient solution of fermentation.

D adds acid for adjusting pH to 1.5 at the isolating clear liquid of scheme C, is heated to 110 ℃, insulation 30min, and pH to 6.0 is regulated in the cooling back, and with residue and solution separating, the clear liquid of acquisition is as the nutrient solution of fermentation.

E adopts the method for papain enzymolysis.In enamel reaction still, add bean cake powder and an amount of water, add the enzyme powder, at 40 ℃, enzymolysis 2h under the condition of pH7.5, with residue and solution separating, the clear liquid of acquisition is as the nutrient solution of fermentation.

The different dregs of beans hydrolysis processs of table 2 are to the influence of schizochytrium limacinum biomass

The hydrolysis scheme	Biomass (g/L*d)	DHA content (%, dry weight)
			??A	??8.25±0.61	??19.6
??B	??4.58±0.47	??11.3
			??C	??1.08±0.12	??4.3
??D	??0.82±0.03	??3.2
			??E	??6.85±0.31	??10.1

Cultivate schizochytrium limacinum acquisition biomass with the hydrolyzed solution (option A) of acid-hydrolyzed method and be up to 33.25g/L, DHA content is 19.6%; The biomass that obtains with basic hydrolysis method (option b) is 18g/L, and DHA content is 11.3%; And only obtained schizochytrium limacinum seldom with traditional soybean protein extraction scheme (scheme C) and improvement project D thereof.Determine that hydrolytic process as shown in Figure 1.

Embodiment 3

Different hydrolysising conditions are to the influence of schizochytrium limacinum biomass and DHA content

After determining the hydrolysis scheme, to hydrolysis time (min), hydrolysis temperature (℃), (M) carried out orthogonal experiment optimization with acid concentration; The experiment gradient is hydrolysis time 15min, 20min, 25min, 100 ℃ of hydrolysis temperatures, 110 ℃, 120 ℃, concentration of hydrochloric acid 0.25mol/L, 0.4mol/L, 1.0mol/L.Add the 600L substratum in the 1000L fermentor tank, transferring pH value is 6.0, and adds VITAMIN, after the sterilization, inserts the bacterial classification seed liquor, cultivates 4 days down for 25 ℃.Centrifugal collection thalline is surveyed its dry weight; Get the part thalline, BF routinely ₃The esterification of-methyl alcohol method is by the percentage composition of DHA in the gas chromatography determination thalline.The results are shown in Table 3.

Table 3 hydrolysising condition is optimized orthogonal design table

Experiment	Hydrolysis time (min)	Hydrolysis temperature (℃)	Concentration of hydrochloric acid (mol/L)	Biomass (g/L*d)	DHA content (%, dry weight)
						??1	??15	??100	??0.25	??8.76±0.43	??14.6
??2	??15	??110	??0.4	??6.43±0.59	??15.7
						??3	??15	??120	??1.0	??7.86±0.72	??17.5
??4	??20	??100	??0.4	??6.98±0.21	??15.6
						??5	??20	??110	??1.0	??10.60±0.24	??12.8
??6	??20	??120	??0.25	??7.23±0.48	??11.7
						??7	??25	??100	??1.0	??4.53±0.32	??10.2

Experiment	Hydrolysis time (min)	Hydrolysis temperature (℃)	Concentration of hydrochloric acid (mol/L)	Biomass (g/L*d)	DHA content (%, dry weight)
						??8	??25	??110	??0.25	??11.16±0.55	??6.3
??9	??25	??120	??0.4	??7.52±0.36	??8.6

Utilize the 5000L enamel reaction still, descending hydrolysis temperature, concentration of hydrochloric acid and the hydrolysis time of being followed successively by of influence of three experiment factor pair schizochytrium limacinum biomasss, optimum hydrolysising condition is: 110 ℃, 0.25mol/L hydrochloric acid, hydrolysis 20min.The soybean meal hydrolysate that obtains is as the nitrogenous source of fermentative production schizochytrium limacinum, add again glucose as carbon source after, obtain schizochytrium limacinum dry powder 33.76g/L through the 100L fermentation culture.

Embodiment 4

Mixing speed is to the growth of schizochytrium limacinum and the influence of DHA content

Adopt the 1000L fermentor tank, the mixing speed of adjusting fermentor tank is respectively 100rpm, 150rpm, 200rpm, 250rpm and 300rpm.With seed liquor in the switching of 10% ratio after, 25 ℃ of condition bottom fermentations were cultivated 4 days, surveyed its biomass, DHA content, observed mixing speed to the growth of schizochytrium limacinum and the influence of DHA content.The results are shown in Table 4.

Table 4 mixing speed is to the growth of schizochytrium limacinum and the influence of DHA content

Experiment	Mixing speed (rpm)	Biomass (g/L*d)	DHA content (% dry weight)
				??1	??100	??2.37±0.22	??9.7
??2	??150	??7.85±0.41	??13.2
				??3	??200	??8.76±0.64	??18.8
??4	??250	??8.39±1.15	??20.6
				??5	??300	??8.49±1.23	??18.6

Along with the increase of mixing speed, cellular biomass and DHA content all raise, and are up to 8.76g/L*d and 20.6%.When rotating speed was elevated to 300rpm, cellular biomass and DHA content all descended slightly, and this may be that excessive shearing force causes.

Embodiment 5

The growth of ventilation comparison schizochytrium limacinum and the influence of DHA content

Adopt the 1000L fermentor tank, regulate the fermentation ventilation than being respectively 0.5,1.0,1.5 and 2.0L min ^-1L ^-1(VVM).With seed liquor in the switching of 10% ratio after, 25 ℃ of condition bottom fermentations were cultivated 4 days, surveyed its biomass, DHA content, observed the growth of ventilation comparison schizochytrium limacinum and the influence of DHA content.The results are shown in Table 5.

The growth of table 5 ventilation comparison schizochytrium limacinum and the influence of DHA content

Experiment	Ventilation is than (L min -1L -1)	Biomass (g/L*d)	DHA content (%, dry weight)
				??1	??0.5	??3.27±0.51	??8.9
??2	??1.0	??6.36±0.29	??15.2
				??3	??1.5	??9.02±1.10	??20.5
??4	??2.0	??8.69±1.33	??19.4

It is a vital link in the microorganism heterotrophic fermentation process that competent oxygen is provided, and it all has remarkable influence for the accumulation of fat in the growth of aerobic microbiological, the cell and unsaturated fatty acids synthetic.Along with the increase of ventilation ratio, cellular biomass and DHA content all raise gradually, and ventilation is than being 1.5L min ^-1L ^-1The time, having obtained production peak is 9.02g/L*d and 20.5%.

Embodiment 6

The substratum concentration and the fermentation condition that adopt above embodiment to optimize utilize soybean meal hydrolysate to carry out the fermentative production of schizochytrium limacinum as nitrogenous source.Fermentor tank system 100L-1000L-15000L three cascade supervention ferment.

Glucose 70g/L

Dregs of beans 40g/L

Sodium Glutamate 20g/L

Seawater: distilled water (V: V)=1: 1

Initial pH 5.5-6

After the fermentation ends, the result of 15000L fermentor tank is:

The result of table 6 15000L fermentor tank

Biomass (g/L*d)	??8.52±0.65
		DHA content (%, dry bacterial powder weight)	??18.55％

After the 15000L fermentation, the output that obtains schizochytrium limacinum is 34.08g/L, and DHA content is 6.33g/L.

Table 7 15000L fermentation obtains the composition analysis result of schizochytrium limacinum dry powder

Composition	Content (%, dry bacterial powder weight)
		Protein	??18.4
Total fat	??50.9
		Moisture	??5.35

The schizochytrium limacinum total lipid content that obtains through this method fermentation is 50.9%, is higher than its protein content far away, has accumulated lipid acid in the cell efficiently.

Table 8 15000L fermentation obtains the amino acid analysis result of schizochytrium limacinum dry powder

Amino acid	Content (%, dry bacterial powder weight)
		Aspartic acid	??1.14
Threonine	??0.44
		Serine	??0.78
L-glutamic acid	??0.97
		Glycine	??0.70
L-Ala	??1.03
		Xie Ansuan	??1.85
Isoleucine	??0.46
		Leucine	??0.77
Tyrosine	??0.35
		Phenylalanine	??0.54
Methionin	??0.77
		Histidine	??0.39
Arginine	??0.63
		Proline(Pro)	??0.77
Summation	??11.59

Table 8 has illustrated the amino acid composition of schizochytrium limacinum, contains the necessary aminoacid component of various human bodies in the schizochytrium limacinum.

Table 9 15000L fermentation obtains the fatty acid analysis result of schizochytrium limacinum dry powder

Lipid acid	Content (%, total fatty acids)
		??C12:0	??0.18±0.04
??C14:0	??8.45±0.43
		??C15:0	??1.02±0.32
??C16:0	??36.97±0.51
		??C17:0	??0.45±0.09
??C18:0	??1.63±0.36
		??C18:2?n-6	??0.24±0.07
??C18:3?n-3	??0.49±0.06
		??C18:3?n-6	??0.37±0.12
??C20:0	??0.34±0.05
		??C21:0	??0.33±0.07
??C20:3?n-6	??0.25±0.10
		??C20:4?n-6	??0.47±0.06
??C22:0	??0.18±0.03
		??C20:5?n-3	??0.56±0.16
??C22:5?n-6	??7.81±0.34
		??C22:6?n-3	??38.54±0.51
??Saturated	??49.55
		??Unsaturated	??48.73

Table 9 has listed that the one-tenth of various lipid acid is grouped in the schizochytrium limacinum, and unsaturated fatty acids has accounted for about half of total fatty acids in the cell, and particularly DHA content has accounted for the 38-39% of total fatty acids.

Embodiment 7

Different dregs of beans are made hydrolyzed solution and are done the comparison that nitrogenous source is cultivated schizochytrium limacinum to the influence of schizochytrium limacinum growth and lipid acid composition and with yeast extract

Soya-bean oil production at present has squeezing and leaches two kinds of technologies.Because the technology difference, some difference of the composition of dregs of beans, the schizochytrium limacinum component of producing as major nitrogen source with it may there are differences.Under the same conditions, respectively the dregs of beans of producing with different process (H, L P) make the nitrogenous source of hydrolyzed solution as the schizochytrium limacinum fermentative production, finish back detection of biological amount and lipid acid and form and compare.Table 10-13.

Fermentation condition: add the 600L substratum in the 1000L fermentor tank, the inoculum size by 10% inserts seed liquor.Rotating speed is 200r/min, and air flow 1.2VVM, fermented 4 days by 25 ℃.

Leach dregs of beans high temperature modification (writing a Chinese character in simplified form H), the perfume (or spice) grain and oil company limited of speeding in Boxing County, Shandong Province provides.

Leach dregs of beans low temperature modification (writing a Chinese character in simplified form L), the perfume (or spice) grain and oil company limited of speeding in Boxing County, Shandong Province provides.

Squeezing dregs of beans high temperature modification (writing a Chinese character in simplified form P), source, Ji, Wuqiang County, Hebei province grain and oil company limited provides.

The biochemical composition of three kinds of dregs of beans of table 10

Table 10 has provided the biochemical composition of the dregs of beans of three kinds of various processes acquisitions, and Protein content all accounts for more than 40% of gross weight in three kinds of dregs of beans, is the ideal protein source.

The amino acid composition analysis of three kinds of dregs of beans of table 11 and yeast extract

Table 11 has compared the amino acid of dregs of beans and yeast extract and has formed difference, and amino acid whose content will be less than yeast extract in the dregs of beans.

The different dregs of beans of table 12 are made the influence of hydrolyzed solution to schizochytrium limacinum growth and DHA content

Experiment	The nitrogenous source type	Biomass (g/L*d)	DHA content (% dry weight)
				??1	??YEAST??EXTRACT	??6.23±0.57	??14.3
??2	??H	??9.37±0.63	??21.6
				??3	??P	??7.96±0.49	??18.2
??4	??L	??6.02±0.65	??13.5

Make the influence of hydrolyzed solution and yeast extract by more different dregs of beans to schizochytrium limacinum growth and DHA content, as can be seen: when leaching dregs of beans high temperature modification (writing a Chinese character in simplified form H) and making behind the hydrolyzed solution nitrogenous source as the schizochytrium limacinum fermentative production, the biomass of schizochytrium limacinum can reach 37.5g/L, DHA content can reach 21.6% of dry weight, and 8.1g/L.The output and the DHA content that are higher than schizochytrium limacinum when using yeast extract as nitrogenous source.

The different dregs of beans of table 13 are made the influence that hydrolyzed solution is formed schizochytrium limacinum lipid acid

By table 13 as can be seen: in the schizochytrium limacinum of soybean meal hydrolysate as nitrogen source fermentation production acquisition, the content of unsaturated fatty acids and the content of DHA all will be higher than the content of these indexs when doing nitrogenous source with yeast extract, illustrate with soybean meal hydrolysate and help schizochytrium limacinum accumulate unsaturated fatty acids and D HA as nitrogenous source, help reaching the production purpose, increase economic efficiency.

The above only is preferred embodiment of the present invention, is not to be the restriction of the present invention being made other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not break away to any simple modification, equivalent variations and remodeling that above embodiment did, still belongs to the protection domain of technical solution of the present invention according to technical spirit of the present invention.

Claims (8)

1. the fermentation manufacturing technique of a schizochytrium limacinum is characterized in that with dregs of beans comprising the steps: as the major nitrogen source fermentative production

1) dregs of beans hydrolysis

Adding mass ratio in hydrolytic decomposition pot is 1: the dregs of beans of 5-10 and water, add concentrated hydrochloric acid regulator solution pH to 1.0-3.0, and be heated to 100 ℃-120 ℃, pressure rises to 0.04-0.1MPa, soaking time 10～40min, pH to 5.0-7.0 is regulated with alkali in the cooling back; In hydrolytic decomposition pot, add the 5-10 seawater doubly of dregs of beans quality again, promptly get dregs of beans hydrolysis material;

2) press filtration

The said hydrolyzed material is squeezed into plate-and-frame filter press, carry out solid-liquid separation, the effective filtration area of plate-and-frame filter press is 80-100m ², adopt 300-350 order filter cloth, filtration temperature 60-80 ℃, filter pressure 0.10-0.30Mpa; Gained filtrate is soybean meal hydrolysate, collects all filtrates and is stored in the storage tank, is used for the fermentative production of next step schizochytrium limacinum;

3) preparation of fermention medium

As follows with dregs of beans: glucose concn 60-90g/L, dregs of beans concentration 20-80g/L, concentration of sodium glutamate 10-50g/L, the volume ratio 1 of seawater and distilled water: 1-2 as the fermentative production prescription of major nitrogen source fermentation schizochytrium limacinum;

4) setting of fermentor tank system

Staged fermentor tank system, in each grade fermentor tank system, schizochytrium limacinum seed inoculum size is 5%-10%; Leavening temperature is 24-26 ℃, and pH is 5.5-7.0, stirs and blowing air fermentation time 3-4 days;

5) collection of schizochytrium limacinum and drying

After the fermentation ends, the schizochytrium limacinum fermented liquid separates with whizzer, separates back material solid content at 5%-15%; Material is squeezed into spray-drying tower, and the steam output of spray-drying tower is 200-300kg/h, and inlet temperature is 175-190 ℃, and temperature out is 70-90 ℃; The dry powder that spray-dried back obtains is schizochytrium limacinum dry powder, and lucifuge, vacuum cold are preserved fast.

2. fermentation manufacturing technique according to claim 1 is characterized in that: preferred Heating temperature is 105～115 ℃ in described dregs of beans hydrolysing step, soaking time 15～30min.

3. fermentation manufacturing technique according to claim 1 and 2 is characterized in that: carry out eardrum and operate the filtrate of removing in the filter residue after the press filtration step is finished, working pressure is 0.30-0.45Mpa.

4. fermentation manufacturing technique according to claim 3 is characterized in that: the screening formulation of described fermention medium: glucose concn is 70-80g/L, and dregs of beans concentration is 30-60g/L, and concentration of sodium glutamate is 10-40g/L; Preferred 1: 1 of the volume ratio of seawater and distilled water.

5. fermentation manufacturing technique according to claim 1 is characterized in that: described staged fermentor tank system is a 100L-1000L-15000L three grade fermemtation tank body system.

6. fermentation manufacturing technique according to claim 1 or 5 is characterized in that: mend the sugar operation when glucose concn in the fermented liquid during for 20-30g/L, mend at every turn that glucose concn is between the 45-55g/L in the sugared secondary fermentation liquid.

7. fermentation manufacturing technique according to claim 1 or 5, it is characterized in that: mixing speed is 100-300rpm in the described fermentor tank system.

8. fermentation manufacturing technique according to claim 1 or 5, it is characterized in that: ventilation is than being 0.5-2.0Lmin in the described fermentor tank system ^-1L ^-1

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