CN104046661A - Method for preparing biological culture medium obtained by converting rapeseed meal via Neuropara and application of method - Google Patents
Method for preparing biological culture medium obtained by converting rapeseed meal via Neuropara and application of method Download PDFInfo
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Abstract
The invention relates to a method for degrading meal by a fungus and an application of the method. A method for preparing a biological culture medium obtained by converting rapeseed meal via Neuropara is characterized by comprising the following steps: 1) air-drying the residual rapeseed meal left after oil extraction and sterilizing; 2) inoculating Neurospora crassa; 3) preparing into a spore suspension; 4) culturing according to the ratio of the spore suspension to the sterilized rapeseed meal being 1000ml: 500g; 5) then adding water and carrying out hydrolysis; and 6) fermenting hydrolysate, filtering, sterilizing the supernatant, carrying out centrifugation treatment to prepare hydrolysate; and adding water and molasses into the hydrolysate to obtain a biological culture medium, wherein the addition of water is 1-14 times the volume of the prepared hydrolysate and the addition of molasses is 0%-50% of the volume of hydrolyzate. The method is simple in preparation and environment-friendly and the production cost can be significantly reduced. The thallus biomass of the biological culture medium disclosed by the invention is higher than that of the conventional basic culture medium under the same conditions, and the content of grease in the thallus is significantly improved.
Description
Technical field
The present invention relates to a kind of method and application thereof that utilizes fungus degrading grouts (obtaining degradation product, i.e. biological medium).
Background technology
Dregs of rapeseed cake is the residuum after Semen Brassicae campestris oil expression, and the annual dregs of rapeseed cake output of China is about 7,000,000 tons.In dregs of rapeseed cake, the ratio of total protein accounts for 35-45% (quality), and is full price albumen, has hardly limiting amino acid.Rapeseed cake can provide more calcium, iron, violent, phosphorus, selenium and magnesium; Also contain the VITAMIN such as more choline, vitamin H, nicotinic acid, VITMAIN B1 and B2.Both at home and abroad, rapeseed cake is the most general mode of utilizing as the feedstuff protein source of domestic animal, accounts for the 5-10% of rapeseed cake output at present.Often fertilizing the soil it in rural area as organic fertilizer, causes like this significant wastage of this natural protein resource.Dregs of rapeseed cake is a kind of good protein resource, but causes this good protein resource not to be fully used owing to wherein existing some objectionable constituent and antinutritional factor.Both at home and abroad investigator takes several different methods to remove objectionable impurities in grouts to improve its utilization ratio, comprises that physical method, chemical process and biological method carry out detoxification treatment to grouts.Microorganism reduce toxicity method has the advantages such as cost is little, simple to operate, nutritive ingredient loss is little with respect to additive method, and research both at home and abroad is at present more is to adopt the bacterium reduce toxicities such as Geotrichum, aspergillus tubigensis, Rhizopus oligosporus, the main Fodder making of tunning.
Microbial oil is by certain micro-organisms, as mould, yeast, algae etc. are converted into carbohydrate fatty acid triglycercide under certain condition and are stored in thalline, generally can accumulate the microbe species that accounts for more than 20% grease of dry cell weight and be called grease microorganism.The lipid acid of most of microbial oil forms similar to common Vegetable oil lipoprotein, if yeast, mould are mainly palmitinic acid, Zoomeric acid, stearic acid, oleic acid; Minority microorganism, especially marine microalgae, as Crypthecodinium cohnii grease contains rare polyunsaturated fatty acid, as DHA etc.Utilize microorganisms producing grease to there is the restriction that is not subject to season, weather condition, and there is the advantages such as raw materials for production are extensive, with short production cycle.
Polyunsaturated fatty acid (Polyunsaturased fatty acids, PUFAs), as docosahexenoic acid (Docosahexaenoic acid, DHA) etc. be the main component of human central nervous system membranes fat structure, to human body particularly infant's brain and neural growth be absolutely necessary, aspect medical science and nutritive health-care, there is important effect.The efficiency of the synthetic DHA of human body is extremely low, and DHA is to remaining healthy very important.Such lipid acid is mainly derived from fish oil in the market, find lasting, that stability and safety substitutes source is extremely important.Although some micro-algaes that utilize carbonic acid gas to carry out autophyting growth can be synthesized the PUFAs of high level, this class micro algae growth is slow, and culture cycle is long, in large-scale production, is restricted; Minority heterotrophic microalgae can carry out high-density culture by fermentation mode.Crypthecodinium cohnii (Crypthecodinium cohnii) is a kind of heterotrophism marine microalgae, and in its lipid acid, DHA content is up to 30%～50%, and other content of polyunsaturated fatty acid seldom, is considered to the desirable resource of industrial production DHA.The substratum of producing at present Crypthecodinium cohnii is nearly all to utilize glucose etc. as carbon source, and yeast powder etc., as nitrogenous source, are cultivated cost height and limited development.The present invention adopts cheap dish dregs of rice degraded hydrolyzate as nitrogenous source, utilizes the by product molasses of the sucrose course of processing as carbon source simultaneously, Crypthecodinium cohnii is carried out to high density fermentation and produce DHA.Molasses, except the soluble sugar that contains high level, also contain other nutritive ingredients such as vitamin H, VITAMIN, amino acid and mineral substance, are carbon source raw materials more cheap in fermentation industry.This new bio substratum is compared with conventional medium (containing glucose, yeast extract and sea salt), and Crypthecodinium cohnii biomass obviously improves, and DHA content does not reduce.This substratum is the excellent culture medium that a kind of low cost, high density fermentation Crypthecodinium cohnii are produced DHA, has no at present report.
Summary of the invention
The object of this invention is to provide preparation method and the application thereof of the biological medium of the mould conversion dregs of rapeseed cake acquisition of a kind of arteries and veins spore, simple and easy, environmental protection prepared by the method, can significantly reduce cost of manufacture.
For realizing above object, the technical solution adopted in the present invention is: the preparation method of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore obtains, is characterized in that it comprises the steps:
1). remaining dregs of rapeseed cake after oil expression is dried, pulverizer quantitatively takes dregs of rapeseed cake 500g after pulverizing, and by the dregs of rapeseed cake after pulverizing and the mass ratio of water, is 1:1.5-2, in the dregs of rapeseed cake after pulverizing, add water sterilizing, obtained the rapeseed cake after sterilizing;
2). inoculation neurospora crassa (Neurospora crassa):
Neurosporaintemedia (Neurospora crassa) is seeded on the flat board of wheat bran solid medium, in 28 ℃ of incubators, cultivates 3-5 days; Described wheat bran solid culture based component is: the volume ratio=5 ﹕ 1.5:100 of bran skin ﹕ agar agar powder ﹕ water;
3). the flat board of cultured Neurosporaintemedia adds the polysorbas20 aqueous solution (mass volume ratio that 5ml, concentration are 0.1%, tween 20 ﹕ water=0.1g ﹕ 100ml), and add sterilizing granulated glass sphere (as 10), rock gently a moment, mould spores on flat board is eluted, the solution that contains mould spores is drawn out from flat board, and (spore suspension content is: 10 to add 1000ml sterilized water to make spore suspension
5～10
7individual cell/mL), standby;
4) by the proportioning of the rapeseed cake after spore suspension (mould spores suspension) and sterilizing, be 1000ml:500g, spore suspension is poured in the rapeseed cake after sterilizing, in 28 ℃ of incubators, cultivate 3-5 days;
5) then add water to be hydrolyzed, 40～65 ℃ of Water Under solutions 2～6 days, the add-on of water was 2 times of rapeseed cake quality after sterilizing, obtains fermentation hydrolysis liquid;
6) fermentation hydrolysis liquid, through filtering, is got supernatant liquor sterilizing, centrifugal treating, makes hydrolyzed solution;
Said hydrolyzed liquid adds water and molasses, obtains a kind of biological medium; The add-on of water is 1～14 times of the hydrolyzed solution volume made, and the add-on of molasses is 0%～50% of hydrolyzed solution volume.
Described biological medium also comprises the essential raw material of other microorganism growth, and other microorganism growth must raw material add-on be 0%～40% of hydrolyzed solution quality.Above-mentioned other microorganism growth must raw material be: any one in seawater extract, yeast extract, peptone, glucose, glycerine, wood sugar, methyl alcohol, ethanol, semi-lactosi, raffinose etc. or two or more by the mixing of any proportioning.
In described spore suspension, mould is neurospora crassa (Neurospora crassa).With the aspergillus oryzae (A.Oryzoe) that has a report in contrast.
Said hydrolyzed liquid and molasses are used simultaneously, can replace the conventional fermentation raw materials such as glucose and yeast powder for cultivating micro-algae production high value Omega-3 long chain polyunsaturated fatty acids DHA.Biological medium optimization of C/C composites of the present invention is: the molasses of 7wt%～50wt% hydrolyzed solution, substratum final concentration 0wt%～10wt% (best is 2wt%～6wt%), 25g/L seawater extract, water are surplus.Inoculation Crypthecodinium cohnii makes final concentration reach 1.2 * 10
5individual/ml.22 ℃, 180rpm shaking table are cultivated 5 days.
For cultivate Molds and yeasts (rhizopus stolonifer (Rhizopus stolonifer), Trichosporon fermentans (Trichosporon fermentans), rhodotorula glutinis (Rhodotorula glutinis), tangerine woods saccharomyces oleaginosus (Lipomyces kononenkoae), this reaches saccharomyces oleaginosus (Lipomyces starkeyi), mortierella (Mortierella sp.) }, adopt following 2 kinds of biological mediums: 1) described biological medium is: hydrolyzed solution 7wt%, water is surplus.2) described biological medium is: hydrolyzed solution 7wt%, and molasses 6wt%, water is surplus.Control medium (YPD) composition is: 1wt% yeast extract, 2wt% protein extract, 2wt% glucose, water 1L.
In order to cultivate Crypthecodinium cohnii (Crypthecodinium cohnii), with following 3 kinds of biological mediums: 1) described biological medium is: hydrolyzed solution 7wt%, seawater extract 25g/L, water is surplus.2) described biological medium is: hydrolyzed solution 7wt%, and molasses 6wt%, seawater extract 25g/L, water is surplus.3) described biological medium is: hydrolyzed solution 7wt%, and yeast powder 0.4wt%～4wt%, seawater extract 25g/L, water is surplus.Control medium is: yeast extract: 2g/L, glucose: 9g/L, seawater extract: 25g/L, water 1L.
The application of above-mentioned biological medium, is characterized in that it can be used for as substratum the multiple grease microorganism that ferments.
Above-mentioned multiple grease microorganism comprises rhizopus stolonifer (Rhizopus stolonifer), Trichosporon fermentans (Trichosporon fermentans), rhodotorula glutinis (Rhodotorula glutinis), tangerine woods saccharomyces oleaginosus (Lipomyces kononenkoae), this reaches saccharomyces oleaginosus (Lipomyces starkeyi), Crypthecodinium cohnii (Crypthecodinium cohnii) or mortierella (Mortierella sp.).
The biomass (increasing by 1～2 times) that thalline biomass of the present invention obtains under the same conditions higher than conventional minimum medium, and thalline fat content also obviously improves (higher than control medium 13.54%～29.92%).For cultivating marine microalgae---the Crypthecodinium cohnii of high Lipid-producing and polyunsaturated fatty acid, 7%～50% hydrolyzed solution that the Neurospora crassa of take degraded dregs of rapeseed cake obtains is nitrogenous source, the by product molasses that add the sucrose course of processing are carbon source (final concentration is 0wt%～10wt%), cultivate the relative content that 5 days its DHA account for total fatty acids to be: 5.0～18.6%.Method described in the invention is a kind of low consumption energy, simple processing grouts method and the method for utilizing the polyunsaturated fatty acid of its hydrolyzed solution production high added value; Except being conducive to the resources effective utilizations such as agricultural machining by product and reducing the advantages such as environmental pollution, the all right direct fermentation culture based raw material as bacterium, fungi and micro-algae, on microbial oil is produced, application can replace the carbon nitrogen source raw material that cost is relatively high, reduces costs; And can be used as other microorganism fermentation culture and prepare business-like PUFAs.
The present invention has the following advantages
1 Neurosporaintemedia reported for nobody before the fermentative degradation of grouts, and result proof of the present invention is with this bacterium inoculation fermentation, with conventional aspergillus comparison, have that fungal hyphae growth is more luxuriant, degraded more thoroughly and the thalline nutritive ingredient of acquisition more.
2 hydrolyzed solutions that obtain can be directly used in cultivates multiple grease microorganism, comprise rhizopus stolonifer (Rhizopus stolonifer), Trichosporon fermentans (Trichosporon fermentans), rhodotorula glutinis (Rhodotorula glutinis), tangerine woods saccharomyces oleaginosus (Lipomyces kononenkoae), this reaches saccharomyces oleaginosus (Lipomyces starkeyi), Crypthecodinium cohnii (Crypthecodinium cohnii) etc., and can further attempt the cultivation raw material for the microorganism fermentation of other production purposes.
3., while using together with the scrap feed material molasses of said hydrolyzed liquid and sugarcane industry, the biomass of cultivating multiple grease microorganism surpasses the conventional minimum medium of contrast use.Its raw materials cost significantly reduces.
4. said hydrolyzed liquid and molasses are used simultaneously, can replace normal fermentation raw material that the costs such as glucose, yeast extract and peptone are relatively high for the Omega-3 long chain polyunsaturated fatty acids DHA of the micro-algae production of high-density culture high value.Hydrolyzed solution prepared by the Neurosporaintemedia of the take degraded dish dregs of rice is main body substratum, in the substratum that adds different concns molasses, cultivates Crypthecodinium cohnii, records the relative content that DHA accounts for total fatty acids to be: 5.0～18.6%; Crypthecodinium cohnii DHA output is 1.34～9.69mg/L, apparently higher than not adding the DHA output obtaining in the hydrolyzed solution substratum of molasses: 0.34-0.43mg/L.
5. the method is prepared simple and easy, environmental protection, and significantly reduces cost of manufacture.
6. the biomass (increasing by 1～2 times) that thalline biomass of the present invention obtains under the same conditions higher than conventional minimum medium, and thalline fat content also obviously improves (higher than control medium 13.54%～29.92%).
Accompanying drawing explanation
Fig. 1 is the growth figure of mould on wheat bran.A is aspergillus oryzae, and b is neurospora crassa.
Fig. 2 is the hydrolyzed solution figure that different mould degraded grouts obtain.A is that aspergillus oryzae transforms the biological medium (hydrolyzed solution) that dregs of rapeseed cake obtains, and b is that neurospora crassa transforms the biological medium (hydrolyzed solution) that dregs of rapeseed cake obtains.
Embodiment
Embodiment 1
The neurospora crassa (Neurospora crassa) of take is grouts degradation bacteria, and cultivates marine microalgae---Crypthecodinium cohnii with the hydrolyzed solution of its degraded, operates successively according to the following step:
A. the preparation of biological medium (hydrolyzed solution)
After after 1.Jiang oil pressing factory oil expression, remaining dregs of rapeseed cake pulverizer is pulverized, the rapeseed cake taking after 500g pulverizes packs in disposable freshness protection package, adds 1000 ml waters to mix thoroughly, pricks suitable for readingly, and 105 ℃, sterilizing 30 minutes are standby, have obtained the rapeseed cake after sterilizing.
2. Neurosporaintemedia (Neurospora crassa) is seeded on the flat board of wheat bran solid medium, in 28 ℃ of incubators, cultivates 3-5 days; Described wheat bran solid culture based component is: the volume ratio=5 ﹕ 1.5:100 of bran skin ﹕ agar agar powder ﹕ water.
3. the flat board of cultured Neurosporaintemedia adds the polysorbas20 aqueous solution (mass volume ratio that 5ml, concentration are 0.1%, tween 20 ﹕ water=0.1g ﹕ 100ml), and add sterilizing granulated glass sphere (as 10), rock gently a moment, mould spores on flat board is eluted, the solution that contains mould spores is drawn out from flat board, and (spore suspension content is: 10 to add 1000ml sterilized water to make spore suspension
5～10
7individual cell/mL), standby;
4., after the alcohol flushing sterilization of the basin (or other containers) of feeding with 75wt%, the coarse colza meal after sterilizing is encased in charging basin; Then 1000ml spore suspension is poured in the rapeseed cake after 500g sterilizing, fully mixes, cover air-permeable envelope, in 28 ℃ of incubators, cultivate 3-5 days; Middle stirring once.
5. add 1 premium on currency (being advisable to cover dregs of rapeseed cake fermentation material) in charging basin (fermentation basin), put 50 ℃ of condition insulation hydrolysis 48h, its certain interval of time (6h) shakes and makes a movement, and obtains fermentation hydrolysis liquid.
6. fermentation hydrolysis liquid (the rapeseed cake powder being hydrolyzed) is used multilayer filtered through gauze, removes residue, collects filtrate.Filtrate is killed mould spores by sterilizing, and centrifugal removal impurity is collected supernatant liquor (being called hydrolyzed solution).
B. cultivate grease microorganism-Crypthecodinium cohnii
7. in order to cultivate Crypthecodinium cohnii (Crypthecodinium cohnii), with following 3 kinds of biological mediums: 1) described biological medium is: hydrolyzed solution 7wt%, seawater extract 25g/L, water is surplus.2) described biological medium is: hydrolyzed solution 7wt%, and molasses 6wt%, seawater extract 25g/L, water is surplus.3) described biological medium is: hydrolyzed solution 7wt%, and yeast powder 0.4wt%～4wt% (to final concentration 0.4%, 1%, 1.8%, 2.8% and 4wt%), seawater extract 25g/L, water is surplus.Control medium is: yeast extract: 2g/L, glucose: 9g/L, seawater extract: 25g/L, water 1L.
Cultivate respectively Crypthecodinium cohnii; Inoculation Crypthecodinium cohnii makes final concentration reach 1.2 * 10
5individual/ml.22 ℃, 180rpm shaking table are cultivated 5 days.
C. the extraction of microorganism collection and grease
Centrifugal collection frustule, pulverizes after going supernatant final vacuum lyophilization system dry; Adopt chloroform/methanol method to extract grease, gas chromatography determination lipid acid, in Table 4.
As shown in Figure 1, a is aspergillus oryzae in the growth of mould on wheat bran, and b is neurospora crassa.
As shown in Figure 2, a is that aspergillus oryzae transforms the biological medium (hydrolyzed solution) that dregs of rapeseed cake obtains to the hydrolyzed solution that different mould degraded grouts obtain, and b is that neurospora crassa transforms the biological medium (hydrolyzed solution) that dregs of rapeseed cake obtains.
Hydrolyzed solution Major Components, in Table 1.
Table 1. hydrolyzed solution Major Components (embodiment 1):
Note: the mensuration of hydrolyzed solution total reducing sugar (anthrone colorimetry), the mensuration of hydrolyzed solution soluble sugar (phynol method), the mensuration of hydrolyzed solution total nitrogen (Kjeldahl determination).
Table 2. hydrolyzed solution adds the mensuration (g dry cell weight/L) [(embodiment 1) that yeast powder is cultivated Crypthecodinium cohnii biomass yield, described biological medium is: hydrolyzed solution 7wt%, yeast powder 0.4wt%～4wt% is (to final concentration 0.4%, 1%, 1.8%, 2.8% and 4wt%), seawater extract 25g/L, water is surplus]
Crypthecodinium cohnii biomass is to be 1.2 * 10 according to the initial inoculum size of Crypthecodinium cohnii
5individual cell/mL calculates.22 ℃, 180rpm shaking table is cultivated 5 days, and 6000rpm centrifugal collecting cell, goes to weigh dry cell weight the data obtained after the lyophilize of supernatant final vacuum.Crypthecodinium cohnii culture medium prescription: the hydrolyzed solution that basic recipe is 7wt%, seawater extract 25g/L, water is surplus, yeast powder 0,0.4wt%, 1wt%, 1.8wt%, 2.8wt%, 4wt% are added respectively in experiment on the basis of basic recipe.And 3 repetitions are set.Gained dry cell weight is finally obtained mean value.
Interpretation of result: from then on showing data statistics can find out, yeast powder adds in identical situation, the biomass that Crypthecodinium cohnii produces in hydrolyzed solutions in the hydrolysis of different fungies there are differences, and adds the biomass that the hydrolyzed solution of the mould generation hydrolysis of more yeast powder clock pulse born of the same parents obtains higher than aspergillus oryzae hydrolyzed solution.In the hydrolyzed solution that the same fungi hydrolysis dish dregs of rice produce, along with the increase of yeast powder addition, the yield of biomass of Crypthecodinium cohnii is also to increase gradually.
Table 3. hydrolyzed solution interpolation molasses cultivation Crypthecodinium cohnii biomass [(embodiment 1), described biological medium is: hydrolyzed solution 7wt%, molasses 0-9wt%, seawater extract 25g/L, water is surplus]
Note: the hydrolyzed solution that experimental design substratum is 7%, 25g/L seawater extract, adds respectively that concentration of molasses is followed successively by 0%, 1wt%, 3wt%, 6wt%, 9wt%.
Table 4. hydrolyzed solution adds yeast powder and cultivates in Crypthecodinium cohnii cell DHA and account for total fatty acid content (%) [(embodiment 1) described biological medium is: hydrolyzed solution 7wt%, yeast powder 0.4wt%～4wt% is (to final concentration 0.4%, 1%, 1.8%, 2.8% and 4wt%), seawater extract 25g/L, water is surplus]
Hydrolyzed solution prepared by the Neurosporaintemedia of the take degraded dish dregs of rice is main body substratum, in adding yeast powder and adding respectively the substratum of 0.4,1.8,2.8,4g/L yeast powder, does not cultivate Crypthecodinium cohnii, records the relative content that DHA accounts for total fatty acids to be: 7.5～18.5%.
Table 5. hydrolyzed solution cultivation Crypthecodinium cohnii production DHA output and content [(embodiment 1), described biological medium is: hydrolyzed solution 7wt%, molasses 0-9wt%, seawater extract 25g/L, water is surplus]
Hydrolyzed solution prepared by the Neurosporaintemedia of the take degraded dish dregs of rice is main body substratum, in the substratum that adds different concns molasses, cultivates Crypthecodinium cohnii, records the relative content that DHA accounts for total fatty acids to be: 22.5～33.7%; Crypthecodinium cohnii DHA output is 5.63～29.15mg/L, apparently higher than not adding the DHA output obtaining in the hydrolyzed solution substratum of molasses: 1.96mg/L; Adding the relative content that Crypthecodinium cohnii DHA in the substratum of different concns molasses accounts for dry cell weight is: 5.4～9.8mgDHA/g stem cell is 3.07mgDHA/g stem cell and do not add the relative content that DHA in the hydrolyzed solution substratum of molasses accounts for dry cell weight.
Embodiment 2
Take Neurosporaintemedia as grouts degradation bacteria, with its degraded hydrolyzed solution, cultivate marine microalgae---Crypthecodinium cohnii, according to the following step, operate successively:
A. the preparation of biological medium (hydrolyzed solution)
After remaining dregs of rapeseed cake pulverizer is pulverized after 1.Jiang oil pressing factory oil expression, take 500g rapeseed cake and pack in disposable freshness protection package, add 1000 ml waters to mix thoroughly, prick suitable for readingly, 105 ℃, sterilizing 30 minutes are standby.
2. Neurosporaintemedia (Neurospora crassa) is seeded on the flat board of wheat bran solid medium, in 28 ℃ of incubators, cultivates 3-5 days; Described wheat bran solid culture based component is: the volume ratio=5 ﹕ 1.5:100 of bran skin ﹕ agar agar powder ﹕ water.
3. the flat board of cultured Neurosporaintemedia adds the polysorbas20 aqueous solution (mass volume ratio that 5ml, concentration are 0.1%, tween 20 ﹕ water=0.1g ﹕ 100ml), and add sterilizing granulated glass sphere (as 10), rock gently a moment, mould spores on flat board is eluted, the solution that contains mould spores is drawn out from flat board, and (spore suspension content is: 10 to add 1000ml sterilized water to make spore suspension
5～10
7individual cell/mL), standby;
4., after the alcohol flushing sterilization of the basin (or other containers) of feeding with 75wt%, the rapeseed cake after 500g sterilizing is encased in charging basin.Then 1000ml spore suspension is poured in rapeseed cake, fully mixes, cover air-permeable envelope, in 28 ℃ of incubators, cultivate 3-5 days, middle stirring once.
5. add 1 premium on currency (being advisable to cover dregs of rapeseed cake fermentation material) in charging basin (fermentation basin), put 50 ℃ of condition insulation hydrolysis 48h, its certain interval of time (6h) shakes and makes a movement, and obtains fermentation hydrolysis liquid.
6. fermentation hydrolysis liquid multilayer filtered through gauze, removes residue, collects filtrate.Filtrate is killed mould spores by sterilizing, centrifugal removal impurity, collection supernatant liquor (being called fermentation hydrolysis liquid).
B. cultivate grease microorganism-yeast
7. adopt following 2 kinds of biological mediums: 1) described biological medium is: hydrolyzed solution 7wt%, water is surplus.2) described biological medium is: hydrolyzed solution 7wt%, and molasses 6wt%, water is surplus.
Cultivate respectively saccharomyces oleaginosus (comprise Trichosporon fermentans, rhodotorula glutinis, tangerine woods saccharomyces oleaginosus, this reach saccharomyces oleaginosus), inoculation yeast makes final concentration reach 1.2 * 10
5individual/ml.28 ℃, 180rpm shaking table are cultivated 5 days.
C. the extraction of microorganism collection and grease
Centrifugal collecting cell, pulverizes after going supernatant final vacuum lyophilization system dry; Chloroform/methanol method is extracted grease, gas chromatography determination lipid acid.
Table 6. hydrolyzed solution is cultivated the yield of biomass (g/L) (embodiment 2, embodiment 3) of saccharomyces oleaginosus and fungi
Note: chosen four kinds of typical saccharomyces oleaginosuses, only added respectively sterilizing after the hydrolyzed solution of 7wt%, inoculated respectively four primary yeasts, make cell OD600 at 0.1 (YPD), 28 ℃, rotating speed is that 180rpm shaking table was cultivated after 3 days, after centrifugal collecting cell vacuum lyophilization, weighs dry cell weight.
The total fatty acid content of table 7. hydrolyzed solution culturing yeast (mg/g stem cell is heavy) (embodiment 2)
Embodiment 3
A. the preparation of biological medium (hydrolyzed solution)
Take Neurosporaintemedia as grouts degradation bacteria, with its degraded hydrolyzed solution, cultivate marine microalgae---Crypthecodinium cohnii, according to the following step, operate successively:
After remaining dregs of rapeseed cake pulverizer is pulverized after 1.Jiang oil pressing factory oil expression, take 500g rapeseed cake and pack in disposable freshness protection package, add 750 ml waters to mix thoroughly, prick suitable for readingly, 105 ℃, sterilizing 30 minutes are standby.
2. Neurosporaintemedia (Neurospora crassa) is seeded on the flat board of wheat bran solid medium, in 28 ℃ of incubators, cultivates 3-5 days; Described wheat bran solid culture based component is: the volume ratio=5 ﹕ 1.5:100 of bran skin ﹕ agar agar powder ﹕ water.
3. the flat board of cultured Neurosporaintemedia adds the polysorbas20 aqueous solution (mass volume ratio that 5ml, concentration are 0.1%, tween 20 ﹕ water=0.1g ﹕ 100ml), and add sterilizing granulated glass sphere (as 10), rock gently a moment, mould spores on flat board is eluted, the solution that contains mould spores is drawn out from flat board, and (spore suspension content is: 10 to add 1000ml sterilized water to make spore suspension
5～10
7individual cell/mL), standby;
4., after the alcohol flushing sterilization of the basin (or other containers) of feeding with 75wt%, the coarse colza meal after sterilizing is encased in charging basin.Then 1000ml spore suspension is poured in the rapeseed cake after 500g sterilizing, fully mixes, cover air-permeable envelope, in 28 ℃ of incubators, cultivate 3～5 days, middle stirring once.
5. add 1 premium on currency (being advisable to cover dregs of rapeseed cake fermentation material) in fermentation basin, put 40～65 ℃ of condition insulation hydrolysis 6 days, its certain interval of time (6h) shakes and makes a movement, and obtains fermentation hydrolysis liquid.
6. fermentation hydrolysis liquid (or claim be hydrolyzed rapeseed cake powder) is used multilayer filtered through gauze, removes residue, collection filtrate.Filtrate is killed mould spores by sterilizing, centrifugal removal impurity, collection supernatant liquor (being called hydrolyzed solution).
B. cultivate grease microorganism-mould
7. adopt following 2 kinds of biological mediums: 1) described biological medium is: hydrolyzed solution 7wt%, water is surplus.2) described biological medium is: hydrolyzed solution 7wt%, and molasses 6wt%, water is surplus.
Cultivate respectively mould (comprising mortierella and Rhizopus stolonifer), mould inoculum size is approximately every 100mL substratum, and to add diameter be 2 of the agar blocks that the growth of the punch tool size of 3cm has mould.28 ℃, 180rpm shaking table are cultivated 5 days.
C. the extraction of microorganism collection and grease
Filtered through gauze is collected mycelia, after vacuum freeze-drying system is dry, pulverizes; Chloroform/methanol method is extracted grease, gas chromatography determination lipid acid.
The microorganism explanation the present invention relates to:
For transforming the mould of dregs of rapeseed cake:
Neurospora crassa (Neurospora crassa)
Aspergillus oryzae (Aspergillus oryzae)
(1) take the grease microorganism that grouts hydrolyzed solution is nutrition:
Rhizopus stolonifer (Rhizopus stolonifer)
Trichosporon fermentans (Trichosporon fermentans)
Rhodotorula glutinis (Rhodotorula glutinis)
Tangerine woods saccharomyces oleaginosus (Lipomyces kononenkoae)
This reaches saccharomyces oleaginosus (Lipomyces starkeyi)
Crypthecodinium cohnii (Crypthecodinium cohnii)
Contrast minimum medium composition
Lipid-producing Yeast and mould substratum (YPD) in contrast: 1% yeast extract, 2% protein extract, 2% glucose, water 1L.
Crypthecodinium cohnii is conventional medium (glucose 9g/L, yeast extract 2g/L, sea salt 25g/L, water 1L) for contrast.
Claims (10)
1. the preparation method of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore obtains, is characterized in that it comprises the steps:
1). remaining dregs of rapeseed cake after oil expression is dried, and pulverizer quantitatively takes dregs of rapeseed cake after pulverizing, and by the dregs of rapeseed cake after pulverizing, is 1:1.5-2 with the mass ratio of water, in the dregs of rapeseed cake after pulverizing, adds water sterilizing, has obtained the rapeseed cake after sterilizing;
2). inoculation neurospora crassa (Neurospora crassa):
Neurosporaintemedia (Neurospora crassa) is seeded on the flat board of wheat bran solid medium, in 28 ℃ of incubators, cultivates 3-5 days; Described wheat bran solid culture based component is: the volume ratio=5 ﹕ 1.5:100 of bran skin ﹕ agar agar powder ﹕ water;
3). the flat board of cultured Neurosporaintemedia adds the polysorbas20 aqueous solution that 5ml, concentration are 0.1%, and adds sterilizing granulated glass sphere, rocks, mould spores on flat board is eluted, the solution that contains mould spores is drawn out from flat board, added 1000ml water to make spore suspension, standby;
4) by the proportioning of the rapeseed cake after spore suspension and sterilizing, be 1000ml:500g, spore suspension is poured in the rapeseed cake after sterilizing, in 28 ℃ of incubators, cultivate 3-5 days;
5) then add water to be hydrolyzed, 40～65 ℃ of Water Under solutions 2～6 days, the add-on of water was 2 times of rapeseed cake quality after sterilizing, obtains fermentation hydrolysis liquid;
6) fermentation hydrolysis liquid, through filtering, is got supernatant liquor sterilizing, centrifugal treating, makes hydrolyzed solution;
Said hydrolyzed liquid adds water and molasses, obtains a kind of biological medium; The add-on of water is 1～14 times of the hydrolyzed solution volume made, and the add-on of molasses is 0%～50% of hydrolyzed solution volume.
2. the preparation method of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore according to claim 1 obtains, it is characterized in that: described biological medium also comprises the essential raw material of other microorganism growth, and other microorganism growth must raw material add-on be 0%～40% of hydrolyzed solution quality; Above-mentioned other microorganism growth must raw material be: any one in seawater extract, yeast extract, peptone, glucose, glycerine, wood sugar, methyl alcohol, ethanol, semi-lactosi, raffinose etc. or two or more by the mixing of any proportioning.
3. the preparation method of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore according to claim 1 obtains, is characterized in that: described biological medium is: the molasses of 7wt%～50wt% hydrolyzed solution, 0wt%～10wt%, 25g/L seawater extract, water are surplus.
4. the preparation method of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore according to claim 1 obtains, is characterized in that: described biological medium is: hydrolyzed solution 7wt%, water is surplus.
5. the preparation method of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore according to claim 1 obtains, is characterized in that: described biological medium is: hydrolyzed solution 7wt%, and molasses 6wt%, water is surplus.
6. the preparation method of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore according to claim 1 obtains, is characterized in that: described biological medium is: hydrolyzed solution 7wt%, and seawater extract 25g/L, water is surplus.
7. the preparation method of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore according to claim 1 obtains, is characterized in that: described biological medium is: hydrolyzed solution 7wt%, and molasses 6wt%, seawater extract 25g/L, water is surplus.
8. the preparation method of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore according to claim 1 obtains, is characterized in that: described biological medium is: hydrolyzed solution 7wt%, and yeast powder 0.4wt%～4wt%, seawater extract 25g/L, water is surplus.
9. the application of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore according to claim 1 obtains, is characterized in that it is as the substratum multiple grease microorganism that is used for fermenting.
10. the application of the biological medium that the mould conversion dregs of rapeseed cake of arteries and veins spore according to claim 9 obtains, is characterized in that: above-mentioned multiple grease microorganism comprises rhizopus stolonifer (Rhizopus stolonifer), Trichosporon fermentans (Trichosporon fermentans), rhodotorula glutinis (Rhodotorula glutinis), tangerine woods saccharomyces oleaginosus (Lipomyces kononenkoae), this reaches saccharomyces oleaginosus (Lipomyces starkeyi), Crypthecodinium cohnii (Crypthecodinium cohnii) or mortierella (Mortierella sp.).
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