CN105586371A - Method for producing microbial oil through oil-producing microorganisms adopting jerusalem artichoke as raw materials - Google Patents
Method for producing microbial oil through oil-producing microorganisms adopting jerusalem artichoke as raw materials Download PDFInfo
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Abstract
The invention discloses a method for cultivating oil-producing microorganisms adopting jerusalem artichoke as raw materials. The method comprises the following steps of preparing a seed culture medium, proliferating and cultivating thallus, preparing an oil-producing culture medium, culturing thallus oil in an accumulated mode, extracting oil-producing microorganism oil and the like. Inulin is adopted as the raw materials for preparing the culture medium, the cost is low, and the method is applicable to large-scale industrial application; according to characteristics of a jerusalem artichoke hydrolysate and different processing modes, the content of nutrient elements is adjusted, the culture technology for phosphorus-limiting/sulfur-control combined two-stage culture of oil-producing microorganisms is developed, the thallus proliferation speed and the oil accumulation rate of the oil-producing microorganisms are remarkably improved, and the oil yield is remarkably improved. Due to the application, the biodiesel oil raw material sources are enlarged, the production period can be remarkably shortened, the production cost is lowered, and the production efficiency is improved.
Description
Technical field
The present invention relates to oil-containing microorganism and cultivate and microbial grease production technical field, specifically, relate toTaking inulin as raw material, the regulation and control of limit phosphorus/sulphur cultivate oil-containing microorganism and utilize this quasi-microorganism raw in conjunction with two-phase methodThe method of producing microbial grease.
Background technology
Microbial grease (microbialoil) claim again Unicell Oils and Fats (singlecelloil, SCO), be by yeast,It is carbon that the oleaginous microorganisms such as mould, bacterium and algae utilize carbohydrate, hydrocarbon and common greaseSource, nitrogenous source, be aided with the grease that inorganic salts are produced under certain condition. With respect to animal and plant fat, microorganismThe grease production cycle is short, not limited by season and weather, and raw material sources are wide, substantially the occupying volume money of ploughing outward notSource, is easy to realize large-scale production, is considered to very potential new oil resource. Therefore, microbial oilFat not only may be served as the substitute of edible oil or other functional grease, also may be at regeneratable liquors fuelIn Biodiesel development, play a significant role.
Oleaginous microorganism oil and fat accumulation process is the bioprocess that a height relies on growing environment, especially battalionSupport availability. Early stage research shows, excessive and other nutrient of the carbon source of oleaginous microorganism in culture mediumUnder not enough condition, unnecessary carbon source can be saved as to grease in born of the same parents, these limitative nutrients comprise nitrogen,Phosphorus, zinc, iron and magnesium etc. Up to now, nitrogenous source restriction is the most general hand that inspires microbial accumulation greaseSection, adopts higher C/N recently to realize conventionally. If the C/N when in culture medium is than rising to 133.5 from 83.5Time, the fat content of Mortierella isabellina is increased to 56.0% from original 50.0%; Cunninghamella echinulata(Cunninghamellaechinulata) fat content from 36.0% being increased to 47.0% (PapanikolaouS.,Etal.JournalofAppliedMicrobiology, 2004,97 (4): 867-875.). In addition, also someone attemptsAdopt the modes such as P elements restriction or element sulphur restriction, improve the ability of microbial accumulation grease. For example WuThink of state etc. accumulates oily new method at the red winter spore yeast of research circle, adopts the mode of P elements restriction, regulates and controls micro-lifeThe content of thing grease, result shows, in the time that C/P rises to 9552 from 72 the content of microbial grease from21.2% rises to 62.1%; Adopt element sulphur ways to restrain can obtain similar results, when C/S is from 150Be raised to the content from 20.8% to 55.8% of 18310 o'clock microbial greases. Natural material is often all rich in nitrogen, byThan the difficulty of dephosphorization or desulfurization is larger, cost is higher, therefore adopt P elements restriction and element sulphur in denitrogenationRestriction substitutes the restriction of nitrogen element, the mode of a kind of effective regulating and controlling microbial oil and fat accumulation of can yet be regarded as.
Jerusalem artichoke has another name called Jerusalem artichoke, Jerusalem artichoke, is suitable for being grown in beach ground, salt-soda soil; Output is large, and its stem tuber is dryMaterial per mu yield can reach 1.2 tons, and cauline leaf dry weight more than 1.3 tons, exceedes the biomass per unit area yield water of corn and wheatFlat. In jerusalem artichoke stem tuber, inulin content is very high, can account for 20% left and right of weight in wet base, 80% left and right of dry weight, and chrysanthemumPowder is polyfructosan, is easy to be converted into fermentable fructose, can directly be utilized by microorganism. Therefore, adoptJerusalem artichoke is microorganism fermentation raw material production biodiesel, can not realize and earn ground with grain, meets again the biological bavin of ChinaThe industrial policy of fry dried food ingredients exhibition. But jerusalem artichoke is applied to microbial grease and produces and face that nitrogen content is too high, thallineThe problem such as poor growth, target product yield are not high, is unfavorable for large-scale industrial production.
Summary of the invention
The object of the invention is to, develop a kind of oleaginous microorganism cultural method taking jerusalem artichoke as raw material, the partyMethod can improve thalline oil and fat accumulation speed, realizes the efficient synthetic of grease; Solve Jerusalem artichoke raw material and denitrogenate elementDifficulty, cannot adopt the regulation and control of limit nitrogen to accumulate the problem of grease, further improves grease production intensity and substrate and turnsRate, realizes effective utilization of cultivating raw material, reduces production costs, and improves microbe oil fermentation technology warpThe object of Ji property, realizes large-scale industrial production.
For achieving the above object, the invention provides a kind of oleaginous microorganism taking jerusalem artichoke as raw material and produce micro-lifeThe method of thing grease, comprises the steps:
S1, preparation jerusalem artichoke hydrolyzate: inulin is mixed 1:1～10 in mass ratio with water, adopt 3mol/L hydrochloric acidRegulate pH to 1～4; Under saturated vapor, be hydrolyzed 10～40min, regulate pH to neutral, obtain jerusalem artichoke hydrolyzate;
S2, prepare seed culture medium: get 1/2nd of jerusalem artichoke hydrolyzate that step S1 makes, add nitrogenous sourceTo 1～20g/L, add potassium dihydrogen phosphate to 1.0g/L, add MgSO4·7H2O to 0.5g/L, sterilizing,To the seed culture medium of microorganism;
S3, growing microorganism are cultivated: with 10% (V/V) inoculum concentration access oleaginous microorganism seed liquor(OD600nm=1～10), in the seed culture medium making in step S1, at 20～40 DEG C, rotating speed beUnder 40～800rpm condition, 12～96h is cultivated in ventilation, and thalline is collected in centrifugation, is the product after propagationOil microbial cells;
Above-mentioned growing microorganism incubation is carried out in shaking table, and the rotating speed of shaking table is 40～800rpm;
S4, prepare produce oil culture medium: the jerusalem artichoke hydrolyzate remaining 1/2nd that step S1 is made takes offPhosphorus processing, obtains the dephosphorization produce oil culture medium that carbon and P elements molar ratio (C/P) are greater than 70;
Or the jerusalem artichoke hydrolyzate remaining 1/2nd that step S1 is made carries out desulfurization processing, obtain carbonThe sulphur removal produce oil culture medium that is greater than 150 with element sulphur molar ratio (C/S);
S5, thalline oil and fat accumulation are cultivated: the oleaginous microorganism thalline after the propagation that step S3 is made is inoculated inIn the dephosphorization produce oil culture medium that step S4 makes or sulphur removal produce oil culture medium; At 20～40 DEG C, pH value be3.0～8.0, rotating speed is to cultivate 48～168h under 40～800rpm condition, collects thalline, is the produce oil of oil-containingMicrobial cells, dries to constant weight in 105 DEG C, obtains oleaginous microorganism dry mycelium;
Above-mentioned growing microorganism incubation is carried out in shaking table, and the rotating speed of shaking table is 40～800rpm;
S6, extraction oleaginous microorganism grease: the oil in the oleaginous microorganism dry mycelium that extraction step S5 obtainsFat, obtains oleaginous microorganism grease.
Under optimal way, the granularity of inulin is 50～200 orders described in step S1.
Under optimal way, nitrogenous source is yeast extract, peptone, NH described in step S24NO3、(NH4)2SO4In one or more combination.
Under optimal way, described in step S3 oleaginous microorganism be described oleaginous microorganism for after fermented and cultured,In extracellular microbial, fat content is greater than eucaryon or the prokaryotic micro-organisms of its dry cell weight 20wt%; Comprise that produce oil is thinBacterium, produce oil fungi, produce oil mould, oil-producing microalgae.
Above-mentioned produce oil bacterium, produce oil fungi, produce oil mould, oil-producing microalgae comprise artificial and natural mutation bacterial strain.
Under optimum way, described produce oil bacterium be Arthrobacter belong to, Rhodococcus belong to orMycobacterium belongs to;
Described produce oil fungi be Rhodosporidium belong to, Cryptococcuscur belong to, Yarrowia belong to,Rhodotorula belongs to, Rhizopus belongs to, Lipomyces belongs to, Trichosporon belongs to, Mortierella belongs to,Mucor belongs to or Cunninghamella belongs to;
Described produce oil mould is that Mortierella belongs to, Cunninghamella belongs to, Mucor belongs to or AspergillusBelong to;
Described oil-producing microalgae be Crypthecodinium belong to, Botryococcus belong to, Chlorella belong to,Nannochloropsis belongs to, Haliphthoros belongs to or Schizochytrium belongs to.
Under optimal way, dephosphorization treatment adopts chemical precipitation method described in step S4.
Concrete operation method bibliography: Xu Jianyu, universe, palace, Pan Mingfu. several coagulant deep phosphorous removal performancesComparative study. Guangxi light industry, 2009, (1), 89-91; Huang is relied on oneself, Xiao Jingjing, Li Mi. and chemical precipitation-magnetic flocculation is darkSpend the research of quick dephosphorization. Wuhan University Of Technology's journal, 2009,32 (1), 102-105.
Under optimal way, desulfurization is processed and is adopted chemical precipitation method described in step S4.
Concrete operation method bibliography: Su Qiaohong, Li Shuhui. sulfur-containing waste water treatment process experimental study. ZhejiangWork, 2008,5:4-7; Peng Xuhong. the practice of wastewater with high concentration of sulfide treatment process. Bohai University's journal (natural scienceVersion), 2007,1:21-23; Xi Shenglan, Ni Jianmin, Li Zeqing, Zhou Mi, Mo Jiansong. dyeing waste water wet desulfurizing processResearch. Guangdong chemical industry, 2011,1:149-150.
Under optimal way, the oleaginous microorganism that step S6 adopts acid heat-organic solvent method extraction step S4 to obtainGrease in dry mycelium.
Under optimum way, step S6 concrete operations are:
In S61, the oleaginous microorganism dry mycelium that obtains to step S5 by oleaginous microorganism dry mycelium every gram described10ml concentration is that the ratio of 4mol/L hydrochloric acid adds hydrochloric acid, 78 DEG C of water bath processing 1.5h;
S62, add the chloroform/methanol solvent extraction that the volume ratio of 2 times of volumes is 1:1 after cooling, collect organicPhase;
S63, remaining step S62 water is extracted once with equal-volume chloroform again, collect organic phase;
S64, the organic phase that step S62 and step S63 are collected merge, and add the chlorination of equal-volume 0.1wt%Sodium solution, mixes, centrifugal, collects organic phase;
S65, the organic phase anhydrous sodium sulfate drying that step S64 is collected, solvent removed by evaporation at reduced pressure,To oleaginous microorganism grease.
Technique effect of the present invention is:
1, to adopt inulin be that raw material is prepared culture medium in the present invention, cheap, is suitable for large-scale industrial application.
2, the present invention is according to the feature of jerusalem artichoke hydrolyzate, by different processing modes, adjusts nutrientContent, the oleaginous microorganism culture technique that the regulation and control of exploitation rising limit phosphorus/sulphur were cultivated in conjunction with two stages, significantly improves bacteriumThe oil and fat accumulation speed of body multiplication rate and oleaginous microorganism, grease yield obviously improves.
3, the present invention adopts the removal of jerusalem artichoke powder phosphorus/element sulphur to significantly improve the availability of hydrolysis of inulin liquid,Improve microbe oil fermentation Technical Economy.
4, the application extension of the inventive method the glyceride stock source of biodiesel, can significantly shorten and produce weekPhase, reduces production costs, and has improved production efficiency.
5, the inventive method is simple, can significantly improve grease production intensity and substrate conversion efficiency, and realization hasEffect is utilized the raw materials such as jerusalem artichoke, reaches and reduces costs, and improves the object of microbe oil fermentation Technical Economy.
Detailed description of the invention
Below by several concrete embodiments, the present invention will be further described.
Comparative example 1
(1) preparation of culture medium: get 100 order inulin 100g inulin and water in mass ratio 1:10 mix, addWater constant volume, to 1000ml, adopts 3M salt acid for adjusting pH to 3.6; Under saturated vapor, be hydrolyzed 30min, adjustJoint pH, to neutral, obtains jerusalem artichoke hydrolyzate; Add dusty yeast 10g/L, potassium dihydrogen phosphate is to 1.0g/L,MgSO4·7H2O to 0.5g/L, sterilizing;
(2) fermented and cultured: with 10% (V/V) (OD600nm=7.8) inoculum concentration is by viscosity trichosporon cutaneumTrichosporonfermentansCICC1368 (purchased from Chinese common micro-organisms culture presevation administrative center) connectsIn the culture medium making in step (1), 28 DEG C, 100rpm, adjusts a pH value every 24h, makes to cultivateThe pH of base maintains between 5-7, and 72h stops fermentation, and centrifugation, obtains oleaginous microorganism thalline, inDry to constant weight for 105 DEG C, weigh, represent thalline biomass with g dry mycelium/L zymotic fluid, obtain biomass and be8.0g/L。
(3) extract oleaginous microorganism grease: the oleaginous microorganism dry mycelium that step (2) is obtained adopts acidHeat-organic solvent method extracts grease, weighs, and calculates oil quantity and fat content, with g grease/L zymotic fluid tableShow oil quantity, fat content is the percentage that oil quantity accounts for biomass, obtains altogether grease 1.44g/L, calculatesThalline fat content 18%.
Embodiment 1
(1) prepare seed culture medium: get 100 order inulin 50g inulin and water in mass ratio 1:10 mix, addWater constant volume, to 500ml, adopts 3mol/L salt acid for adjusting pH to 3.6; Under saturated vapor, be hydrolyzed 30min, adjustJoint pH, to neutral, obtains jerusalem artichoke hydrolyzate, prepares a jerusalem artichoke hydrolyzate according to same method again, in order to stepSuddenly in (3), use; Get a copy of it jerusalem artichoke hydrolyzate, add dusty yeast to 10g/L, potassium dihydrogen phosphate extremely1.0g/L，MgSO4·7H2O to 0.5g/L, sterilizing, obtains the seed culture medium of microorganism;
(2) growing microorganism is cultivated: with 10% (V/V) (OD600nm=7.8) inoculum concentration is by viscosity silk sporeYeast TrichosporonfermentansCICC1368 is (in Chinese common micro-organisms culture presevation managementThe heart) be connected in the seed culture medium that step (1) makes, 28 DEG C, 100rpm, cultivates 24h, centrifugation,Obtain oleaginous microorganism thalline;
(3) produce oil culture medium preparation: another part of jerusalem artichoke hydrolyzate in step (1) carried out to dephosphorization treatment,With Ca (OH)2Regulate pH value to 10.2, obtain the dephosphorization culture medium of C/P=20970, after sterilizing as micro-Biological produce oil culture medium;
(4) thalline oil and fat accumulation is cultivated: the oleaginous microorganism thalline obtaining in step (2) is inoculated inProduce oil culture medium in step (3), 28 DEG C, 100rpm, adjusts a pH value every 24h, makes culture mediumPH maintains between 5-7, after cultivation 48h, stops fermentation, centrifugation, and the produce oil of collecting acquisition oil-containing is micro-Biomass, dries to constant weight in 105 DEG C, weighs, and represents thalline biomass with g dry mycelium/L zymotic fluid,Biomass is 10.0g/L;
(5) extract oleaginous microorganism grease: the oleaginous microorganism dry mycelium that step (4) is obtained uses acidHeat-organic solvent method extracts grease, weighs, and calculates oil quantity and fat content, with g grease/L zymotic fluid tableShow oil quantity, fat content is the percentage that oil quantity accounts for biomass, obtains altogether grease 2.5g/L, calculates bacteriumBody fat content 25%.
Relatively found that of comparative example 1 and embodiment 1, is used the inulin of same quality, according to the present inventionTechnical scheme, biomass has improved 50%, grease yield has improved 74%. Therefore, with prior art sideCase is compared, and the present invention has grease yield and the production efficiency of raising, the significant advantage such as reduces costs.
Comparative example 2
(1) preparation of culture medium: get 50 order inulin 140g inulin and water in mass ratio 1:4.3 mix, addWater constant volume, to 600ml, adopts 3M salt acid for adjusting pH to 2.0; Under saturated vapor, be hydrolyzed 20min, regulatePH, to neutral, obtains jerusalem artichoke hydrolyzate; Add peptone 5g/L, potassium dihydrogen phosphate is to 1.0g/L,MgSO4·7H2O to 0.5g/L, sterilizing;
(2) fermented and cultured: with 10% (V/V) (OD600nm=6.5) inoculum concentration is by bending CryptococcusCryptococcuscurvatusATCC20509 (collecting center purchased from US mode bacterial classification) is connected to step (1)In the culture medium making, 24 DEG C, 150rpm, adjusts a pH value every 24h, and the pH of culture medium is maintainedBetween 5-7,144h stops fermentation, and centrifugation, obtains oleaginous microorganism thalline, in 105 DEG C of oven dryTo constant weight, weigh, represent thalline biomass with g dry mycelium/L zymotic fluid, obtaining biomass is 18.0g/L.
(3) extract oleaginous microorganism grease: the oleaginous microorganism dry mycelium that step (2) is obtained adopts acidHeat-organic solvent method extracts grease, weighs, and calculates oil quantity and fat content, with g grease/L zymotic fluid tableShow oil quantity, fat content is the percentage that oil quantity accounts for biomass, obtains altogether grease 3.6g/L, calculatesThalline fat content 20%.
Embodiment 2
(1) prepare seed culture medium: get 50 order inulin 70g inulin and water in mass ratio 1:4.3 mix, addWater constant volume, to 300ml, adopts 3mol/L salt acid for adjusting pH to 2.0; Under saturated vapor, be hydrolyzed 20min, adjustJoint pH, to neutral, obtains jerusalem artichoke hydrolyzate, prepares a jerusalem artichoke hydrolyzate according to same method again, in order to stepSuddenly in (3), use; Get a copy of it jerusalem artichoke hydrolyzate, add peptone 5g/L, potassium dihydrogen phosphate to 1.0g/L，MgSO4·7H2O to 0.5g/L, sterilizing, obtains the seed culture medium of microorganism;
(2) growing microorganism is cultivated: with 10% (V/V) (OD600nm=6.5) inoculum concentration is by hidden bending ballYeast CryptococcuscurvatusATCC20509 (collecting center purchased from US mode bacterial classification) is connected to stepSuddenly in the seed culture medium that (1) makes, 24 DEG C, 150rpm, cultivates 24h, and centrifugation, obtains produce oilMicrobial cells;
(3) produce oil culture medium preparation: another part of jerusalem artichoke hydrolyzate in step (1) carried out to dephosphorization treatment,With Ca (OH)2Regulate pH value to 11.0, obtain the dephosphorization culture medium of C/P=31180, after sterilizing as micro-Biological produce oil culture medium;
(4) thalline oil and fat accumulation is cultivated: the oleaginous microorganism thalline obtaining in step (2) is inoculated inProduce oil culture medium in step (3), 24 DEG C, 150rpm, adjusts a pH value every 24h, makes culture mediumPH maintains between 5-7, after cultivation 96h, stops fermentation, centrifugation, and the produce oil of collecting acquisition oil-containing is micro-Biomass, dries to constant weight in 105 DEG C, weighs, and represents thalline biomass with g dry mycelium/L zymotic fluid,Biomass is 23.0g/L;
(5) extract oleaginous microorganism grease: the oleaginous microorganism dry mycelium that step (4) is obtained uses acidHeat-organic solvent method extracts grease, weighs, and calculates oil quantity and fat content, with g grease/L zymotic fluid tableShow oil quantity, fat content is the percentage that oil quantity accounts for biomass, obtains altogether grease 9.2g/L, calculatesThalline fat content 40%.
Relatively found that of comparative example 2 and embodiment 2, is used the inulin of same quality, according to the present inventionTechnical scheme, biomass has improved 28%, grease yield has improved 74%. The more important thing is, improvingWhen biomass and grease yield, fermentation time has shortened 24h. Therefore, with prior art scheme phaseRatio, the present invention has grease yield and the production efficiency of raising, reduces costs, the significant advantage such as shortening time.
Comparative example 3
(1) preparation of culture medium: get 200 order inulin 200g and water in mass ratio 1:5 mix, add water fixedHold to 1000ml, adopt 3M salt acid for adjusting pH to 2.5; Under saturated vapor, be hydrolyzed 10min, regulate pHTo neutral, obtain jerusalem artichoke hydrolyzate; Add NH4NO35g/L, potassium dihydrogen phosphate is to 1.0g/L,MgSO4·7H2O to 0.5g/L, sterilizing;
(2) fermented and cultured: with 10% (V/V) (OD600nm=5.0) inoculum concentration is by opaque red coccusRhodococcusopacusPD630 (purchased from German microorganism fungus kind preservation center) is connected to step (1) systemIn the culture medium obtaining, 30 DEG C, 200rpm, adjusts a pH value every 24h, and the pH of culture medium is maintainedBetween 5-7,96h stops fermentation, and centrifugation, obtains oleaginous microorganism thalline, in 105 DEG C of oven dry to permanentHeavy, weigh, represent thalline biomass with g dry mycelium/L zymotic fluid, obtaining biomass is 13.6g/L.
(3) extract oleaginous microorganism grease: the oleaginous microorganism dry mycelium that step (2) is obtained uses acidHeat-organic solvent method extracts grease, weighs, and calculates oil quantity and fat content, with g grease/L zymotic fluid tableShow oil quantity, fat content is the percentage that oil quantity accounts for biomass, obtains altogether grease 2.3g/L, calculatesThalline fat content 17%.
Embodiment 3
(1) prepare seed culture medium: get 200 order inulin 100g inulin and water in mass ratio 1:5 mix,Add water constant volume to 500ml, adopt 3mol/L salt acid for adjusting pH to 2.5; Under saturated vapor, be hydrolyzed 10min,Regulate pH to neutral, obtain jerusalem artichoke hydrolyzate, prepare again a jerusalem artichoke hydrolyzate according to same method, prepare againstIn step (3), use; Get a copy of it jerusalem artichoke hydrolyzate, add NH4NO35g/L, potassium dihydrogen phosphate extremely1.0g/L，MgSO4·7H2O to 0.5g/L, sterilizing, obtains the seed culture medium of microorganism;
(2) growing microorganism is cultivated: with 10% (V/V) (OD600nm=5.0) inoculum concentration is by opaque redCoccus RhodococcusopacusPD630 (purchased from German microorganism fungus kind preservation center) is connected to step (1)In the seed culture medium making, 30 DEG C, 200rpm, cultivates 48h, and centrifugation, obtains oleaginous microorganismThalline;
(3) produce oil culture medium preparation: another part of jerusalem artichoke hydrolyzate in step (1) carried out to desulfurization processing,With the processing of element sulphur precipitating reagent, obtain the sulphur removal culture medium of C/S=13320, after sterilizing as production by biologicalOil culture medium;
(4) thalline oil and fat accumulation is cultivated: the oleaginous microorganism thalline obtaining in step (2) is inoculated inProduce oil culture medium in step (3), 30 DEG C, 200rpm, adjusts a pH value every 24h, makes culture mediumPH maintains between 5-7, after cultivation 48h, stops fermentation, centrifugation, and the produce oil of collecting acquisition oil-containing is micro-Biomass, dries to constant weight in 105 DEG C, weighs, and represents thalline biomass with g dry mycelium/L zymotic fluid,Biomass is 19.5g/L;
(5) extract oleaginous microorganism grease: the oleaginous microorganism dry mycelium that step (4) is obtained uses acidHeat-organic solvent method extracts grease, weighs, and calculates oil quantity and fat content, with g grease/L zymotic fluid tableShow oil quantity, fat content is the percentage that oil quantity accounts for biomass, obtains altogether grease 5.1g/L, calculatesThalline fat content 26%.
Relatively found that of comparative example 3 and embodiment 3, is used the inulin of same quality, according to the present inventionTechnical scheme, biomass has improved 43%, grease yield has improved 122%. Therefore, with prior art sideCase is compared, and the present invention has grease yield and the production efficiency of raising, the significant advantage such as reduces costs.
Comparative example 4
(1) preparation of culture medium: get 200 order inulin 120g and water in mass ratio 1:5 mix, add water fixedHold to 600ml, adopt 3M salt acid for adjusting pH to 3.5; Under saturated vapor, be hydrolyzed 15min, regulate pHTo neutral, obtain jerusalem artichoke hydrolyzate; Add (NH4)2SO43g/L, potassium dihydrogen phosphate is to 1.0g/L,MgSO4·7H2O to 0.5g/L, sterilizing;
(2) fermented and cultured: with 10% (V/V) (OD600nm=6.5) inoculum concentration will be good for mould doughtilyGeotrichumrobustumCICC1256 (purchased from Chinese industrial microorganism fungus kind preservation administrative center) is connected toIn the culture medium that step (1) makes, 28 DEG C, 150rpm, adjusts a pH value every 24h, makes culture mediumPH maintain between 5-7,120h stop fermentation, centrifugation, obtains oleaginous microorganism thalline, inDry to constant weight for 105 DEG C, weigh, represent thalline biomass with g dry mycelium/L zymotic fluid, obtain biomass and be21.0g/L。
(3) extract oleaginous microorganism grease: the oleaginous microorganism dry mycelium that step (2) is obtained uses acidHeat-organic solvent method extracts grease, calculates oil quantity and fat content, represents grease with g grease/L zymotic fluidAmount, fat content is the percentage that oil quantity accounts for biomass, obtains altogether grease 5.2g/L, calculates thalline oilFat content 21%.
Embodiment 4
(1) prepare seed culture medium: get 200 order inulin 60g inulin and water in mass ratio 1:5 mix, addWater constant volume, to 300ml, adopts 3M salt acid for adjusting pH to 3.5; Under saturated vapor, be hydrolyzed 15min, regulatePH, to neutral, obtains jerusalem artichoke hydrolyzate, prepares a jerusalem artichoke hydrolyzate according to same method again, in order to step(3) in, use; Get a copy of it jerusalem artichoke hydrolyzate, add (NH4)2SO43g/L, potassium dihydrogen phosphate to 1.0g/L，MgSO4·7H2O to 0.5g/L, sterilizing, obtains the seed culture medium of microorganism;
(2) growing microorganism is cultivated: with 10% (V/V) (OD600nm=6.5) inoculum concentration will be good for mould doughtilyGeotrichumrobustumCICC1256 (purchased from Chinese industrial microorganism fungus kind preservation administrative center) is connected toIn the seed culture medium that step (1) makes, 28 DEG C, 150rpm, cultivates 12h, and centrifugation, is producedOil microbial cells;
(3) produce oil culture medium preparation: another part of jerusalem artichoke hydrolyzate in step (1) carried out to desulfurization processing,With the processing of element sulphur precipitating reagent, obtain the sulphur removal culture medium of C/S=170, after sterilizing as microorganism produce oilCulture medium;
(4) thalline oil and fat accumulation is cultivated: the oleaginous microorganism thalline obtaining in step (2) is inoculated inProduce oil culture medium in step (3), 28 DEG C, 150rpm, adjusts a pH value every 24h, makes culture mediumPH maintains between 5-7, after cultivation 96h, stops fermentation, centrifugation, and the produce oil of collecting acquisition oil-containing is micro-Biomass, dries to constant weight in 105 DEG C, weighs, and represents thalline biomass with g dry mycelium/L zymotic fluid,Biomass is 28.0g/L;
(5) extract oleaginous microorganism grease: the oleaginous microorganism dry mycelium that step (4) is obtained uses acidHeat-organic solvent method extracts grease, weighs, and calculates oil quantity and fat content, with g grease/L zymotic fluid tableShow oil quantity, fat content is the percentage that oil quantity accounts for biomass, obtains altogether grease 12.2g/L, calculatesThalline fat content 44%.
Relatively found that of comparative example 4 and embodiment 4, is used the inulin of same quality, according to the present inventionTechnical scheme, biomass has improved 33%, grease yield has improved 135%. The more important thing is, improvingWhen biomass and grease yield, fermentation time has shortened 12h. Therefore, with prior art scheme phaseRatio, the present invention has grease yield and the production efficiency of raising, reduces costs, the significant advantage such as shortening time.
Comparative example 5
(1) preparation of culture medium: get 80 order inulin 160g and water in mass ratio 1:5 mix, constant volume adds waterTo 800ml, adopt 3M salt acid for adjusting pH to 3.8; Under saturated vapor, be hydrolyzed 15min, regulate pH extremelyNeutrality, obtains jerusalem artichoke hydrolyzate; Add (NH4)2SO43g/L, potassium dihydrogen phosphate is to 1.0g/L,MgSO4·7H2O to 0.5g/L, sterilizing;
(2) fermented and cultured: with 10% (V/V) (OD600nm=6.5) inoculum concentration is by trichosporon cutaneumTrichosporoncutaneumGIM2.67 (purchased from microbial preservation center, Guangdong Province) is connected to step (1) systemIn the culture medium obtaining, 28 DEG C, 200rpm, adjusts a pH value every 24h, and the pH of culture medium is maintainedBetween 5-7,168h stops fermentation, and centrifugation, obtains oleaginous microorganism thalline, in 105 DEG C of oven dry to permanentHeavy, weigh, represent thalline biomass with g dry mycelium/L zymotic fluid, obtaining biomass is 19.8g/L.
(3) extract oleaginous microorganism grease: the oleaginous microorganism dry mycelium that step (2) is obtained uses acidHeat-organic solvent method extracts grease, weighs, and calculates oil quantity and fat content, with g grease/L zymotic fluid tableShow oil quantity, fat content is the percentage that oil quantity accounts for biomass, obtains altogether grease 3.9g/L, calculatesThalline fat content 19.7%.
Embodiment 5
(1) prepare seed culture medium: get 80 order inulin 80g inulin and water in mass ratio 1:5 mix, addWater constant volume, to 400ml, adopts 3M salt acid for adjusting pH to 3.8; Under saturated vapor, be hydrolyzed 15min, regulatePH, to neutral, obtains jerusalem artichoke hydrolyzate, prepares a jerusalem artichoke hydrolyzate according to same method again, in order to step(3) in, use; Get a copy of it jerusalem artichoke hydrolyzate, add (NH4)2SO43g/L, potassium dihydrogen phosphate to 1.0g/L，MgSO4·7H2O to 0.5g/L, sterilizing, obtains the seed culture medium of microorganism;
(2) growing microorganism is cultivated: with 10% (V/V) (OD600nm=6.5) inoculum concentration is by skin shape silk sporeYeast TrichosporoncutaneumGIM2.67 (purchased from microbial preservation center, Guangdong Province) is connected to step (1)In the seed culture medium making, 28 DEG C, 200rpm, cultivates 48h, and centrifugation, obtains oleaginous microorganismThalline;
(3) produce oil culture medium preparation: another part of jerusalem artichoke hydrolyzate in step (1) carried out to desulfurization processing,With the processing of element sulphur precipitating reagent, obtain the sulphur removal culture medium of C/S=190, after sterilizing as microorganism produce oilCulture medium;
(4) thalline oil and fat accumulation is cultivated: the oleaginous microorganism thalline obtaining in step (2) is inoculated inProduce oil culture medium in step (3), 28 DEG C, 150rpm, adjusts a pH value every 24h, makes culture mediumPH maintains between 5-7, after cultivation 100h, stops fermentation, and centrifugation, collects the produce oil that obtains oil-containingMicrobial cells, dries to constant weight in 105 DEG C, weighs, and represents thalline biomass with g dry mycelium/L zymotic fluid,Biomass is 24.5g/L;
(5) extract oleaginous microorganism grease: the oleaginous microorganism dry mycelium that step (4) is obtained uses acidHeat-organic solvent method extracts grease, weighs, and calculates oil quantity and fat content, with g grease/L zymotic fluid tableShow oil quantity, fat content is the percentage that oil quantity accounts for biomass, obtains altogether grease 11.0g/L, calculatesThalline fat content 45%.
Relatively found that of comparative example 5 and embodiment 5, is used the inulin of same quality, according to the present inventionTechnical scheme, biomass has improved 24%, grease yield has improved 182%. The more important thing is, improvingWhen biomass and grease yield, fermentation time has shortened 20h. Therefore, with prior art scheme phaseRatio, the present invention has grease yield and the production efficiency of raising, reduces costs, the significant advantage such as shortening time.
The above, be only preferably detailed description of the invention of the present invention, but not office of protection scope of the present inventionBe limited to this, any be familiar with those skilled in the art the present invention disclose technical scope in, according to thisThe technical scheme of invention and inventive concept thereof are equal to replaces or changes, and all should be encompassed in protection of the present inventionWithin scope.
Claims (8)
1. the oleaginous microorganism taking jerusalem artichoke as raw material is produced a method for microbial grease, it is characterized in that,Comprise the steps:
S1, preparation jerusalem artichoke hydrolyzate: inulin is mixed 1:1～10 in mass ratio with water, adopt 3mol/L hydrochloric acidRegulate pH to 1～4; Under saturated vapor, be hydrolyzed 10～40min, regulate pH to neutral, obtain jerusalem artichoke hydrolyzate;
S2, prepare seed culture medium: get 1/2nd of jerusalem artichoke hydrolyzate that step S1 makes, add nitrogenous sourceTo 1～20g/L, add potassium dihydrogen phosphate to 1.0g/L, add MgSO4·7H2O to 0.5g/L, sterilizing,To the seed culture medium of microorganism;
S3, growing microorganism are cultivated: with 10% (V/V) inoculum concentration access oleaginous microorganism seed liquor(OD600nm=1～10), in the seed culture medium making in step S1, at 20～40 DEG C, rotating speed beUnder 40～800rpm condition, 12～96h is cultivated in ventilation, and thalline is collected in centrifugation, is the product after propagationOil microbial cells;
S4, prepare produce oil culture medium: the jerusalem artichoke hydrolyzate remaining 1/2nd that step S1 is made takes offPhosphorus processing, obtains the dephosphorization produce oil culture medium that carbon and P elements molar ratio are greater than 70;
Or the jerusalem artichoke hydrolyzate remaining 1/2nd that step S1 is made carries out desulfurization processing, obtain carbonThe sulphur removal produce oil culture medium that is greater than 150 with element sulphur molar ratio;
S5, thalline oil and fat accumulation are cultivated: the oleaginous microorganism thalline after the propagation that step S3 is made is inoculated inIn the dephosphorization produce oil culture medium that step S4 makes or sulphur removal produce oil culture medium; At 20～40 DEG C, pH value be3.0～8.0, rotating speed is to cultivate 48～168h under 40～800rpm condition, collects thalline, is the produce oil of oil-containingMicrobial cells, dries to constant weight in 105 DEG C, obtains oleaginous microorganism dry mycelium;
S6, extraction oleaginous microorganism grease: the oil in the oleaginous microorganism dry mycelium that extraction step S5 obtainsFat, obtains oleaginous microorganism grease.
2. the method that the oleaginous microorganism taking jerusalem artichoke as raw material is produced microbial grease according to claim 1,It is characterized in that, the granularity of inulin is 50～200 orders described in step S1.
3. the method that the oleaginous microorganism taking jerusalem artichoke as raw material is produced microbial grease according to claim 1,It is characterized in that, nitrogenous source is yeast extract, peptone, NH described in step S24NO3、(NH4)2SO4InOne or more combination.
4. the method that the oleaginous microorganism taking jerusalem artichoke as raw material is produced microbial grease according to claim 1,It is characterized in that, described in step S3 oleaginous microorganism be described oleaginous microorganism for after fermented and cultured, micro-lifeIn thing born of the same parents, fat content is greater than eucaryon or the prokaryotic micro-organisms of its dry cell weight 20wt%; Comprise produce oil bacterium,Produce oil fungi, produce oil mould, oil-producing microalgae.
5. the method that the oleaginous microorganism taking jerusalem artichoke as raw material is produced microbial grease according to claim 4,It is characterized in that,
Described produce oil bacterium is that Arthrobacter belongs to, Rhodococcus belongs to or Mycobacterium belongs to;
Described produce oil fungi be Rhodosporidium belong to, Cryptococcuscur belong to, Yarrowia belong to,Rhodotorula belongs to, Rhizopus belongs to, Lipomyces belongs to, Trichosporon belongs to, Mortierella belongs to,Mucor belongs to or Cunninghamella belongs to;
Described produce oil mould is that Mortierella belongs to, Cunninghamella belongs to, Mucor belongs to or AspergillusBelong to;
Described oil-producing microalgae be Crypthecodinium belong to, Botryococcus belong to, Chlorella belong to,Nannochloropsis belongs to, Haliphthoros belongs to or Schizochytrium belongs to.
6. the method that the oleaginous microorganism taking jerusalem artichoke as raw material is produced microbial grease according to claim 1,It is characterized in that,
Described in step S4, dephosphorization treatment adopts chemical precipitation method;
Described in step S4, desulfurization is processed and is adopted chemical precipitation method.
7. the method that the oleaginous microorganism taking jerusalem artichoke as raw material is produced microbial grease according to claim 1,It is characterized in that the dry bacterium of oleaginous microorganism that step S6 adopts acid heat-organic solvent method extraction step S5 to obtainGrease in body.
8. the method that the oleaginous microorganism taking jerusalem artichoke as raw material is produced microbial grease according to claim 7,It is characterized in that, step S6 concrete operations are:
In S61, the oleaginous microorganism dry mycelium that obtains to step S5 by oleaginous microorganism dry mycelium every gram described10ml concentration is that the ratio of 4mol/L hydrochloric acid adds hydrochloric acid, 78 DEG C of water bath processing 1.5h;
S62, add the chloroform/methanol solvent extraction that the volume ratio of 2 times of volumes is 1:1 after cooling, collect organicPhase;
S63, remaining step S62 water is extracted once with equal-volume chloroform again, collect organic phase;
S64, the organic phase that step S62 and step S63 are collected merge, and add the chlorination of equal-volume 0.1wt%Sodium solution, mixes, centrifugal, collects organic phase;
S65, the organic phase anhydrous sodium sulfate drying that step S64 is collected, solvent removed by evaporation at reduced pressure,To oleaginous microorganism grease.
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