CN102533879A - Microbial oil extraction method - Google Patents
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Abstract
The invention discloses a microbial oil extraction method, and belongs to the field of bioengineering downstream technology. Oil-producing microorganism fermented liquor is used as a raw material, and is subjected to enzymolysis and extraction by organic solvent to obtain the microbial oil, wherein the extraction rate is up to 100% maximally. The microbial oil extraction method disclosed by the invention does not need concentration of the fermented liquor, recovery of strains, drying of the strains and other pretreatment operations, has mild enzymolysis conditions, short treatment time, low extraction temperature, high extraction rate, low requirements on equipment and low toxicity of the used organic solvent, greatly reduces the power and energy consumptions in the microbial oil extraction process, lowers the process cost and alleviates the environmental pollution and damage to human health. According to the method, a new way is provided for mass production and extraction of the microbial oil.
Description
Technical field
The present invention relates to a kind of process for extracting of microbial oil, concretely, is earlier with enzyme the fermentation liquid of oleaginous microorganism to be carried out pre-treatment, obtains grease with the organic solvent lixiviate then, belongs to biotechnology downstream technical field.
Background technology
Many mikrobes like yeast, mould, little algae, bacterium etc., can be converted into grease with glucide under certain condition to be stored in the thalline, and this grease is called microbial oil.In cell, can accumulate the bacterial strain that grease surpasses dried cell weight 20%, be called oleaginous microorganism.The fat content of some oleaginous microorganisms can reach more than 70% of dried cell weight (Ratledge C. Acta Biotechnology, 1991,11 (5), 429).The lipid acid of microbial oil is formed similar with general Vegetable oil lipoprotein, is lipid acid with C16, C18, is main like palmitinic acid, Triple Pressed Stearic Acid, oleic acid and linolic acid.In addition, contain a large amount of functional fatty acids in the grease of some bacterial strains, like linolenic acid, arachidonic acid, timnodonic acid, docosahexenoic acid etc.Compare with Vegetable oil lipoprotein production, the microbe oil fermentation cycle is short, does not receive the influence of place, season, climate change etc., but continuous production; And the oleaginous microorganism microorganism resource is abundant, can utilize and transform various agricultural waste wood Mierocrystalline celluloses and allogenic material material thereof, to efficiently utilizing renewable resources, improving the ecological environment, develop the biomass energy industry and have special meaning.Therefore, utilizing microbe transformation method to obtain grease has a high potential.
As how the oleaginous microorganism culture is a raw material, efficiently, separation and Extraction grease at low cost, is one of bottleneck of realizing the microbial oil large-scale production.For obtaining intracellular grease, from the oleaginous microorganism fermentation liquid, reclaim thalline earlier usually, carry out drying, extract microbial oil with reference to the Vegetable oil lipoprotein extraction process then.Be raw material with the oil-containing dry mycelium in the current document, and the process for extracting of employing such as milling process (how to conquer east Chen Tao. microbial oil is learned. Beijing: Chemical Industry Press; 2005), pressure solvent method (White PM, Potter TL, Strickland TC. Journal of Agricultural and Food Chemistry; 2009,57 (16), 7171), UW secondary solvent extraction (Vicente G; Bautista LF, Rodr í guez R, et al. Biochemical Engineering Journal; 2009,48 (1), 22), microwave-assisted solvent extraction (Young JC
.Journal of Agricultural and Food Chemistry; 1995; 43 (11), 2904), the supercutical fluid method (how to conquer east Chen Tao. microbial oil is learned. Beijing: Chemical Industry Press; 2005) and soxhlet extraction (Li Chao. the food analysis philosophy and technique. Beijing: scientific and technical literature press, 1987).Also having with the oleaginous microorganism wet thallus in the document is raw material, adopt solvent extraction method or acid heat method extract greasy report (Li Zhifeng opens the tinkling of pieces of jade. microbiology circular, 2001,28 (6), 72).The common feature of aforesaid method is at first from the oleaginous microorganism fermentation liquid, to isolate thalline through technology such as centrifugal or filtrations.Yet many oleaginous microorganisms are unicellular organism, and cell size is little, and the thalline water cut is high.In the oleaginous microorganism late stage of culture, because a large amount of greases of intracellular accumulation, cell density reduces, and fermentation liquid viscosity is big.Therefore, adopt centrifugal or filtering technique Separation and Recovery thalline difficulty relatively from fermentation liquid, running cost high (Lee AK, Lewis DM, Ashman PJ. Journal of Applied Physiology, 2009,21 (5), 559).Obviously, be the material extraction grease to be rich in greasy microbial cells, there are following one or more defectives, comprise complex process, equipment requirements height, high, the use high toxicity organic solvent of energy consumption.
With the oleaginous microorganism concentrated broth is that the grease process for extracting of raw material has high pressure homogenate method (CN101323865) etc.; This method need concentrate fermented liquid; And high pressure homogenate smudge cells wall technique is high to equipment requirements; Energy expenditure is big, and the grease extraction cost is high, is not suitable for the microbial oil suitability for industrialized production.With the oleaginous microorganism fermentation liquid is that the grease process for extracting of raw material has the direct extraction of fermented liquid organic solvent (CN101824440A) etc.These methods use high toxicity solvent such as chloroform etc. just can obtain higher extraction rate under higher extraction temperature, though the little solvent of toxicity like No. four solvents, No. six extraction solvent wet goods at high temperature (> 50 oC) extraction rate also not high (< 55%) down.These method processing safeties are poor, and are big to HUMAN HEALTH and environmental damage.
Summary of the invention
The invention provides a kind of is raw material with the oleaginous microorganism fermentation liquid, and enzyme is assisted the greasy method of organic solvent lixiviate.Present method raw material pre-treatment process is simple, and mild condition, energy consumption are low.Because enzyme is handled and effectively removed the greasy obstacle of solvent extraction, can use the organic solvent that toxicity is low, environment is more friendly.
The present invention is achieved through following technical proposals:
1) in fermentation liquid, adding enzyme is 0.01 g/L～5 g/L to total enzyme concn, and at 20 oC～50 oC, pH 3～8, handle 5 minutes～10 hours;
2) fermentation liquid of handling to step 1) adds organic solvent, and solvent load is 0.2 times～5 times of fermentation liquid volume, handles 10 minutes～5 hours at 20 oC～60 oC;
3) from step 2) reclaim organic phase the mixture handled;
4) organic phase that step 3) is obtained is removed organic solvent by ordinary method, obtains microbial oil.
The enzyme that uses in the technical scheme of the present invention is cellulase, hemicellulase, polygalacturonase, β-1; 3-mannase, beta-glucanase, α-1,3-LSD, proteolytic enzyme, Proteinase K, Phospholipid hydrolase, Snailase, chitinase etc. or the combination of their necessity.Wherein, Cellulase, hemicellulase, polygalacturonase, beta-glucanase, proteolytic enzyme from Ningxia jade of the He family Bioisystech Co., Ltd buy, be respectively 50,000 U/g, 1,200,000 U/g, 3.5 ten thousand U/g, 1,200,000 U/g, 800,000 U/g than vigor, Proteinase K is buied from precious biotechnology (Dalian) ltd; Than vigor is 3.0 ten thousand U/g; Phospholipid hydrolase is buied from Novi letter (China) Investment Co., Ltd, is 1.0 ten thousand U/g than vigor, Snailase from Beijing the prosperous Bioisystech Co., Ltd of ancient cooking vessel state buy; β-1,3-mannase be the contriver belong to the laboratory with pichia spp (
Pichia pastorisX-33) be the host, with document (Sugino H, Furuichi S, Murao S; Et al. Bioscience Biotechnology and Biochemistry, 2004,68,757) dna recombinant expression of announcing obtains; It is 0.64 ten thousand U/g than vigor, and α-1,3-LSD are that the contriver belongs to laboratory reference literature (Shalom G, J Pratten; Et al. Protein Expression and Purification, 2008,60 (2), 170) preparation; Than vigor is 0.39 ten thousand U/g, and chitinase is that the contriver belongs to laboratory reference literature (Tsujibo H, H Orikoshi, et al. Journal of Bacteriology; 1993,175 (1), 176) preparation is 1.0 ten thousand U/g than vigor.
The organic solvent that the present invention uses is solubilized grease under the room temperature, liquid state organics that polarity is lower, like sherwood oil (boiling spread 60 oC～90 oC), normal hexane, ETHYLE ACETATE, methylene dichloride, No. six solvent for extraction, No. four solvents, industrial hexane (boiling spread 66 oC～69 oC) etc. or their necessity combination.
It is air distillation or vacuum-evaporation etc. that the present invention removes the method that organic solvent obtains microbial oil mutually from organic solvent.
The fermentation liquid that the present invention uses can also make up through heating, microwave radiation, supersound process etc. or their necessity and handle with before zymin contact, makes oleaginous microorganism endogenous protein inactivation or reduces active purpose to reach.
The oleaginous microorganism that the present invention uses can surpass fungi, little algae, bacterium or the genetic engineering bacterium of dried cell weight 20% (w/w) for thalline fat content after fermentation culture, and they include but not limited to, the produce oil fungi, as the red winter spore yeast of circle (
Rhodosporidium toruloides), white Cryptococcus (
Cryptococcus albidus), crooked Cryptococcus (
Cryptococcus curvatus), inferior sieve separate the fat yeast (
Yarrowia lipolytica), rhodotorula glutinis (
Rhodotorula glutinis), the lactose rhodotorula (
Rhodotorula lactosa), little rhodotorula (
Rhodotorula minuta), tangerine woods saccharomyces oleaginosus (
Lipomyces kononenkoae), skin shape trichosporon (
Trichosporon cutaneum), the fermentable trichosporon (
Trichosporon fermentans), strong mould doughtily (
Geotrichum robustum), Mortierella isabellina (
Mortierella isabellina), a volume branch Mucor (
Mucor circinelloides), little Ke Yinhan mould (
Cunninghamella) and Christian Breton plan endomyces (
Endomycopsis burtonii); The little algae of produce oil, as Blang's grape algae (
Botryococcus braunii), Crypthecodinium cohnii (
Crypthecodinium cohnii), chlorella (
Chlorella protothecoides), Nannochloropsis oceanica (
Nannochloropsis sp.) and schizochytrium limacinum (
Schizochytrium limacinum); The produce oil bacterium, as coryneform bacteria (
Corynebacterium), Nocardia bacteria (
Nocardia), mycobacterium (
Mycobacterium) etc.
The present invention uses the method for the actual fat content reference literature (Li YH, Zhao ZB, Bai FW. Enzyme Microbial Technology, 2007,41,312) of thalline material to measure, and calculates the grease extraction yield of additive method as benchmark.
Embodiment
Following examples have been chosen the fermentation liquid and the enzyme thereof of some typical oleaginous microorganisms and have been handled and the grease leaching process, help to understand this patent, do not use the present invention but be not limited in any form on other produce oil bacterial strain materials.
Comparative Examples 1
According to the described method of document (Li YH, Zhao ZB, Bai FW. Enzyme Microbial Technology, 2007,41,312), cultivate oleaginous yeast
R. toruloidesAS 2.1389 (bacterium source in Chinese common micro-organisms culture presevation administrative center), the density that obtains fermenting is the fermentation liquid of 114 g dry mycelium (CDW)/L, the dry mycelium fat content is 63%.Get above-mentioned fermentation liquid 50 ml, add 50 ml ETHYLE ACETATE again, thorough mixing under 25 oC, lixiviate 1 h, organic phase is taken out in spinning, and evaporating solvent gets microbial oil, and extraction rate is 10%.
Embodiment 1
According to the described method of document (Li YH, Zhao ZB, Bai FW. Enzyme Microbial Technology, 2007,41,312), cultivate oleaginous yeast
R. toruloidesAS 2.1389 (bacterium source in Chinese common micro-organisms culture presevation administrative center), the density that obtains fermenting is the fermentation liquid of 114 g dry mycelium (CDW)/L, the dry mycelium fat content is 63%.Get above-mentioned fermentation liquid 50 ml, microwave treatment under normal pressure (800 W, 60 s).Add β-1,3-mannase to final concentration is 0.28 g/L, handles 0.5 h down at pH 5.0,25 oC; Add 50 ml ETHYLE ACETATE again, thorough mixing under 25 oC, lixiviate 1 h, organic phase is taken out in spinning, and evaporating solvent gets microbial oil, and extraction rate is 95%.
The experimental result of comparing embodiment 1 and Comparative Examples 1 shows fermentation liquid through microwave and β-1, and the 3-mannase can extract grease with ETHYLE ACETATE after handling very effectively; And directly from sweet mash liquid, be difficult to the extraction separation grease with ETHYLE ACETATE.Therefore, the pre-treatment process of fermentation liquid has unusual effect to the microbial oil extraction separation.
Embodiment 2
Other operational conditions are with embodiment 1, but save in the leaching process with β-1, and the 3-mannase is handled the step of fermentation liquid.Extraction rate is 35%.Comparing embodiment 1 and embodiment 2 are explained with β-1, and the 3-mannase is handled and significantly improved the grease extraction yield.
Embodiment 3
Other operational conditions are with embodiment 1, but save the step of microwave treatment fermentation liquid in the leaching process.Extraction rate is 30%.
Comparing embodiment 1 and embodiment 3; Explanation is carried out microwave treatment before enzyme is handled, can significantly improve the grease extraction yield, and its reason possibly be that microwave treatment causes oleaginous microorganism endogenous protein inactivation or active significantly reduction; More help β-1,3-mannase performance effect.
Embodiment 4
Other operational conditions are with embodiment 1, but in the processing mode that enzyme is handled the primary fermentation mash are: processing is 2 minutes 121 oC under.Extraction rate is 93%.
Comparing embodiment 1 and embodiment 4 are explained at β-1, and the 3-mannase carries out pyroprocessing before handling, and can obtain and the proximate effect of microwave treatment.
Embodiment 5
Other operational conditions are with embodiment 1, but in the processing mode that enzyme is handled the primary fermentation mash are: processing is 10 minutes 70 oC under.Extraction rate is 87%.
Comparing embodiment 1 and embodiment 4, embodiment 5; Pyroprocessing is carried out in explanation before enzyme is handled temperature and time can change in the scope at broad; As long as can make oleaginous microorganism endogenous protein inactivation or active significantly reduction, grease is extracted obtain good effect.
Embodiment 6
According to the described method of document (Li YH, Zhao ZB, Bai FW. Enzyme Microbial Technology, 2007,41,312), cultivate oleaginous yeast
R. toruloidesAS 2.1389 (bacterium source in Chinese common micro-organisms culture presevation administrative center), the density that obtains fermenting is the fermentation liquid of 15 g CDW/L, the dry mycelium fat content is 35%.Get above-mentioned fermentation liquid 50 ml, microwave treatment under normal pressure (500 W, 1 min).Add β-1,3-mannase to final concentration is 0.01 g/L, handles 10 h down at pH 5.0,25 oC; Add 100 ml ETHYLE ACETATE again, thorough mixing under 25 oC, lixiviate 1 h, organic phase is taken out in spinning, and evaporating solvent gets microbial oil, and extraction rate is 100%.
Embodiment 7
Cultivate oleaginous yeast according to the described method of document (Ykema A, Verbree EC, Kater MM, Smit H. Applied Microbiology and Biotechnology, 1988,29,211)
C. curvatusATCC 20509 (bacterium source is in U.S. representative microbial DSMZ), the density that obtains fermenting is the fermentation liquid of 90 g CDW/L, the dry mycelium fat content is 40%.Get above-mentioned fermented liquid 50 ml, under 121 oC, handled 5 minutes.Adding Snailase to final concentration is 2.0 g/L, handles 2.0 h down at pH 6.5,37 oC; Add 100 ml methylene dichloride again, thorough mixing under 20 oC, lixiviate 1 h, organic phase is taken out in spinning, and evaporating solvent gets microbial oil, and extraction rate is 96%.
Embodiment 8
According to the described method of document (Li YH, Zhao ZB, Bai FW. Enzyme Microbial Technology, 2007,41,312), cultivate oleaginous yeast
R. glutinisAS 2.703 (bacterium source in Chinese common micro-organisms culture presevation administrative center), the density that obtains fermenting is the fermentation liquid of 105 g CDW/L, the dry mycelium fat content is 60%.Get above-mentioned fermentation liquid 50 ml, heat treated 2 h under 50 oC.Add β-1,3-mannase to final concentration is 0.30 g/L, handles 1.0 h down at pH 5.0,37 oC; Transfer pH to 7.5, adding Proteinase K to its final concentration is 0.05 g/L, adds 250 ml ETHYLE ACETATE again, thorough mixing under 40 oC, and lixiviate 10 min, organic phase is taken out in spinning, and evaporating solvent gets microbial oil, and extraction rate is 85%.
Embodiment 9
According to the described method of document (Li YH, Zhao ZB, Bai FW. Enzyme Microbial Technology, 2007,41,312), cultivate oleaginous yeast
L. starkeyiAS 2.1560 (bacterium source in Chinese common micro-organisms culture presevation administrative center), the density that obtains fermenting is the fermentation liquid of 61 g CDW/L, the dry mycelium fat content is 58%.Get above-mentioned fermentation liquid 50 ml, supersound process 20 min.Adding beta-glucanase to final concentration is 0.10 g/L, handles 1.0 h down at pH 5.0,25 oC; Transfer pH to 7.0, adding polygalacturonase to its final concentration is 0.05 g/L, under 25 oC, continues to handle 30 min; Add 25 ml normal hexanes and 50 ml sherwood oils again, thorough mixing under 25 oC, lixiviate 5.0 h, organic phase is taken out in spinning, and evaporating solvent gets microbial oil, and extraction rate is 90%.
Embodiment 10
According to the described method of document (Li YH, Zhao ZB, Bai FW. Enzyme Microbial Technology, 2007,41,312), cultivate oleaginous yeast
T. cutaneumAS 2.571 (bacterium source in Chinese common micro-organisms culture presevation administrative center), the density that obtains fermenting is the fermentation liquid of 81 g CDW/L, the dry mycelium fat content is 53%.Get above-mentioned fermentation liquid 50 ml, microwave treatment (400 W, 3 min).Add polygalacturonase, α-1,3-LSD and chitinase to its final concentration is respectively 0.40 g/L, 0.10 g/L and 0.08 g/L, handles 1.0 h down at pH 5.0,37 oC; Add industrial hexane 150 ml again, thorough mixing under 60 oC, lixiviate 20 min, organic phase is taken out in spinning, and evaporating solvent gets microbial oil, and extraction rate is 78%.
Embodiment 11
According to the described method of document (Li YH, Zhao ZB, Bai FW. Enzyme Microbial Technology, 2007,41,312), cultivate oleaginous yeast
M. isabellinaAS 3.3410 (bacterium source in Chinese common micro-organisms culture presevation administrative center), the density that obtains fermenting is the fermentation liquid of 70 g CDW/L, the dry mycelium fat content is 55%.Get above-mentioned fermentation liquid 50 ml, heat treated 10 min under 90 oC.Adding beta-glucanase to final concentration is that 0.23 g/L and Snailase to final concentration are 0.60 g/L, handles 1.0 h down at pH 6.0,37 oC; Add ETHYLE ACETATE 100 ml again, thorough mixing under 50 oC, lixiviate 30 min, organic phase is taken out in spinning, and evaporating solvent gets microbial oil, and extraction rate is 90%.
Embodiment 12
Cultivate the little algae of produce oil according to the described method of document (Xiong W, Li XF, Xiang JY, et al. Applied Microbiology and Biotechnology, 2008,78,29)
C. cohniiATCC 30556 (bacterium source is in U.S. representative microbial DSMZ), the density that obtains fermenting is the fermentation liquid of 50 g CDW/L, the dry mycelium fat content is 52%.Get above-mentioned fermented liquid 50 ml, microwave treatment (800 W, 30 s).Adding cellulase to its final concentration is that 1.0 g/L and Snailase to its final concentration are 4.0 g/L, handles 2.0 h down at pH 7.0,50 oC; Add No. four solvent 20 ml and ETHYLE ACETATE 80 ml again, thorough mixing under 40 oC, lixiviate 1 h, organic phase is taken out in spinning, and evaporating solvent gets microbial oil, and extraction rate is 93%.
Embodiment 13
Cultivate the little algae of produce oil according to the described method of document (Xiong W, Li XF, Xiang JY, et al. Applied Microbiology and Biotechnology, 2008,78,29)
C. protothecoidesCS-41 (bacterium source is in the little algae of Australian CSIRO research centre), the density that obtains fermenting is the fermentation liquid of 18 g CDW/L, the dry mycelium fat content is 49%.Get above-mentioned fermented liquid 50 ml, heat treated 1 h under 60 oC.Adding cellulase to its final concentration is that 0.3 g/L and hemicellulase to its final concentration are 0.10 g/L, handles 1.5 h down at pH 7.0,37 oC; Add ETHYLE ACETATE 100 ml, thorough mixing under 40 oC, lixiviate 1 h, organic phase is taken out in spinning, and evaporating solvent gets microbial oil, and extraction rate is 89%.
The invention has the beneficial effects as follows:
Compare with the grease process for extracting that with dry mycelium, wet thallus, concentrated broth is raw material; The present invention does not need concentrated broth, reclaims operations such as thalline, dry thalline; Overcome the technical barrier that directly from the high viscosity fermentation mash, reclaims thalline; Simplify process step, practiced thrift energy consumption, reduced cost; And, through grease in effective extraction cell, can also increase cell density, help later stage solid-liquid separation operation and bacterium slag to reclaim;
Compare with methods such as milling process, pressure solvent method, UW secondary solvent extraction, microwave-assisted solvent extraction, supercutical fluid method, acid heat method or high pressure homogenization methods; The present invention passes through the enzyme process fracturing cell walls, mild condition, and energy expenditure is little; Low for equipment requirements, cost is low; Little to components such as grease, protein destruction simultaneously, the oil quality that obtains is high, and the bacterium slag is of high nutritive value;
Compare with the acid heat method with solvent extraction method, solvent for use toxicity of the present invention is little, and residual few at water, solvent loss is few, and more friendly to environment, process more cleans;
Compare with the direct extraction of fermentation liquid organic solvent, the present invention is owing to use enzyme to handle the obstacle of effectively having removed extract oil fat, and the organic solvent toxicity of use is low, environment is more friendly; And extraction temperature is low; Extracting effect is good, good operation safety, consuming little energy.
In a word, the present invention's technology is effectively simple, and facility investment is few, and energy consumption is low, has improved the Technological Economy property of microbe oil fermentation, helps large-scale industrial production.
Claims (8)
1. microbial oil process for extracting is characterized in that: at first handle the oleaginous microorganism fermentation liquid with enzyme, and the fermentation liquid of handling with organic solvent lixiviate enzyme then, the Separation and Recovery organic phase except that desolvating, obtains microbial oil again.
2. according to the said method of claim 1; It is characterized in that: said enzyme is cellulase, hemicellulase, polygalacturonase, β-1; 3-mannase, beta-glucanase, α-1, a kind of or necessity more than the two kinds combination in 3-LSD, proteolytic enzyme, Proteinase K, Phospholipid hydrolase, Snailase, the chitinase.
3. according to claim 1 or 2 said methods, it is characterized in that: the enzyme treatment condition are mash pH=3～8, temperature 20 oC～50 oC, 5 minutes～10 hours time, enzyme dosage 0.01 g/L～5 g/L.
4. according to the said method of claim 1; It is characterized in that: the liquid state organics that said organic solvent is a solubilized grease under the room temperature, polarity is low is a kind of in boiling spread 60 oC～90 oC sherwood oils, normal hexane, ETHYLE ACETATE, methylene dichloride, No. six solvent for extraction, No. four solvents, the boiling spread 66 oC～69 oC industrial hexane or necessity combination more than two kinds.
5. according to the said method of claim 1, it is characterized in that: said extracting condition is temperature 20 oC～60 oC, and 10 minutes～5 hours time, the volume ratio of organic solvent and fermentation liquid is 1:5～5:1.
6. according to the said method of claim 1, it is characterized in that: fermentation liquid can pass through inactivation treatment before enzyme is handled, and its ablation method is a kind of or necessity more than the two kinds combination in heating, microwave radiation, the ultrasonication.
7. according to the described method of claim 1, it is characterized in that: described oleaginous microorganism fermentation liquid is the suspension liquid that contains thalline that produce oil fungi, bacterium or little algae obtain under the liquid culture condition.
8. according to the described method of claim 1, it is characterized in that: described oleaginous microorganism can surpass fungi, little algae or the bacterium of dried cell weight 20% (w/w) for thalline fat content after fermentation culture.
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