CN102533879B - 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 extracting method of microbial oil, concretely, is first with enzyme, the fermentation liquid of oleaginous microorganism to be carried out to pre-treatment, then with the organic solvent lixiviate, obtains grease, belongs to biotechnology downstream technical field.
Background technology
Many microorganisms, as yeast, mould, micro-algae, bacterium etc., can be converted into carbohydrate grease under certain condition to be stored in thalline, and this grease is called microbial oil.Can accumulate the bacterial strain that grease surpasses dry cell weight 20% in cell, be called oleaginous microorganism.The fat content of some oleaginous microorganisms can reach more than 70% of dry cell weight (Ratledge C. Acta Biotechnology, 1991,11 (5), 429).The lipid acid of microbial oil forms similar to general Vegetable oil lipoprotein, with C16, C18, is lipid acid, as palmitinic acid, stearic acid, oleic acid and linolic acid are main.In addition, contain a large amount of functional fatty acids in the grease of some bacterial strains, as linolenic acid, arachidonic acid, timnodonic acid, docosahexenoic acid etc.With Vegetable oil lipoprotein production, compare, the microbe oil fermentation cycle is short, is not subject to the impact of place, season, climate change etc., can produce continuously; 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, improve the ecological environment, Developing Biomass Energy Industry has special meaning.Therefore, utilizing microbe transformation method to obtain grease has a high potential.
As oleaginous microorganism culture how is raw material, efficiently, separation and Extraction grease at low cost, be one of bottleneck of realizing the microbial oil large-scale production.For obtaining intracellular grease, usually first from the oleaginous microorganism fermentation liquid, reclaim thalline, carry out drying, then with reference to the Vegetable oil lipoprotein extraction process, extract microbial oil.The oil-containing dry mycelium take in current document as raw material, how the extracting method adopted (conquers east as milling process, 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), ultrasonic wave 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), how supercritical fluid method (conquers 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).The oleaginous microorganism of take wet thallus is also arranged as raw material in document, the report of employing solvent extraction method or acid heat method extraction grease (Li Zhifeng, a tinkling of pieces of jade. microbiology circular, 2001,28 (6), 72).The common feature of aforesaid method is at first by technology such as centrifugal or filtrations, from the oleaginous microorganism fermentation liquid, to isolate thalline.Yet many oleaginous microorganisms are unicellular organism, cell size is little, and the thalline water content is high.In the oleaginous microorganism late stage of culture, due to a large amount of greases of intracellular accumulation, cell density reduces, and fermentation liquid viscosity is large.Therefore, adopt centrifugal or filtering technique Separation and Recovery thalline from fermentation liquid is more difficult, running cost high (Lee AK, Lewis DM, Ashman PJ. Journal of Applied Physiology, 2009,21 (5), 559).Obviously, the grease-contained microbial cells of the richness of take is the material extraction grease, has following one or more defects, comprises complex process, equipment requirements is high, energy consumption is high, use the high toxicity organic solvent.
The grease extracting method that the oleaginous microorganism concentrated broth of take is raw material has high pressure homogenate method (CN101323865) etc., the method need to be concentrated fermented liquid, and high pressure homogenate smudge cells wall technique is high to equipment requirements, energy expenditure is large, the grease extraction cost is high, is not suitable for the microbial oil suitability for industrialized production.The grease extracting method that the oleaginous microorganism fermentation liquid of take is raw material has fermented liquid organic solvent direct extraction (CN101824440A) etc.These methods are used high toxicity solvent just can obtain higher extraction rate as chloroform etc. under higher extraction temperature, though the little solvent of toxicity as No. four solvents, No. six extraction solvent wet goods at high temperature (> 50 oC) lower extraction rate also not high (<55%).These method processing safeties are poor, large to HUMAN HEALTH and environmental damage.
Summary of the invention
The invention provides and a kind ofly take the oleaginous microorganism fermentation liquid as raw material, the method for the auxiliary organic solvent lixiviate grease of enzyme.Present method raw material pretreatment process is simple, and mild condition, energy consumption are low.Because enzyme is processed the obstacle of efficient solution except the solvent extraction grease, can use the organic solvent that toxicity is low, environment is more friendly.
The present invention is achieved by following technical proposals:
1) to adding enzyme in fermentation liquid, to total enzyme concn, be 0.01 g/L～5 g/L, at 20 oC～50 oC, pH 3～8, process 5 minutes～10 hours;
2) fermentation liquid of processing to step 1) adds organic solvent, and solvent load is 0.2 times～5 times of fermentation liquid volume, at 20 oC～60 oC, processes 10 minutes～5 hours;
3) from step 2) reclaim organic phase the mixture processed;
4) organic phase step 3) obtained is removed organic solvent according to a conventional method, obtains microbial oil.
The enzyme used in technical scheme of the present invention is cellulase, hemicellulase, polygalacturonase, β-1,3-mannonase beta-glucanase, α-1,3-dextranase, proteolytic enzyme, Proteinase K, Phospholipid hydrolase, helicase, chitinase etc. or the combination of their necessity.Wherein, cellulase, hemicellulase, polygalacturonase, beta-glucanase, proteolytic enzyme is buied from Ningxia jade of the He family Bioisystech Co., Ltd, be respectively 50,000 U/g than vigor, 1200000 U/g, 3.5 ten thousand U/g, 1200000 U/g, 800000 U/g, Proteinase K is buied from precious biotechnology (Dalian) company limited, than vigor, be 3.0 ten thousand U/g, Phospholipid hydrolase is buied from letter (China) Investment Co., Ltd of Novi, than vigor, be 1.0 ten thousand U/g, helicase is buied from the prosperous Bioisystech Co., Ltd of Beijing ancient cooking vessel state, β-1, the 3-mannase be laboratory, contriver place with pichia spp (
Pichia pastorisX-33) be the host, by 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, α-1, the 3-dextranase is laboratory, contriver place reference literature (Shalom G, J Pratten, et al. Protein Expression and Purification, 2008, 60 (2), 170) preparation, than vigor, be 0.39 ten thousand U/g, chitinase is laboratory, contriver place reference literature (Tsujibo H, H Orikoshi, et al. Journal of Bacteriology, 1993, 175 (1), 176) preparation, than vigor, be 1.0 ten thousand U/g.
The organic solvent that the present invention uses is solubilized grease under room temperature, liquid state organics that polarity is lower, as sherwood oil (boiling spread 60 oC～90 oC), normal hexane, ethyl acetate, methylene dichloride, No. six solvent for extraction, No. four solvents, industrial hexane (boiling spread 66 oC～69 oC) etc. or their necessity combination.
The present invention removes the method that organic solvent obtains microbial oil mutually from organic solvent be air distillation or vacuum-evaporation etc.
The fermentation liquid that the present invention uses, with before zymin contact, can also combine and be processed by heating, microwave radiation, supersound process etc. or their necessity, to reach, makes oleaginous microorganism endogenous protein inactivation or falls SA purpose.
The oleaginous microorganism that the present invention uses can surpass dry cell weight 20%(w/w for thalline fat content after fermentation culture) fungi, micro-algae, bacterium or genetic engineering bacterium, they include but not limited to, the produce oil fungi, as justify red winter spore yeast (
Rhodosporidium toruloides), white Cryptococcus (
Cryptococcus albidus), crooked Cryptococcus (
Cryptococcus curvatus), inferior sieve solution fat yeast (
Yarrowia lipolytica), rhodotorula glutinis (
Rhodotorula glutinis), the lactose rhodotorula (
Rhodotorula lactosa), little rhodotorula (
Rhodotorula minuta), tangerine woods saccharomyces oleaginosus (
Lipomyces kononenkoae), trichosporon cutaneum (
Trichosporon cutaneum), Trichosporon fermentans (
Trichosporon fermentans), strong doughtily mould (
Geotrichum robustum), Mortierella isabellina (
Mortierella isabellina), a volume branch Mucor (
Mucor circinelloides), Cunninghamella sp (
Cunninghamella) and the Christian Breton endomycopsi.sp (
Endomycopsis burtonii); Oil-producing microalgae, as Botryococcus braunii (
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 as the grease extraction yield of benchmark additive method.
Embodiment
Following examples have been chosen fermentation liquid and the enzyme thereof of some typical oleaginous microorganisms and have been processed and the grease leaching process, contribute to understand this patent, but be not limited in any form on other produce oil bacterial strain materials, do not apply the present invention.
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 is in Chinese common micro-organisms culture presevation administrative center), the fermentation liquid that the density that obtains fermenting is 114 g dry myceliums (CDW)/L, the dry mycelium fat content is 63%.Get above-mentioned fermentation liquid 50 ml, then add 50 ml ethyl acetate, fully mix under 25 oC, lixiviate 1 h, organic phase is taken out in centrifugation, and evaporating solvent obtains 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 is in Chinese common micro-organisms culture presevation administrative center), the fermentation liquid that the density that obtains fermenting is 114 g dry myceliums (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, processes 0.5 h under pH 5.0,25 oC; Add again 50 ml ethyl acetate, fully mix under 25 oC, lixiviate 1 h, organic phase is taken out in centrifugation, and evaporating solvent obtains microbial oil, and extraction rate is 95%.
The experimental result of comparing embodiment 1 and Comparative Examples 1, show that fermentation liquid is through microwave and β-1, and the 3-mannase can extract grease very effectively by ethyl acetate after processing; And directly from sweet mash liquid, be difficult to extract separate grease by ethyl acetate.Therefore, the pretreatment process of fermentation liquid extracts to separate to microbial oil and has unusual effect.
Embodiment 2
Other operational conditions, with embodiment 1, are used β-1 but save in leaching process, and the 3-mannase is processed the step of fermentation liquid.Extraction rate is 35%.Comparing embodiment 1 and embodiment 2 illustrate that with β-1 the 3-mannase is processed 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 leaching process.Extraction rate is 30%.
Comparing embodiment 1 and embodiment 3, explanation is carried out microwave treatment before enzyme is processed, and can significantly improve the grease extraction yield, and its reason may be that microwave treatment causes oleaginous microorganism endogenous protein inactivation or active significantly reduction, more be conducive to β-1,3-mannase performance effect.
Embodiment 4
Other operational conditions are with embodiment 1, but in the processing mode that enzyme is processed the primary fermentation mash are: under 121 oC, process 2 minutes.Extraction rate is 93%.
Comparing embodiment 1 and embodiment 4, illustrate in β-1, and the 3-mannase carries out pyroprocessing before processing, and can obtain the effect approximate with microwave treatment.
Embodiment 5
Other operational conditions are with embodiment 1, but in the processing mode that enzyme is processed the primary fermentation mash are: under 70 oC, process 10 minutes.Extraction rate is 87%.
Comparing embodiment 1 and embodiment 4, embodiment 5, the temperature and time that pyroprocessing is carried out in explanation before enzyme is processed can change in wider scope, as long as can make oleaginous microorganism endogenous protein inactivation or active significantly reduction, can make grease extract and 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 is in Chinese common micro-organisms culture presevation administrative center), the fermentation liquid that the density that obtains fermenting is 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, processes 10 h under pH 5.0,25 oC; Add again 100 ml ethyl acetate, fully mix under 25 oC, lixiviate 1 h, organic phase is taken out in centrifugation, and evaporating solvent obtains 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 fermentation liquid that the density that obtains fermenting is 90 g CDW/L, the dry mycelium fat content is 40%.Get above-mentioned fermented liquid 50 ml, process 5 minutes under 121 oC.Adding helicase to final concentration is 2.0 g/L, processes 2.0 h under pH 6.5,37 oC; Add again 100 ml methylene dichloride, fully mix under 20 oC, lixiviate 1 h, organic phase is taken out in centrifugation, and evaporating solvent obtains 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 is in Chinese common micro-organisms culture presevation administrative center), the fermentation liquid that the density that obtains fermenting is 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, processes 1.0 h under pH 5.0,37 oC; Adjust pH to 7.5, adding Proteinase K to its final concentration is 0.05 g/L, then adds 250 ml ethyl acetate, under 40 oC, fully mix, and lixiviate 10 min, organic phase is taken out in centrifugation, and evaporating solvent obtains 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 is in Chinese common micro-organisms culture presevation administrative center), the fermentation liquid that the density that obtains fermenting is 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, processes 1.0 h under pH 5.0,25 oC; Adjust pH to 7.0, adding polygalacturonase to its final concentration is 0.05 g/L, continues to process 30 min under 25 oC; Add again 25 ml normal hexanes and 50 ml sherwood oils, fully mix under 25 oC, lixiviate 5.0 h, organic phase is taken out in centrifugation, and evaporating solvent obtains 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 is in Chinese common micro-organisms culture presevation administrative center), the fermentation liquid that the density that obtains fermenting is 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-dextranase and chitinase to its final concentration is respectively 0.40 g/L, 0.10 g/L and 0.08 g/L, processes 1.0 h under pH 5.0,37 oC; Add again industrial hexane 150 ml, fully mix under 60 oC, lixiviate 20 min, organic phase is taken out in centrifugation, and evaporating solvent obtains 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 is in Chinese common micro-organisms culture presevation administrative center), the fermentation liquid that the density that obtains fermenting is 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 helicase to final concentration are 0.60 g/L, processes 1.0 h under pH 6.0,37 oC; Add again ethyl acetate 100 ml, fully mix under 50 oC, lixiviate 30 min, organic phase is taken out in centrifugation, and evaporating solvent obtains microbial oil, and extraction rate is 90%.
Embodiment 12
Cultivate oil-producing microalgae 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 fermentation liquid that the density that obtains fermenting is 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 helicase to its final concentration are 4.0 g/L, processes 2.0 h under pH 7.0,50 oC; Add again No. four solvent 20 ml and ethyl acetate 80 ml, fully mix under 40 oC, lixiviate 1 h, organic phase is taken out in centrifugation, and evaporating solvent obtains microbial oil, and extraction rate is 93%.
Embodiment 13
Cultivate oil-producing microalgae according to the described method of document (Xiong W, Li XF, Xiang JY, et al. Applied Microbiology and Biotechnology, 2008,78,29)
C. protothecoidesThe CS-41(bacterium source is in the micro-algae of Australian CSIRO research centre), the fermentation liquid that the density that obtains fermenting is 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, processes 1.5 h under pH 7.0,37 oC; Add ethyl acetate 100 ml, fully mix under 40 oC, lixiviate 1 h, organic phase is taken out in centrifugation, and evaporating solvent obtains microbial oil, and extraction rate is 89%.
The invention has the beneficial effects as follows:
With take the grease extracting method that dry mycelium, wet thallus, concentrated broth be raw material and compare, the present invention does not need concentrated broth, reclaims the operations such as thalline, dry thalline, overcome and directly from the high viscosity fermentation mash, reclaimed the technical barrier of thalline, simplified processing step, save energy consumption, reduced cost; And, by grease in effective extraction cell, can also increase cell density, contribute to later stage solid-liquid separation operation and bacterium slag to reclaim;
With methods such as milling process, pressure solvent method, ultrasonic wave secondary solvent extraction, microwave-assisted solvent extraction, supercritical fluid method, acid heat method or high pressure homogenization methods, compare, 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; Simultaneously little to the components such as grease, protein destruction, the oil quality obtained is high, and the bacterium slag is of high nutritive value;
With solvent extraction method, with the acid heat method, compare, solvent for use toxicity of the present invention is little, and residual few at water, solvent loss is few, more friendly to environment, and process is more clean;
With fermentation liquid organic solvent direct extraction, compare, the present invention is owing to using enzyme to process the obstacle of efficient solution except the extracting grease, 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 technology of the present invention is simply effective, and facility investment is few, and energy consumption is low, has improved the Technical Economy of microbe oil fermentation, is conducive to large-scale industrial production.
Claims (6)
1. a microbial oil extraction method, it is characterized in that: the fermentation liquid of the red winter spore yeast of oleaginous yeast circle, rhodotorula glutinis, lactose rhodotorula or little rhodotorula is carried out to inactivation treatment, use again β-1 in 20 ℃～50 ℃ scopes of temperature, 3-mannonase polygalacturonase, α-1, necessity combination of one or two or more kinds in 3-dextranase, Phospholipid hydrolase, chitinase is carried out enzyme and is processed 5 minutes～10 hours, then the fermentation liquid of processing with organic solvent lixiviate enzyme, the Separation and Recovery organic phase, except desolventizing, obtain microbial oil.
2. according to the described method of claim 1, it is characterized in that: the enzyme treatment condition are mash pH=3～8, enzyme dosage 0.01g/L～5g/L.
3. according to the described method of claim 1, it is characterized in that: the liquid state organics that described organic solvent is solubilized grease under room temperature, polarity is low is one or two or more kinds necessity combination in 60 ℃～90 ℃ sherwood oils of boiling spread, normal hexane, ethyl acetate, methylene dichloride, No. six solvent for extraction, No. four solvents, 66 ℃～69 ℃ industrial hexane of boiling spread.
4. according to the described method of claim 1, it is characterized in that: described extracting condition is 20 ℃～60 ℃ of temperature, and 10 minutes～5 hours time, the volume ratio of organic solvent and fermentation liquid is 1:5～5:1.
5. according to the described method of claim 1, it is characterized in that: the inactivation treatment method is one or two or more kinds necessity combination in heating, microwave radiation, ultrasonication.
6. it is characterized in that in accordance with the method for claim 1: the fermentation liquid of described oleaginous microorganism is the suspension liquid that contains thalline that oleaginous yeast obtains under the liquid culture condition.
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