CN102234670A - Method for producing bacterial cellulose through solid state fermentation by using inert adsorption carrier - Google Patents
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Abstract
The invention discloses a method for producing bacterial cellulose through solid state fermentation by using inert adsorption carriers. According to the invention, steam exploded straws are adopted as a raw material. The method employs biomass resources with low cost and wide sources, such that a problem of high carbon source cost in prior arts is overcome, and the product provided by the invention can be widely applied as a bio-based material with high added value. According to the invention, the straw resource is processed through a pretreatment process of steam explosion, such that cellulose and hemicellulose components of the straws can be fully utilized, and accessibility and utilance of cellulase is improved. With the method of solid state fermentation by using inert adsorption carriers, a problem of oxygen dissolving difficulty during a standing cultivating process of bacterial cellulose fermentation is solved, and shearing and damaging effects to the strains during a dynamic fermentation process can be avoided. The carriers can be repeatedly utilized after being cleaned.
Description
Technical field
The present invention relates to field of fermentation engineering, particularly a kind of is raw material with the steam puffed stalk, the method that adopts inertia absorption carrier solid state fermentation to produce bacteria cellulose.
Background technology
Mierocrystalline cellulose be the abundantest on the earth, with the closest biosynthesizing material of human lives's relation.Mierocrystalline cellulose extensively exists in the plants such as trees, cotton, also has to be present in some bacteriums and indivedual rudimentary animal on a small quantity, and Mierocrystalline cellulose is the chain polymer that D-glucose is formed by connecting with β-1,4 glycosidic link, has (C
6H
10O
5) composition of n, be the main component of vascular plant, lichen and a part of alga cells wall.Synthetic cellulose is the function that has of plant just, and certain micro-organisms is synthetic cellulose efficiently also, and acetobacter xylinum (Acetobacter xylinumX-2 is called for short Ax) is exactly outstanding representative wherein.Discovery and chemical qualitativity by acetobacter xylinum synthetic bacteria cellulose have been done reported first as far back as 1886 by Brown, and to it characteristic understanding and industrial applications after 100 years just and with realization.Compare with the traditional plant Mierocrystalline cellulose, bacterial fibers have many premium propertiess, as the nanometer fineness of high purity, high-polymerization degree, high-crystallinity, high-hydrophilic, high Young's modulus, high strength and fiber.Because these characteristics make it become a kind of very potential novel biomaterial at field of light industry such as food, medicine, chemical industry, nonwoven fabrics.
The composition of substratum has very big influence to cellulose output in the efficient fermenting bacteria Mierocrystalline cellulose of the acetobacter xylinum process.The cellulosic substratum of acetobacter xylinum fermenting bacteria generally comprises: as the glucose (or fructose) of carbon source, yeast extract (or peptone), organic acid (as lactic acid, acetic acid, citric acid), inorganic salt as nitrogenous source (as Na
2HPO
4Or contain Ga
2+, Mg
2+Salt), also add caffeine, vitamin H etc. sometimes, seek the fermention medium of best proportioning by certain optimization method, can improve cellulose output greatly.But production cost can improve greatly in this process, thus at present generally with it as high value added product.For carrying out scale operation, just essentially seek raw materials low-cost, wide material sources, wait the fermentative production Mierocrystalline cellulose as rice bran or degrease rice bran, corn leaching solution, sugar cane juice, waste molasses, but these raw materials are because the low output that causes of purity is difficult to improve.
Crop material is important Biological resources in the farm crop production system.According to incomplete estimation, the whole world can produce the stalk of nearly 2,000,000,000 t every year.It mainly is made up of Mierocrystalline cellulose, hemicellulose and xylogen three parts, and after suitable pre-treatment, content of cellulose increases in the wheat stalk, and the imporosity between Mierocrystalline cellulose, hemicellulose and xylogen is destroyed.Utilize cellulase can make Mierocrystalline cellulose, hydrolysis of hemicellulose be soluble sugar, and then the soluble sugar that generates be converted into clean fuel by microbial fermentation---alcohol or other high value-added product (as bacteria cellulose).Along with the enhancing of people to the consciousness of the necessity of the sense of urgency of fossil energy crisis and environment protection, people are more and more surging to the research and the application enthusiasm of reproducible, environmentally friendly bio-based materials.And bacteria cellulose is as the polysaccharide polymer that has huge commercial promise in a kind of industrial production, if can be the raw material that sets out with biomass resources low-cost, wide material sources, carry out scale operation, the bio-based materials that then can be used as a kind of high added value is used widely.
Aspect zymotechnique, the cultural method of bacteria cellulose roughly is divided into stationary method and dynamic method in industrial production at present, and both all belong to the category of liquid submerged fermentation.Stationary method is meant that acetobacter xylinum leaves standstill cultivation, produces cellulose membrane on the fermented liquid surface.Yet, can limit effective transmission of oxygen owing to cellulosic formation in the static cultivation process, thereby further limit cellulosic synthetic.Dynamic method is to ventilate to cultivating acetobacter xylinum in mechanical agitator tank or air lift type biochemical reactor, can solve the problem of dissolved oxygen difficulty to a certain extent.But, in dynamic fermenting process, being subjected to the influence of shearing force, thalline is easily to the direction sudden change of not producing bacteria cellulose, thus reduction output.
Therefore improving zymotechnique obtains the ideal cellulose output and has become bacteria cellulose applied research focus.For example, report usage level fermentor tank (horizontal fermentors), in culturing process, can feed competent oxygen-rich air, improve the oxygen turnover ratio, the Okiyarm of or employing two-step fermenting---Japan and Shirage adopt this method to optimize the Mierocrystalline cellulose production process, compare remarkable in economical benefits with traditional industry.Though these methods all increase on output for traditional fermentation process, these methods all can not well solve oxygen supply and reduce bacterial classification shear damage between contradiction.Therefore under study for action, should leave standstill by abundant combination and cultivate and problem that the advantage of ventilation stir culture avoids both to exist.
Since the sixties in this century, a kind of new fermentation mode occurred, it is a solid phase in the solid ferment process with the inert solid carrier, to be adsorbed on nutrient solution on the carrier as nutrition, its solid state fermentation that appears as provides new thinking, has widened application prospect.Solid-state carrier in the inert support solid state fermentation provides the huge surface of microorganism growth, make microorganism to need under the condition of bubbling air or oxygen on a small quantity or not in feeding, from the external world, obtain the required oxygen of self growth, more help the metabolic environment of microorganism growth thereby provide.
According to the characteristics of problem in the bacteria cellulose fermentative production and inertia absorption carrier solid state fermentation, the present invention utilizes steam puffed stalk as raw material, adopts plasticized polyurethane strand foam or polyvinyl chloride plastic strand foam as the inertia absorption carrier, the solid state fermentation bacteria cellulose.Found biomass resources low-cost, wide material sources for the scale operation of bacteria cellulose on the one hand, overcome the oxygen supply problem in the ordinary liquid standing for fermentation process simultaneously and solved the problem of bacterial classification shearing damage in the dynamic fermenting process.
Summary of the invention
In order to solve the drawback of various processes in traditional bacteria cellulose fermentative production---there is the insufficient problem of oxygen supply in liquid standing for fermentation method, and the dynamic agitation fermentation process that can improve the dissolved oxygen situation can cause the shearing damage of fermented bacterium---and provide a kind of inertia absorption carrier solid state fermentation to produce the method for bacteria cellulose.Solid-state carrier in the inert support solid state fermentation provides the huge surface of microorganism growth on the one hand, make microorganism to need under the condition of bubbling air or oxygen on a small quantity or not in feeding, from the external world, obtain the required oxygen of self growth, solved the insufficient problem of dissolved oxygen; The shearing force that the solid state fermentation culture condition that leaves standstill is on the other hand avoided is for the damaging action of bacterial classification.
Technical scheme of the present invention is as follows:
Method of producing bacteria cellulose provided by the invention with inertia absorption carrier solid fermentation and straw puffing, it may further comprise the steps: the 1) preparation of steam puffed stalk enzymolysis solution; 2) preparation acetobacter xylinum seed culture fluid; 3) preparation acetobacter xylinum fermention medium, inoculation, and mix co-cultivation with the inertia absorption carrier and produce bacteria cellulose; 4) separation and Extraction of fermentation after product bacteria cellulose.
Its concrete technology is as follows:
1) be to carry out the quick-fried pre-treatment of vapour under the condition of 0.9～1.9MPa, dimension pressure time 1～5min with water content at 10～25% straw vapor pressure in the self-control steam blasting device, air-dry; Or be to carry out under the condition of 0.9～1.9MPa after steam explosion handles at vapor pressure at 10～25% straw with water content, through air-dry standby after twice washing;
With steam puffed stalk and the distilled water solid-to-liquid ratio of steam puffed stalk according to 1: 6～1: 12, the cellulase consumption of the quick-fried stalk of 20～40FPU/g net gas, enzymolysis 48～72h; After enzymolysis finished, through 4000r/min, 30min was centrifugal, obtains the steam puffed stalk enzymolysis solution, needed adjustment system pH to 4.5～4.8 before the enzymolysis.
2) will be inoculated in the acetobacter xylinum slant medium available from acetobacter xylinum (Acetobacter xylinum) the CGMCC No.1.1812 at Chinese microbial preservation management committee common micro-organisms preservation center, 30 ℃ of following inoculation culture 1d, being inoculated in seed culture medium, to be cultured to the bacteria suspension optical density(OD) constant, obtains the acetobacter xylinum seed culture fluid;
The slant culture based component is: glucose 20g/L, beef extract-peptone 10g/L, yeast powder 5g/L, citric acid 1.15g/L, Na
2HPO
412H
2O 6.8g/L, MgSO
45H
2O 0.51g/L, agar 20g/L, pH value 6.8;
Seed culture medium: glucose 20g/L, beef extract-peptone 10g/L, yeast powder 5g/L, citric acid 1.15g/L, Na
2HPO
412H
2O 6.8g/L, MgSO
45H
2O 0.51g/L, pH value 6.8;
3) get exsiccant inertia absorption carrier and put into the 150mL Erlenmeyer flask, the carrier piling height is 1～5cm, and also the cooling back was standby in 20 minutes in 121 ℃ of sterilizations; Behind the culture medium inoculated, press 1: 6～1: 18 mixed with the inertia absorption carrier, and under aseptic condition, make abundant, the evenly absorption of substratum, be placed on 25～35 ℃ of cultivation 4～7d in the fixed temperature and humidity incubator with glass stick extruding inertia absorption carrier
Fermention medium is based on seed culture medium, and its composition is: adjusting glucose concn is the steam puffed stalk enzymolysis solution of 20g/L, beef extract-peptone 10g/L, yeast powder 5g/L, citric acid 1.15g/L, Na
2HPO
412H
2O 6.8g/L g/L, MgSO
45H
2O 0.51g/L, pH value 6.8.
4) obtain fermented liquid by overstocking after the fermentation ends, to add long-pending 95% ethanol sedimentation of triploid and add 4g/LNaOH solution after fully and boil suction filtration after 30 minutes, water washes to pH and is neutral in the suction filtration process, promptly gets the product bacteria cellulose.
Beneficial effect of the present invention is:
1. bacteria cellulose is the polysaccharide polymer that has huge commercial promise in a kind of industrial production, be the raw material that sets out with biomass resource such as wheat stalks low-cost, wide material sources, the scale operation bacteria cellulose is applied its bio-based materials that can be used as a kind of high added value;
2. solved the influence of dissolved oxygen restriction for fermented-producing bacteria cellulose.The liquid standing for fermentation method of general employing in traditional bacteria cellulose fermentative production, promptly the product cellulose membrane produces on the fermented liquid surface.Yet effective transmission of oxygen can be restricted in this process, thereby makes dissolved oxygen become the limiting factor of bacteria cellulose fermentative production.And cultivate acetic bacteria at mechanical agitator tank, though can solve the problem of dissolved oxygen restriction to a certain extent, in the later stage of fermentation, fermented liquid presents the feature of pseudoplastic fluid, even the dead band that high-power stirring also often exists dissolved oxygen to be difficult to arrive.Solid-state carrier in the inert support solid state fermentation provides the huge surface of microorganism growth, make microorganism to need under the condition of bubbling air or oxygen on a small quantity or not in feeding, from the external world, obtain the required oxygen of self growth, solved the insufficient problem of dissolved oxygen; Product output improves 2～3 times, and the carbon source transformation efficiency improves.
3. need not stir in the fermenting process, the shearing force that the solid state fermentation culture condition that leaves standstill is avoided is for the damaging action of bacterial classification.
4. the inertia absorption carrier adopts urethane foam or polyvinyl chloride foam, is easy to clean, and is with low cost, can reuse.
5. when improving bacteria cellulose output, make fermentation period shorten to 4d from 10d.
6. through fourier infrared (FT-IR) assay products structure, the strong and standard cellulose spectrogram in spectrogram peak position that obtains and peak matches.Through the molecular weight of viscosity method mensuration fermentation gained bacteria cellulose, the products therefrom molecular weight is about common 1/3 of the liquid fermentation method bacteria cellulose polymerization degree that leaves standstill.High molecular weight bacteria cellulose indissoluble in usual vehicle, make its exploitation aspect medicinal carrier material, and the progress of structure activity relationship is slow, so, the low relative molecular mass bacteria cellulose that synthesizing water-solubility is good relatively has breakthrough in the hope of the research to bacteria cellulose, and further does some element tasks for it is developed as drug carrier material.
Embodiment
Below by embodiment technical scheme of the present invention is described further.
Embodiment 1:
Be to carry out the quick-fried pre-treatment of vapour under the condition of 1.0MPa, dimension pressure time 3.0min with water content at 15% wheat stalk vapor pressure in the self-control steam blasting device, air-dry; With steam puffed stalk and the distilled water solid-to-liquid ratio of steam puffed stalk according to 1: 8, the cellulase consumption of the quick-fried stalk of 40FPU/g net gas, enzymolysis 72h; After enzymolysis finished, through 4000r/min, 30min was centrifugal, obtains the steam puffed stalk enzymolysis solution.Need adjustment system pH to 4.8 before the enzymolysis.Measure through HPLC, glucose concn is 32.8g/L in the enzymolysis solution, and wood sugar is 8.6g/L.
Get the 2g exsiccant length of side and be the urethane foam cubes of 0.7cm and put into the 150mL Erlenmeyer flask, 121 ℃ of sterilization 20min and cooling.
According to following configuration fermention medium: dilution enzymolysis solution to glucose concn is 20g/L, according to beef extract-peptone 10g/L, and yeast powder 5g/L, citric acid 1.15g/L, Na
2HPO
412H
2O 6.8g/L, MgSO
45H
2O 0.51g/L adds other nutritive ingredients, regulates pH value to 6.8.
Behind the culture medium inoculated, both are mixed, under aseptic condition, make abundant, the evenly absorption of substratum, be placed in the fixed temperature and humidity incubator and cultivate 4d with glass stick extruding urethane foam according to 1: 10 carrier/substratum solid-to-liquid ratio.In the Erlenmeyer flask of fermentation, add distilled water after the fermentation ends in fermented liquid and 1: 1 ratio of distilled water, then the extracting solution in the urethane foam is extruded or come out by centrifugation, add long-pending 95% ethanol sedimentation of triploid fully after, after add 4%NaOH solution and boil suction filtration after 30 minutes, water flushing in the suction filtration process.Product output reaches 6.57g/L.
Embodiment 2:
Be to carry out the quick-fried pre-treatment of vapour under the condition of 1.5MPa, dimension pressure time 5.0min with water content at 20% maize straw vapor pressure in the self-control steam blasting device, air-dry; With steam puffed stalk and the distilled water solid-to-liquid ratio of steam puffed stalk according to 1: 10, the cellulase consumption of the quick-fried stalk of 20CFU/g net gas, enzymolysis 48h; After enzymolysis finished, through 4000r/min, 30min was centrifugal, obtains the steam puffed stalk enzymolysis solution.Need adjustment system pH to 4.5 before the enzymolysis.Measure through HPLC, glucose concn is 21.2g/L in the enzymolysis solution, and wood sugar is 6.5g/L.
Get the 2g exsiccant length of side and be the polyvinyl chloride foam cubes of 0.7cm and put into 150 milliliters of Erlenmeyer flasks, 121 ℃ of sterilizations 20 minutes and cooling.
According to following configuration fermention medium: dilution enzymolysis solution to glucose concn is 20g/L, beef extract-peptone 10g/L, yeast powder 5g/L, citric acid 1.15g/L, Na
2HPO
412H
2O 6.8g/L, MgSO
45H
2O 0.51g/L, pH value 6.8.
Behind the culture medium inoculated, both are mixed, under aseptic condition, make abundant, the evenly absorption of substratum, be placed in the fixed temperature and humidity incubator and cultivate 4d with glass stick extruding polyvinyl chloride foam according to 1: 16 carrier/substratum solid-to-liquid ratio.After the fermentation ends extracting solution in the polyvinyl chloride foam is extruded or is come out by centrifugation, add long-pending 95% ethanol sedimentation of triploid fully after, after add 4%NaOH solution and boil suction filtration after 30 minutes, water flushing in the suction filtration process.Product output reaches 7.32g/L.
Embodiment 3:
Be to carry out the quick-fried pre-treatment of vapour under the condition of 1.0MPa, dimension pressure time 2.0min with water content at 20% wheat straw vapor pressure in the self-control steam blasting device, air-dry; With steam puffed stalk and the distilled water solid-to-liquid ratio of steam puffed stalk according to 1: 8, the cellulase consumption of the quick-fried stalk of 30FPU/g net gas, enzymolysis 48h; After enzymolysis finished, through 4000r/min, 30min was centrifugal, obtains the steam puffed stalk enzymolysis solution.Need adjustment system pH to 4.5 before the enzymolysis.Measure through HPLC, glucose concn is 28.4g/L in the enzymolysis solution, and wood sugar is 8.5g/L.
Get the 2g exsiccant length of side and be the urethane foam cubes of 1.0cm and put into the 150mL Erlenmeyer flask, 121 ℃ of sterilization 20min and cooling.
According to following configuration fermention medium: glucose 20g/L, beef extract-peptone 10g/L, yeast powder 5g/L, citric acid 1.15g/L, Na
2HPO
412H
2O 6.8g/L g/L, MgSO
45H
2O 0.51g/L, pH value 6.8.Behind the culture medium inoculated, both are mixed, under aseptic condition, make abundant, the evenly absorption of substratum, be placed in the fixed temperature and humidity incubator and cultivate 4d with glass stick extruding urethane foam according to 1: 16 carrier/substratum solid-to-liquid ratio.After the fermentation ends extracting solution in the urethane foam is extruded or is come out by centrifugation, add long-pending 95% ethanol sedimentation of triploid fully after, after add 4%NaOH solution and boil suction filtration after 30 minutes, water flushing in the suction filtration process.Product output reaches 5.26g/L.
Claims (7)
1. the method for an inertia absorption carrier solid state fermentation bacteria cellulose, it may further comprise the steps:
1) preparation of steam puffed stalk enzymolysis solution;
2) preparation acetobacter xylinum seed culture fluid;
3) preparation acetobacter xylinum fermention medium is inoculated, and produces bacteria cellulose with inertia absorption carrier mixed culture;
4) separation and Extraction of fermentation after product bacteria cellulose.
2. according to method described in the claim 1, it is characterized in that step 1) is to carry out the quick-fried pre-treatment of vapour under the condition of 0.9～1.9MPa, dimension pressure time 1～5min at 10～25% straw vapor pressure in the self-control steam blasting device with water content, and is air-dry; Or be to carry out under the condition of 0.9～1.9MPa after steam explosion handles at vapor pressure at 10～25% straw with water content, through air-dry standby after twice washing; With steam puffed stalk and the distilled water solid-to-liquid ratio of steam puffed stalk according to 1: 6～1: 12, the cellulase consumption of the quick-fried stalk of 20～40FPU/g net gas, enzymolysis 48～72h; After enzymolysis finished, through 4000r/min, 30min was centrifugal, obtains the steam puffed stalk enzymolysis solution, needed adjustment system pH to 4.5～4.8 before the enzymolysis.
3. according to method described in the claim 1, it is characterized in that, step 2) acetobacter xylinum (Acetobacterxylinum) CGMCC No.1.1812 is inoculated in the acetobacter xylinum slant medium, cultivate 1d for 30 ℃, be inoculated in that to be cultured to the bacteria suspension optical density(OD) in the seed culture medium constant, obtain the acetobacter xylinum seed culture fluid.
4. according to method described in the claim 1, it is characterized in that step 3) is got exsiccant inertia absorption carrier and put into the 150mL Erlenmeyer flask, the carrier piling height is 1～5cm, and also the cooling back was standby in 20 minutes in 121 ℃ of sterilizations; Behind the culture medium inoculated, press 1: 6～1: 18 mixed with the inertia absorption carrier, and under aseptic condition, make abundant, the evenly absorption of substratum, be placed on 25～35 ℃ of cultivation 4～7d in the fixed temperature and humidity incubator with glass stick extruding inertia absorption carrier.
5. according to method described in the claim 1, it is characterized in that, obtain fermented liquid by extruding after the step 4) fermentation ends, the NaOH solution that the ethanol sedimentation that adds long-pending 95% volume fraction of triploid adds 4g/L after fully boils suction filtration after 30 minutes, water washes to pH and is neutral in the suction filtration process, promptly gets the product bacteria cellulose.
6. according to method described in the claim 1, its feature is that also the described steam puffed stalk of step 1) comprises that the quick-fried wheat stalk of vapour, steam exploded wheat straw are or/and the quick-fried maize straw of vapour.
7. according to method described in the claim 1, its feature also is step 2) described in the inertia absorption carrier be urethane foam or polyvinyl chloride foam.
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| CN104026507A (en) * | 2014-06-16 | 2014-09-10 | 河北欧亚匡食品集团有限公司 | Method for producing Chinese date fruit by utilizing waste preserved date liquid glucose |
| CN107312808A (en) * | 2016-04-26 | 2017-11-03 | 财团法人食品工业发展研究所 | Method for producing cellooligosaccharide |
| CN109055457A (en) * | 2018-10-08 | 2018-12-21 | 天津科技大学 | A method of bacteria cellulose is produced using grape skin |
| CN115627277A (en) * | 2022-11-18 | 2023-01-20 | 中国科学院过程工程研究所 | Method for performing solid state fermentation on 2, 3-butanediol by high-solid-state enzymolysis of straws |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104026507A (en) * | 2014-06-16 | 2014-09-10 | 河北欧亚匡食品集团有限公司 | Method for producing Chinese date fruit by utilizing waste preserved date liquid glucose |
| CN107312808A (en) * | 2016-04-26 | 2017-11-03 | 财团法人食品工业发展研究所 | Method for producing cellooligosaccharide |
| CN109055457A (en) * | 2018-10-08 | 2018-12-21 | 天津科技大学 | A method of bacteria cellulose is produced using grape skin |
| CN115627277A (en) * | 2022-11-18 | 2023-01-20 | 中国科学院过程工程研究所 | Method for performing solid state fermentation on 2, 3-butanediol by high-solid-state enzymolysis of straws |
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