CN102876756B - Process for co-producing lactic acid with lower polyxylose - Google Patents
Process for co-producing lactic acid with lower polyxylose Download PDFInfo
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Abstract
The invention discloses a process for co-producing lactic acid with lower polyxylose. Solid waste residues generated in the process of preparing the lower polyxylose with corns serve as a carbon source and are fermented to prepare the lactic acid. The method includes the steps: (1) taking pretreated corncobs as raw materials and taking fungus xylanase as an initial enzyme preparation to prepare the lower polyxylose; and (2) taking the solid waste residues generated in the production process of the step (1) as raw materials and taking fungus cellulase and beta-glucosidase as enzyme preparations to prepare the lactic acid by means of rhizopus oryzae fermentation. In the method, alkali extraction is performed for hemicellulose of the corncobs by means of endo-xylanase selective enzymatic hydrolysis to prepare the lower polyxylose, the waste residues are produced by the lower polyxylose to prepare the lactic acid, and a low-cost new way is provided for the process for preparing the lower polyxylose by a microbial enzyme method and lactic acid fermentation.
Description
Technical field
The invention belongs to microorganism enzymolysis sugar refining and microorganism fermentation field in biological chemistry, be specifically related to utilize raw material corn cob to produce the technique of xylo-oligosaccharide coproduction lactic acid.
Background technology
Oligose is the low polymerization degree carbohydrate being formed by connecting by glycosidic link by 2～10 monose.Oligomeric sugar ordinary oligosaccharide and functional oligose two classes, functional oligose refers to have special biological function, especially can significantly promote bifidus bacillus propagation in human or animal's enteron aisle, be of value to a class oligose of human or animal's health, i.e. so-called bifidus factor.Except promoting in enteron aisle the probiotics proliferated specificallies such as bifidus bacillus, another important biological function of functional oligose is the immunity system stimulating in human or animal body, thereby improves the immunizing power of human or animal body.
Xylo-oligosaccharide claims again wood oligose, the a kind of of functional oligose, by 2～7 wood sugar molecules with β-1, the general name of the polymkeric substance that 4-glycosidic link is formed by connecting, be " performance is the most stable, propagation bifidus bacillus potency ratio the highest " beneficial bacteria factor in current all oligose, be called as " superpower bifidus factor ".That utilizes that endo-xylanase hydrolyzed xylan substrate obtains take the mixture that xylo-bioses, xylotriose be main component, so the key of enzymolysis process is the adaptability of zytase to substrate, selects suitable zytase.Biological enzyme DeR speed is easy to control, specificity is strong and by product is few.Enzyme process preparation is the main method of producing at present xylo-oligosaccharide, is the method most with prospects for commercial application.
After pretreatment, extract is rich in hemicellulose to corn cob, and hemicellulose is a kind of plant resources of abundant inexhaustible, nexhaustible reproducibility.Hemicellulose is by multiple glycosyl (xylosyl, glucosyl group, mannose group, galactosyl, aralino and rhamanopyranosyl etc.); alditol acidic group (galacturonic acidic group and glucal acidic group etc.) and ethanoyl form, and in molecule often with the general name of the complex plycan of side chain.Xylan is the important component part of hemicellulose, to connect β-D-pyranose form wood sugar cell formation main chain by β-Isosorbide-5-Nitrae-glycosidic link, when its source or branch degree difference, on main chain or side chain, with multiple different substituting group, so the structural changes scope of xylan molecule is larger.Inscribe beta-xylanase Main Function is in macromole xylan with compared with the oligose of long-chain, and primary product is xylo-oligosaccharide.Xylo-oligosaccharide becomes the functional foodstuff receiving much concern now.
After pretreatment, hemicellulose component is used for preparing xylo-oligosaccharide to corn cob, and xylo-oligosaccharide is produced waste residue and generally mixed with coal, as fuel, or makes toilet paper.
Lactic acid, formal name used at school Lactic acid is one of three large organic acids of generally acknowledging in the world.Due to the developing of lactic acid and deep processed product Application Areas, supply falls short of demand in worldwide to have made it, and market has openings expands rapidly.Pfansteihl, except possessing the character of common lactic acid, also has unique biochemistry to be worth in addition, is widely used in the industries such as food, medicine, chemical industry.Human body in human body, only contains LDH, so can only utilize Pfansteihl.At present fermentative Production Pfansteihl has obtained rapid popularization, and one of its reason is the glucose, starch of raw materials for production horn of plenty etc.But with glucose, starch, be that raw material production cost is still higher, limited the application of Pfansteihl, especially limited poly(lactic acid) application at field of Environment Protection as degradable plastics.Around reducing Pfansteihl production cost, take, select cheapness, Mierocrystalline cellulose becomes one of study hotspot as raw material production Pfansteihl widely.Utilize xylo-oligosaccharide to produce waste residue and prepare lactic acid, realized effective utilization of waste resource.
Summary of the invention
The invention technical problem to be solved of this technology, is to provide a kind of technique of xylo-oligosaccharide coproduction lactic acid.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A technique for xylo-oligosaccharide coproduction lactic acid, it is carbon source that the corn cob of take is prepared the solid slag producing in xylo-oligosaccharide process, lactic acid is prepared in fermentation.The method comprises the steps:
(1) take through pretreated corn cob is raw material, take fungi zytase as initial enzyme preparation xylo-oligosaccharide;
(2) take the solid slag producing in step (1) production process is raw material again, take fungal cellulase and beta-glucosidase as zymin, through Rhizopus oryzae fermentation, prepares lactic acid.
Step (1), pre-treatment can be the method that alkali extracting etc. can separate hemicellulose and Mierocrystalline cellulose, can adopt the pretreatment process of physics, chemistry, biology or above several method combined utilization, object is that the hemicellulose components of separating can be used to prepare xylo-oligosaccharide, and xylo-oligosaccharide is produced waste residue and is used for preparing lactic acid.
Step (1), fungi zytase can directly be bought on market, or adopts the zytase self-control of Trichodermareesei (Trichoderma reesei), aspergillus niger (Aspergillus niger), viride (Trichoderma viride) or koning trichoderma (Trichoderma koningii) secretion.
The concrete grammar of step (1) see [Xu Yong, Chen Mu, remaining generation Yuan, etc. xylanase hydrolysis is produced the research of xylo-oligosaccharide. chemistry of forest product and industrial .2002,22 (2): 57-60.].The all solids waste residue producing in step production process may be used to the fermentation of lactic acid.
Step (2), specifically has two kinds of methods:
Method I, substep saccharification and fermentation.The solid slag producing in step (1) production process is mixed with fungal cellulase and beta-glucosidase, add water, being mixed to solid-liquid weight ratio is 1:5～20, and regulating pH value is 5.0 ± 0.5, stirs hydrolysis 48 ~ 96h at 50 ± 5 ℃; Again hydrolyzate is stirred at 40 ℃ to 48 ~ 96h through Rhizopus oryzae fermenting lactic acid, then make through decolouring.
Wherein, described hydrolyzate can be after hydrolysis reaction without the liquid of solid-liquid separation and the mixture of solid, can be also that the supernatant liquor obtaining through solid-liquid separation after hydrolysis reaction is enzymolysis glucose solution.
Method II, synchronous saccharification and fermentation.The solid slag producing in step (1) production process is mixed with fungal cellulase and beta-glucosidase, add water and bacterial classification Rhizopus oryzae, being mixed to solid-liquid weight ratio is 1:5～20, regulating pH value is 5.0 ± 0.5, at 40 ℃, stir hydrolysis 48 ~ 96h and realize simultaneous saccharification and fermentation production lactic acid, then make through decolouring.
In method I and II, for every g Mierocrystalline cellulose, the consumption of cellulase is 15FPIU/g, and the consumption of beta-glucosidase is 8IU/g.Cellulose amount in solid slag can be according to American National renewable energy source laboratory (NREL) method quantitative analysis.
In method I and II, the adjusting of pH can be used pH damping fluid or acid, can be phosphoric acid/sodium phosphate buffer, acetic acid/sodium-acetate buffer, citric acid/sodium citrate damping fluid or sulfuric acid, hydrochloric acid etc.
In method I and II, fungal cellulase and beta-glucosidase can directly be bought on market, or by cellulase and the beta-glucosidase self-control of the Trichoderma (Trichoderma) of fungi, Aspergillus (Aspergillus), Penicillium (Penicillium) and Fusarium (Fusarium) secretion.
In method I and II, described Rhizopus oryzae can be the particle that carrier-free immobilization forms.
In method I and II, described lactic acid decolouring adopts activated carbon decolorizing method, can use Powdered Activated Carbon or granulated active carbon.
In method I and II, stirring can be replaced with shaking table vibration.
In method I and II, need to add nitrogenous source and the required inorganic salt that ferment in the process of fermenting lactic acid, the material of interpolation and consumption those skilled in the art can determine according to lactic acid fermented prior art.
Beneficial effect of the present invention: the inventive method is prepared xylo-oligosaccharide by the hemicellulose of endo-xylanase directional enzymatic alkali extracting corn cob, xylo-oligosaccharide is produced waste residue and is prepared lactic acid, for microbial enzyme method is prepared xylo-oligosaccharide technique and lactic fermentation provides a new way cheaply.
Accompanying drawing explanation
Fig. 1 is that Rhizopus oryzae utilizes xylo-oligosaccharide production waste residue enzymolysis solution substep diastatic fermentation to prepare lactic acid; Lactic acid yield (%)=lactic acid concn/(initial sugared concentration-remaining sugar concentration) * 100%.
Fig. 2 is that Rhizopus oryzae utilizes xylo-oligosaccharide production waste residue band slag enzymolysis solution substep diastatic fermentation to prepare lactic acid; Lactic acid yield (%)=lactic acid concn/(initial sugared concentration-remaining sugar concentration) * 100%.
Fig. 3 is that Rhizopus oryzae utilizes xylo-oligosaccharide production waste residue simultaneous saccharification and fermentation to prepare lactic acid; Lactic acid yield (g/g)=lactic acid concn/(over dry * content of cellulose).
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described content of embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
The product zytase that embodiment mentions, cellulase, beta-glucosidase microorganism and by the method for producing the fermentation of zytase microorganism and prepare zytase, the method of cellulase is prepared in the fermentation of cellulase-producing microorganism, and the method that beta-glucosidase is prepared in the fermentation of product beta-glucosidase microorganism is the technology of well known to a person skilled in the art.
Embodiment 1:
The preparation of cellulase:
1. Trichodermareesei mycelium medium component (g/L): glucose 10.0; Peptone 1.0; Ammonium sulfate 1.4; Urea 0.3; Potassium primary phosphate 2.0; Calcium Chloride Powder Anhydrous 0.3; Magnesium sulfate heptahydrate 0.3; Iron vitriol 0.005; Seven water manganous sulfates 0.0016; Zinc Sulphate Heptahydrate 0.0014; Cobalt chloride 0.002.Substratum regulates pH value to 4.8 with the citrate buffer solution of 1mol/L.
2. trichoderma reesei cellulase synthetic medium composition (g/L): glucose 1.0; Paper pulp 10.0; Peptone 1.0; Ammonium sulfate 1.4; Urea 0.3; Potassium primary phosphate 2.0; Calcium Chloride Powder Anhydrous 0.3; Magnesium sulfate heptahydrate 0.3; Iron vitriol 0.005; Seven water manganous sulfates 0.0016; Zinc Sulphate Heptahydrate 0.0014; Cobalt chloride 0.002.Substratum regulates pH value to 4.8 with the citrate buffer solution of 1mol/L.
3. the cultivation of Trichodermareesei mycelium: 50mL Mycelium culture base is placed in 250mL triangular flask, sterilizing 15min at 121 ℃, be cooled to room temperature, access is preserved in the Trichodermareesei spore of test tube slant in right amount, and it is standby cultivate 36h under 30 ± 1 ℃, 170r/min condition after that shaking flask is placed in constant-temperature table.
4. the preparation of trichoderma reesei cellulase: cultivate based on sterilizing 15min at 121 ℃, be cooled to room temperature, access the Trichodermareesei mycelium of above-mentioned cultivation 36h, be placed in constant-temperature table and cultivate under 170r/min condition, culture temperature first day is controlled at 30 ± 1 ℃, is controlled at 28 ± 1 ℃ later.
5. cultivate 4 days, with whizzer, centrifugal 10min under 3000r/min condition, by separated the removing of the solid matter in nutrient solution (thalline and unemployed paper pulp fiber), obtains cellulose enzyme liquid.
The preparation of beta-glucosidase:
1. the female medium component (g/L) of aspergillus niger kind: glucose 1.0; Peptone 5.Every bottle adds micro-0.05ml, Mandels nutritive salt dope 5ml, 2 of tween 80s.Substratum regulates pH value to 4.8 with the citrate buffer solution of 1mol/L.
2. Aspergillus Niger beta-glucosidase medium component (g/L): corn cob 35; Wheat bran 15; Glucose 1.0; Potassium primary phosphate 2.0; Calcium chloride 0.3; Magnesium sulfate 0.3; Ammonium sulfate 2.11; Urea 1.9; Peptone 5.Every bottle adds micro-0.05mL, 2 of tween 80s.Each triangle bottle graft 5mL kind is female, 1mL nitrogenous source.Substratum regulates pH value to 4.8 with the citrate buffer solution of 1mol/L.
3. black-koji mould filament culture condition: 50mL Mycelium culture base is placed in 250mL triangular flask, sterilizing 15min at 121 ℃, be cooled to room temperature, access is preserved in the aspergillus niger spore of test tube slant in right amount, and it is standby cultivate 36h under 30 ± 1 ℃, 170r/min condition after that shaking flask is placed in constant-temperature table.
4. the preparation of aspergillus niger beta-glucosidase: cultivate based on sterilizing 15min at 121 ℃, be cooled to room temperature, access the black-koji mould filament of above-mentioned cultivation 36h, be placed in constant-temperature table and cultivate under 170r/min condition, culture temperature first day is controlled at 30 ± 1 ℃, is controlled at 28 ± 1 ℃ later.
5. cultivate 10 days, with whizzer, centrifugal 10min under 3000r/min condition, by separated the removing of the solid matter in nutrient solution (thalline and unemployed corn cob, wheat bran), obtains beta-glucosidase enzyme liquid.
The mensuration of filter paper enzyme activity: adopt the standard method that international theory and applied chemistry association (IUPAC) are recommended to measure.Experiment condition: substrate Whatman No.1 filter paper 50mg, the enzyme liquid and the damping fluid that add suitable extension rate, making pH value of reaction system is 4.8, in 50 ℃, the reciprocating constant temperature oscillator of oscillation frequency 80r/min, reacts 60min, the glucose amount that assaying reaction generates.A filter paper enzyme activity unit of force (FPIU) is defined as the enzyme amount that per minute under standard reaction condition generates 1 μ mol glucose.
The mensuration of endoglucanase vigor: endoglucanase vigor represents with carboxymethylcelluloenzyme enzyme activity conventionally.Experiment condition: suspension liquid of carboxyl methyl cellulose substrate 1%(w/v), the enzyme liquid and the damping fluid that add suitable extension rate, making pH value of reaction system is 4.8, in 50 ℃, the reciprocating constant temperature oscillator of oscillation frequency 80r/min, reacts 30min, the glucose amount that assaying reaction generates.The unit of a carboxymethylcelluloenzyme enzyme activity (IU) is defined as the enzyme amount that per minute under standard reaction condition generates 1 μ mol glucose.
The mensuration of beta-glucoside enzyme activity: adopt p-nitrophenyl-β-D-Glucose glycosides (pNPG) for substrate mensuration.0.1mL is after suitably the enzyme liquid of dilution mixes with the pNPG solution (citric acid-Sodium phosphate dibasic damping fluid that is 4.8 by 0.05mol/L, pH value is prepared) of 0.9mL 5mmol/L, adds immediately the Na of 2mL 1mol/L in 50 ℃, the reciprocating constant temperature oscillator of oscillation frequency 80r/min after reacting 10min
2cO
3solution termination reaction, then add 10mL distilled water, shake up.Under 400nm, measure absorbancy.With 0.1mL distilled water, replace enzyme liquid to make blank.Each sample is done 2～3 Duplicate Samples, averages.A beta-glucoside enzyme activity unit (IU) is defined as per minute under standard reaction condition and generates the required enzyme amount of 1 μ mol p-NP.
Also can adopt with the bacterial classification of Trichoderma (Trichoderma), Aspergillus (Aspergillus), Penicillium (Penicillium) and Fusarium (Fusarium) similar method preparation to obtain cellulase, beta-glucosidase.
Existing patent is shown in the preparation of endo-xylanase and separation and purification.
Embodiment 2: xylo-oligosaccharide is produced and the obtaining of cellulosic material.
Corn cob extracts the residue after xylan, and main component is Mierocrystalline cellulose, and its content is 71.87%(butt), moisture content is 72.94%, is a kind of good cellulose resource.Meanwhile, the process of corn cob alkali extracting xylan is in fact also a pretreated process of cellulose raw material alkali.Therefore, corn cob is after alkali extracting, and the raw material without pre-treatment just can be used as lactic acid-producing again, directly carries out the hydrolysis of cellulase.The acquisition process of its raw material is as follows:
1. take 50gNaOH and be fully dissolved in 702mL distilled water, adding grinding particle size is the air-dry corn cob 112g(over dry 100g of 0.5～1cm), at 90 ℃, react 3h.
2. above-mentioned reactant vacuum filtration is removed Xylan extractive, by 500mL water filtering and washing, discards washings.Xylan extractive is used for preparing xylo-oligosaccharide.
3. in above-mentioned filter residue, add 300mL distilled water, with 72%(w/v) sulfuric acid be neutralized to pH value 5.0 ± 0.5, vacuum filtration, and with 3 washings of 900mL distillation moisture, suction filtration, obtain xylo-oligosaccharide and produce waste residue.
It is exactly the needed lactic acid-producing raw material of this technology that this xylo-oligosaccharide is produced waste residue; Pretreated object is to extract xylan, improve the accessibility of Mierocrystalline cellulose to cellulase in cellulose raw material, what can adopt physics, chemistry, biology or above several method combined utilization can be by the effectively separated pretreatment process of hemicellulose, Mierocrystalline cellulose simultaneously.
Embodiment 3: substep diastatic fermentation is prepared lactic acid.
1. Rhizopus oryzae utilizes the xylo-oligosaccharide production waste residue substep diastatic fermentation of embodiment 2 to prepare lactic acid.Xylo-oligosaccharide is produced waste residue and is placed on 4 ℃ of refrigerator equilibrium water contents two days, after survey moisture, extracting, surveys three elements.The xylo-oligosaccharide that takes respectively embodiment 2 is produced the heavy 5g of waste residue 18.48g(over dry, Mierocrystalline cellulose 3.59g wherein, moisture 13.48g) in 250mL triangular flask, add cellulase solution 0.45g(enzyme dosage 15FPIU/g Mierocrystalline cellulose), beta-glucosidase 0.08g(enzyme dosage 8IU/g Mierocrystalline cellulose), add damping fluid and distilled water, hierarchy of control solid-liquid weight ratio is 1:20, is that water volume is 100g, pH5.0 ± 0.5, is placed in shaking table enzymolysis 48h under 50 ± 5 ℃ of conditions of temperature of 150r/min.
2. sample 1mL, deactivation 5min in 100 ℃ of water-baths, centrifugal 5min under 10000r/min, gets the suitably rear concentration of measuring fermentable sugars wherein of dilution of supernatant liquor.
3. enzymolysis solution centrifuging and taking enzymolysis clear liquid.
4. basic medium (g/L) ferments: glucose 50; Ammonium sulfate 3; The independent sterilizing of calcium carbonate 10(); Potassium primary phosphate 0.30; Magnesium sulfate heptahydrate 0.75; Zinc Sulphate Heptahydrate 0.20; Nature pH.Get a fresh slant activation bacterial classification, to slant medium, add 10mL sterilized water (at twice), with transfering loop, scrape spore, make spore suspension.The triangular flask of 250mL packs 50mL fermentation basic medium into, and access spore suspension 5mL is placed in 30 ℃, the shaking table of 170r/min and cultivates 12h.
5. fermention medium (g/L): it is carbon source that the xylo-oligosaccharide of take is produced waste residue enzymolysis solution; Ammonium sulfate 2; The independent sterilizing of calcium carbonate 30(); Potassium primary phosphate 0.30; Magnesium sulfate heptahydrate 0.75; Zinc Sulphate Heptahydrate 0.20; Nature pH.After fermentation 0h, add calcium carbonate (grinding).Above-mentioned substratum is all at 0.1MPa, sterilizing 15min under 121 ℃ of conditions.The triangular flask of 250mL packs 100mL fermention medium into, and every bottle graft enters 5mL fermentation basic medium, is placed in 40 ℃, the shaking table of 170r/min and cultivates 48h.Fermented liquid is got to 1mL in the centrifuge tube of 1.5mL, deactivation 5min in 100 ℃ of water-baths, 72% the sulfuric acid that adds 100 μ l after taking off, centrifugal 5min under 10000rpm, then get supernatant liquor distilled water diluting, with syringe, by the film of 0.22 μ m, squeeze in chromatogram bottle, fermentable sugars concentration in enzymolysis solution, for lactic acid, high performance liquid chromatography (HPLC) detects again.Chromatographic condition is as follows: chromatographic instrument: Agillent1100 high performance liquid chromatograph; Chromatographic column: Bio-Rad Aminex HPX-87H; Moving phase: 0.005mol/L sulfuric acid, flow velocity: 0.6mL/min; Column temperature: 55 ℃; Detector: differential refraction detector; Sample size: 10 μ L.External standard method.Take that after solid-liquid separation, to obtain supernatant liquor be enzymolysis glucose solution and prepare lactic acid as carbon source substep diastatic fermentation and the results are shown in Figure 1.The substep diastatic fermentation of take without the band slag enzymolysis solution of solid-liquid separation as carbon source is prepared lactic acid and be the results are shown in Figure 2.
Embodiment 4: substep diastatic fermentation is prepared lactic acid.
Rhizopus oryzae utilizes the xylo-oligosaccharide production waste residue substep diastatic fermentation of embodiment 2 to prepare lactic acid.Xylo-oligosaccharide is produced waste residue and is placed on 4 ℃ of refrigerator equilibrium water contents two days, after survey moisture, extracting, surveys three elements.The xylo-oligosaccharide that takes respectively embodiment 2 is produced the heavy 10g of waste residue 36.95g(over dry, Mierocrystalline cellulose 7.19g wherein, moisture 26.95g) in 250mL triangular flask, add cellulase solution 0.90g(enzyme dosage 15FPIU/g Mierocrystalline cellulose), beta-glucosidase 0.16g(enzyme dosage 8IU/g Mierocrystalline cellulose), add damping fluid and distilled water, hierarchy of control solid-liquid weight ratio is 1:10, is that water volume is 100g, pH5.0 ± 0.5, is placed in shaking table enzymolysis 60h under 50 ± 5 ℃ of conditions of temperature of 150r/min.
Fermentable sugars, Plasma lactate method and fermentation basic medium, fermention medium form sees embodiment 3.
Embodiment 5: substep diastatic fermentation is prepared lactic acid.
Rhizopus oryzae utilizes the xylo-oligosaccharide production waste residue substep diastatic fermentation of embodiment 2 to prepare lactic acid.Xylo-oligosaccharide is produced waste residue and is placed on 4 ℃ of refrigerator equilibrium water contents two days, after survey moisture, extracting, surveys three elements.The xylo-oligosaccharide that takes respectively embodiment 2 is produced the heavy 20g of waste residue 73.91g(over dry, Mierocrystalline cellulose 14.37g wherein, moisture 53.91g) in 250mL triangular flask, add cellulase solution 1.80g(enzyme dosage 15FPIU/g Mierocrystalline cellulose), beta-glucosidase 0.32g(enzyme dosage 8IU/g Mierocrystalline cellulose), add damping fluid and distilled water, hierarchy of control solid-liquid weight ratio is 1:5, is that water volume is 100g, pH5.0 ± 0.5, is placed in shaking table enzymolysis 96h under 50 ± 5 ℃ of conditions of temperature of 150r/min.
Fermentable sugars, Plasma lactate method and fermentation basic medium, fermention medium form sees embodiment 3.
Embodiment 6: simultaneous saccharification and fermentation is prepared lactic acid.
The xylo-oligosaccharide of take is produced waste residue and is prepared lactic acid as carbon source simultaneous saccharification and fermentation (SSF), fermentation basic medium is as embodiment 3, take xylo-oligosaccharide and produce the heavy 5g of waste residue 18.48g(over dry, Mierocrystalline cellulose 3.59g wherein, moisture 13.48g) in 250mL triangular flask, in each triangular flask, add 1mol/L citrate buffer solution to control pH 5.0 ± 0.5, add nutritive salt, distilled water.Nutritive salt (g/L): ammonium sulfate 2; The independent sterilizing of calcium carbonate 30(); Potassium primary phosphate 0.30; Magnesium sulfate heptahydrate 0.75; Zinc Sulphate Heptahydrate 0.20; After fermentation 12h, add calcium carbonate (grinding).0.1MPa, under 121 ℃ of conditions, after sterilizing 15min, add cellulase solution 0.45g(enzyme dosage 15FPIU/g Mierocrystalline cellulose) simultaneously, beta-glucosidase 0.08g(enzyme dosage 8IU/g Mierocrystalline cellulose) and fermentation basic medium, hierarchy of control solid-liquid weight ratio is 1:20, making total liquid volume is 100g.After stirring, be placed in 40 ℃, SSF 48h in the shaking table of 170r/min.Plasma lactate method is as embodiment 3.The results are shown in Figure 3.
Embodiment 7: simultaneous saccharification and fermentation is prepared lactic acid.
The xylo-oligosaccharide of take is produced waste residue and is prepared lactic acid as carbon source simultaneous saccharification and fermentation (SSF), fermentation basic medium is as embodiment 3, take xylo-oligosaccharide and produce the heavy 10g of waste residue 36.95g(over dry, Mierocrystalline cellulose 7.19g wherein, moisture 26.95g) in 250mL triangular flask, in each triangular flask, add 1mol/L citrate buffer solution to control pH 5.0 ± 0.5, add nutritive salt, distilled water.Nutritive salt (g/L): ammonium sulfate 2; The independent sterilizing of calcium carbonate 30(); Potassium primary phosphate 0.30; Magnesium sulfate heptahydrate 0.75; Zinc Sulphate Heptahydrate 0.20; After fermentation 12h, add calcium carbonate (grinding).0.1MPa, under 121 ℃ of conditions, after sterilizing 15min, add cellulase solution 0.90g(enzyme dosage 15FPIU/g Mierocrystalline cellulose) simultaneously, beta-glucosidase 0.16g(enzyme dosage 8IU/g Mierocrystalline cellulose) and fermentation basic medium, hierarchy of control solid-liquid weight ratio is 1:10, making total liquid volume is 100g.After stirring, be placed in 40 ℃, SSF 60h in the shaking table of 170r/min.Plasma lactate method is as embodiment 3.
Embodiment 8: simultaneous saccharification and fermentation is prepared lactic acid.
The xylo-oligosaccharide of take is produced waste residue and is prepared lactic acid as carbon source simultaneous saccharification and fermentation (SSF), fermentation basic medium is as embodiment 3, take the heavy 20g of 73.91g(over dry, Mierocrystalline cellulose 14.37g wherein, moisture 53.91g) in 250mL triangular flask, in each triangular flask, add 1mol/L citrate buffer solution to control pH 5.0 ± 0.5, add nutritive salt, distilled water.Nutritive salt (g/L): ammonium sulfate 2; The independent sterilizing of calcium carbonate 30(); Potassium primary phosphate 0.30; Magnesium sulfate heptahydrate 0.75; Zinc Sulphate Heptahydrate 0.20; After fermentation 12h, add calcium carbonate (grinding).0.1MPa, under 121 ℃ of conditions, after sterilizing 15min, add cellulase solution 1.80g(enzyme dosage 15FPIU/g Mierocrystalline cellulose) simultaneously, beta-glucosidase 0.32g(enzyme dosage 8IU/g Mierocrystalline cellulose) and fermentation basic medium, hierarchy of control solid-liquid weight ratio is 1:5, making total liquid volume is 100g.After stirring, be placed in 40 ℃, SSF 96h in the shaking table of 170r/min.Plasma lactate method is as embodiment 3.
Embodiment 9: carrier-free immobilization.
The xylo-oligosaccharide of take is produced waste residue and is carried out substep diastatic fermentation as carbon source, enzymolysis and fermentation step be as embodiment 3, and after fermentation one is taken turns, bacterial classification adopts the Rhizopus oryzae particle that filtered through gauze obtains after 48h, add the enzymolysis solution of xylo-oligosaccharide production waste residue as fresh culture, many wheels that repeat to ferment are prepared lactic acid.
Embodiment 10: lactic acid decolouring.
Lactic acid decolouring prepared by substep diastatic fermentation and simultaneous saccharification and fermentation, decolorization condition, for react 30min under 50 ℃ of 80r/min, adds granulated active carbon, Powdered Activated Carbon or laccase and decolours.Solid-liquid weight ratio be the system fermentation of 1:20 prepare lactic acid decolorizing effect in Table 1,2.
The research of table 1 substep diastatic fermentation lactic acid decolouring
The research of table 2 simultaneous saccharification and fermentation lactic acid decolouring
Note: A is the front absorbancy at 420nm of decolouring; B is in 420nm absorbancy after decolouring; C is the front lactic acid concn of decolouring; D is the rear lactic acid concn of decolouring.
Claims (5)
1. a method for xylo-oligosaccharide coproduction lactic acid, is characterized in that, the method comprises the steps:
(1) take through pretreated corn cob is raw material, take fungi zytase as initial enzyme preparation xylo-oligosaccharide;
(2) take the solid slag producing in step (1) production process is carbon source again, take fungal cellulase and beta-glucosidase as zymin, through Rhizopus oryzae fermentation, prepares lactic acid;
Step (2), concrete grammar is for to mix the solid slag producing in step (1) production process with fungal cellulase and beta-glucosidase, add water and Rhizopus oryzae, being mixed to solid-liquid weight ratio is 1:5～20, regulating pH value is 5.0 ± 0.5, at 40 ℃, stir hydrolysis 48～96h and realize simultaneous saccharification and fermentation production lactic acid, then make through decolouring.
2. the method for xylo-oligosaccharide coproduction lactic acid according to claim 1, is characterized in that, for every g Mierocrystalline cellulose, the consumption of cellulase is 15FPIU/g, and the consumption of beta-glucosidase is 8IU/g.
3. the method for xylo-oligosaccharide coproduction lactic acid according to claim 1, it is characterized in that, fungal cellulase and beta-glucosidase are cellulase and the beta-glucosidases by the Trichoderma of fungi (Trichoderma), Aspergillus (Aspergillus), Penicillium (Penicillium) and Fusarium (Fusarium) secretion.
4. the method for xylo-oligosaccharide coproduction lactic acid according to claim 1, is characterized in that, described Rhizopus oryzae is the particle that carrier-free immobilization forms.
5. the method for xylo-oligosaccharide coproduction lactic acid according to claim 1, is characterized in that, described lactic acid decolouring adopts activated carbon decolorizing method.
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