CN102876756A - 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 microbial fermentation field in the biological chemistry, be specifically related to utilize the raw material corn cob to produce the technique of xylo-oligosaccharide coproduction lactic acid.
Background technology
Oligose is the low polymerization degree carbohydrate that is 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 can promoting in the enteron aisle probiotics proliferated specifically such as bifidus bacillus, another important biological function of functional oligose is the immunity system that stimulates in the 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 the 4-glycosidic link is formed by connecting, be present in all oligose " performance is the most stable, propagation bifidus bacillus potency ratio the highest " beneficial bacteria factor, be called as " superpower bifidus factor ".The mixture take xylo-bioses, xylotriose as main component that utilizes that endo-xylanase hydrolyzed xylan substrate obtains, so the key of enzymolysis process is that zytase to the adaptability of substrate, namely selects suitable zytase.Biological enzyme DeR speed is easy to control, specificity is strong and by product is few.The enzyme process preparation is the main method of producing at present xylo-oligosaccharide, is the method that has prospects for commercial application most.
Corn cob after pretreatment, extract is rich in hemicellulose, 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 the 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 not simultaneously, then with multiple different substituting group, so xylan molecular structures variation range is larger on main chain or the side chain.Inscribe beta-xylanase Main Function is in the macromole xylan with than the oligose of long-chain, and primary product is xylo-oligosaccharide.Xylo-oligosaccharide becomes the functional foodstuff that receives much concern now.
Corn cob after pretreatment, the hemicellulose component is used for preparing xylo-oligosaccharide, xylo-oligosaccharide is produced waste residue and generally is mixed with coal, is used as fuel, or makes toilet paper.
Lactic acid, formal name used at school Lactic acid are one of three large organic acids of generally acknowledging in the world.Because 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 enlarges rapidly.Pfansteihl also has unique biochemistry to be worth except the character that possesses common lactic acid in addition, is widely used in the industries such as food, medicine, chemical industry.Human body only contains LDH in the human body, so can only utilize Pfansteihl.At present the 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 be that the raw material production cost is still higher with glucose, starch, limited the application of Pfansteihl, especially limited poly(lactic acid) as the application of degradable plastics at field of Environment Protection.Around reducing the Pfansteihl production cost, take select cheapness, Mierocrystalline cellulose becomes one of study hotspot as the 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 of present technique technical problem to be solved provides 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 kind of technique of xylo-oligosaccharide coproduction lactic acid prepares the solid slag that produces in the xylo-oligosaccharide process take corn cob and is carbon source, fermentation preparation lactic acid.The method comprises the steps:
(1) take the pretreated corn cob of process as raw material, take the fungi zytase as initial enzyme preparation xylo-oligosaccharide;
(2) solid slag that produces in step (1) production process again is as raw material, take fungal cellulase and beta-glucosidase as zymin, through Rhizopus oryzae fermentation preparation 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, purpose 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), the fungi zytase can directly be bought in market, perhaps 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 that produces in the 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 that produces in step (1) production process is mixed with fungal cellulase and beta-glucosidase, add entry, being mixed to the solid-liquid weight ratio is 1:5～20, and regulating the pH value is 5.0 ± 0.5, in 50 ± 5 ℃ of lower hydrolysis 48 ~ 96h that stir; Again hydrolyzate is stirred 48 ~ 96h through the Rhizopus oryzae fermenting lactic acid under 40 ℃, make through decolouring again.
Wherein, described hydrolyzate can be behind the hydrolysis reaction without the liquid of solid-liquid separation and the mixture of solid, also can be that the supernatant liquor that obtains through solid-liquid separation behind the hydrolysis reaction is the enzymolysis glucose solution.
Method II, synchronous saccharification and fermentation.The solid slag that produces in step (1) production process is mixed with fungal cellulase and beta-glucosidase, add entry and bacterial classification Rhizopus oryzae, being mixed to the solid-liquid weight ratio is 1:5～20, regulating the pH value is 5.0 ± 0.5, realize simultaneous saccharification and fermentation production lactic acid in 40 ℃ of lower hydrolysis 48 ~ 96h that stir, make through decolouring again.
Among method I and the 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 the solid slag can be according to American National renewable energy source laboratory (NREL) method quantitative analysis.
Among method I and the 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.
Among method I and the II, fungal cellulase and beta-glucosidase can directly be bought in market, or by cellulase and the beta-glucosidase self-control of the Trichoderma (Trichoderma) of fungi, Aspergillus (Aspergillus), Penicillium (Penicillium) and Fusarium (Fusarium) secretion.
Among method I and the II, described Rhizopus oryzae can be the particle that carrier-free immobilization forms.
Among method I and the II, the activated carbon decolorizing method is adopted in described lactic acid decolouring, can use Powdered Activated Carbon or granulated active carbon.
Among method I and the II, stirring can be replaced with the shaking table vibration.
Among method I and the II, need to add nitrogenous source in the process of fermenting lactic acid and the required inorganic salt that ferment, 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 prepares 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 prepares xylo-oligosaccharide technique and lactic fermentation provides a cheaply new way.
Description of drawings
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 that the described content of embodiment only is used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
The product zytase that embodiment mentions, cellulase, beta-glucosidase microorganism reach the method that is prepared zytase by product zytase microbial fermentation, the cellulase-producing microbial fermentation prepares the method for cellulase, and the method that product beta-glucosidase microbial fermentation prepares beta-glucosidase 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 is regulated 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 is regulated pH value to 4.8 with the citrate buffer solution of 1mol/L.
3. the cultivation of Trichodermareesei mycelium: 50mL Mycelium culture base places the 250mL triangular flask, in 121 ℃ of lower sterilization 15min, be cooled to room temperature, access is preserved in the Trichodermareesei spore of test tube slant in right amount, and shaking flask places constant-temperature table for subsequent use after cultivating 36h under 30 ± 1 ℃, 170r/min condition.
4. the preparation of trichoderma reesei cellulase: cultivate based on 121 ℃ of lower sterilization 15min, be cooled to room temperature, access the Trichodermareesei mycelium of above-mentioned cultivation 36h, place constant-temperature table under the 170r/min condition, to cultivate, the culture temperature first day is controlled at 30 ± 1 ℃, is controlled at later on 28 ± 1 ℃.
5. cultivated 4 days, centrifugal 10min under the 3000r/min condition separates the solid matter in the nutrient solution (thalline and unemployed paper pulp fiber) to remove with whizzer, namely gets 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 is regulated 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; Sal epsom 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, the 1mL nitrogenous source.Substratum is regulated pH value to 4.8 with the citrate buffer solution of 1mol/L.
3. black-koji mould filament culture condition: 50mL Mycelium culture base places the 250mL triangular flask, in 121 ℃ of lower sterilization 15min, be cooled to room temperature, access is preserved in the aspergillus niger spore of test tube slant in right amount, and shaking flask places constant-temperature table for subsequent use after cultivating 36h under 30 ± 1 ℃, 170r/min condition.
4. the preparation of aspergillus niger beta-glucosidase: cultivate based on 121 ℃ of lower sterilization 15min, be cooled to room temperature, access the black-koji mould filament of above-mentioned cultivation 36h, place constant-temperature table under the 170r/min condition, to cultivate, the culture temperature first day is controlled at 30 ± 1 ℃, is controlled at later on 28 ± 1 ℃.
5. cultivated 10 days, centrifugal 10min under the 3000r/min condition separates the solid matter in the nutrient solution (thalline and unemployed corn cob, wheat bran) to remove with whizzer, namely gets beta-glucosidase enzyme liquid.
The mensuration of filter paper enzyme activity: the standard method of adopting international theory and applied chemistry association (IUPAC) to recommend is measured.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, reacts 60min, the glucose amount that assaying reaction generates in 50 ℃, the reciprocating type constant temperature oscillator of oscillation frequency 80r/min.A filter paper enzyme activity unit of force (FPIU) is defined as the enzyme amount that per minute under the standard reaction condition generates 1 μ mol glucose.
The mensuration of endoglucanase vigor: the endoglucanase vigor represents with carboxymethylcelluloenzyme enzyme activity usually.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, reacts 30min, the glucose amount that assaying reaction generates in 50 ℃, the reciprocating type constant temperature oscillator of oscillation frequency 80r/min.The unit of a carboxymethylcelluloenzyme enzyme activity (IU) is defined as the enzyme amount that per minute under the standard reaction condition generates 1 μ mol glucose.
The mensuration of beta-glucoside enzyme activity: adopt p-nitrophenyl-β-D-Glucose glycosides (pNPG) to measure for substrate.0.1mL after suitably the pNPG solution of the enzyme liquid of dilution and 0.9mL 5mmol/L (be that 4.8 citric acid-Sodium phosphate dibasic damping fluid prepared by 0.05mol/L, pH value) mixes, add immediately the Na of 2mL 1mol/L after in 50 ℃, the reciprocating type constant temperature oscillator of oscillation frequency 80r/min, reacting 10min
2CO
3The solution termination reaction adds 10mL distilled water again, shakes up.Under 400nm, measure absorbancy.Replace enzyme liquid to make blank with 0.1mL distilled water.Each sample is done 2～3 Duplicate Samples, averages.A beta-glucoside enzyme activity unit (IU) is defined as that per minute generates the required enzyme amount of 1 μ mol p-NP under the standard reaction condition.
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 seen in the preparation of endo-xylanase and separation and purification.
Embodiment 2: the obtaining of xylo-oligosaccharide production and cellulosic material.
Corn cob extracts the residue behind the xylan, and main component is Mierocrystalline cellulose, and its content is the 71.87%(butt), moisture content is 72.94%, is a kind of good cellulose resource.Simultaneously, the process of corn cob alkali extracting xylan in fact also is a pretreated process of cellulose raw material alkali.Therefore, corn cob need not the raw material that again pre-treatment just can be used as lactic acid-producing after the alkali extracting, directly carries out the hydrolysis of cellulase.The acquisition process of its raw material is as follows:
1. take by weighing 50gNaOH and fully be dissolved in the 702mL distilled water, adding grinding particle size is the air-dry corn cob 112g(over dry 100g of 0.5～1cm), in 90 ℃ of lower reaction 3h.
2. above-mentioned reactant vacuum filtration is removed Xylan extractive, with 500mL water filtering and washing, discards washings.Xylan extractive is used for preparing xylo-oligosaccharide.
3. add 300mL distilled water in the above-mentioned filter residue, use 72%(w/v) sulfuric acid be neutralized to pH value 5.0 ± 0.5, vacuum filtration, and with 900mL distillation moisture wash for 3 times, suction filtration, obtain xylo-oligosaccharide production waste residue.
It is exactly the needed lactic acid-producing raw material of present technique that this xylo-oligosaccharide is produced waste residue; Pretreated purpose is to extract xylan, improve simultaneously in the cellulose raw material Mierocrystalline cellulose to the accessibility of cellulase, that can adopt physics, chemistry, biology or above several method combined utilization can be with the pretreatment process of hemicellulose, Mierocrystalline cellulose effective separation.
Embodiment 3: the substep diastatic fermentation prepares 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, surveys three elements after survey moisture, the extracting.The xylo-oligosaccharide that takes by weighing respectively embodiment 2 is produced the heavy 5g of waste residue 18.48g(over dry, Mierocrystalline cellulose 3.59g wherein, moisture 13.48g) in the 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, places shaking table enzymolysis 48h under 50 ± 5 ℃ of conditions of temperature of 150r/min.
2. sampling 1mL, deactivation 5min in 100 ℃ of water-baths, centrifugal 5min under the 10000r/min gets the concentration that supernatant liquor is suitably measured fermentable sugars wherein after the dilution.
3. enzymolysis solution centrifuging and taking enzymolysis clear liquid.
4. basic medium (g/L) ferments: glucose 50; Ammonium sulfate 3; Calcium carbonate 10(sterilizes separately); Potassium primary phosphate 0.30; Magnesium sulfate heptahydrate 0.75; Zinc Sulphate Heptahydrate 0.20; Nature pH.Get a fresh slant activation bacterial classification, add 10mL sterilized water (at twice) to slant medium, scrape spore with transfering loop, make spore suspension.The triangular flask of the 250mL 50mL fermentation basic medium of packing into, access spore suspension 5mL places 30 ℃, the shaking table of 170r/min to cultivate 12h.
5. fermention medium (g/L): produce the waste residue enzymolysis solution as carbon source take xylo-oligosaccharide; Ammonium sulfate 2; Calcium carbonate 30(sterilizes separately); Potassium primary phosphate 0.30; Magnesium sulfate heptahydrate 0.75; Zinc Sulphate Heptahydrate 0.20; Nature pH.Add calcium carbonate (grinding) behind the fermentation 0h.Above-mentioned substratum is all at 0.1MPa, and 15min sterilizes under 121 ℃ of conditions.The triangular flask of the 250mL 100mL fermention medium of packing into, every bottle graft enter 5mL fermentation basic medium, place 40 ℃, the shaking table of 170r/min to cultivate 48h.Fermented liquid is got 1mL in the centrifuge tube of 1.5mL, deactivation 5min in 100 ℃ of water-baths, take off 72% the sulfuric acid of rear adding 100 μ l, centrifugal 5min under 10000rpm, then get the supernatant liquor distilled water diluting, squeeze in the chromatogram bottle by the film of 0.22 μ m with syringe, fermentable sugars concentration, lactic acid detect with high performance liquid chromatography (HPLC) in the enzymolysis solution again.Chromatographic condition is as follows: chromatographic instrument: the 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.Being that the enzymolysis glucose solution the results are shown in Figure 1 as carbon source substep diastatic fermentation prepares lactic acid through obtaining supernatant liquor after the solid-liquid separation.As preparing lactic acid, carbon source substep diastatic fermentation the results are shown in Figure 2 take the band slag enzymolysis solution without solid-liquid separation.
Embodiment 4: the substep diastatic fermentation prepares 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, surveys three elements after survey moisture, the extracting.The xylo-oligosaccharide that takes by weighing respectively embodiment 2 is produced the heavy 10g of waste residue 36.95g(over dry, Mierocrystalline cellulose 7.19g wherein, moisture 26.95g) in the 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, places 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: the substep diastatic fermentation prepares 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, surveys three elements after survey moisture, the extracting.The xylo-oligosaccharide that takes by weighing respectively embodiment 2 is produced the heavy 20g of waste residue 73.91g(over dry, Mierocrystalline cellulose 14.37g wherein, moisture 53.91g) in the 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, places 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 prepares lactic acid.
Produce waste residue as carbon source simultaneous saccharification and fermentation (SSF) preparation lactic acid take xylo-oligosaccharide, fermentation basic medium such as embodiment 3, take by weighing xylo-oligosaccharide and produce the heavy 5g of waste residue 18.48g(over dry, Mierocrystalline cellulose 3.59g wherein, moisture 13.48g) in the 250mL triangular flask, add 1mol/L citrate buffer solution control pH 5.0 ± 0.5 in each triangular flask, add nutritive salt, distilled water.Nutritive salt (g/L): ammonium sulfate 2; Calcium carbonate 30(sterilizes separately); Potassium primary phosphate 0.30; Magnesium sulfate heptahydrate 0.75; Zinc Sulphate Heptahydrate 0.20; Add calcium carbonate (grinding) behind the fermentation 12h.0.1MPa, add simultaneously cellulase solution 0.45g(enzyme dosage 15FPIU/g Mierocrystalline cellulose behind the sterilization 15min under 121 ℃ of conditions), beta-glucosidase 0.08g(enzyme dosage 8IU/g Mierocrystalline cellulose) and the fermentation basic medium, hierarchy of control solid-liquid weight ratio is 1:20, making total liquid volume is 100g.After stirring, place 40 ℃, SSF 48h in the shaking table of 170r/min.Plasma lactate method such as embodiment 3.The results are shown in Figure 3.
Embodiment 7: simultaneous saccharification and fermentation prepares lactic acid.
Produce waste residue as carbon source simultaneous saccharification and fermentation (SSF) preparation lactic acid take xylo-oligosaccharide, fermentation basic medium such as embodiment 3, take by weighing xylo-oligosaccharide and produce the heavy 10g of waste residue 36.95g(over dry, Mierocrystalline cellulose 7.19g wherein, moisture 26.95g) in the 250mL triangular flask, add 1mol/L citrate buffer solution control pH 5.0 ± 0.5 in each triangular flask, add nutritive salt, distilled water.Nutritive salt (g/L): ammonium sulfate 2; Calcium carbonate 30(sterilizes separately); Potassium primary phosphate 0.30; Magnesium sulfate heptahydrate 0.75; Zinc Sulphate Heptahydrate 0.20; Add calcium carbonate (grinding) behind the fermentation 12h.0.1MPa, add simultaneously cellulase solution 0.90g(enzyme dosage 15FPIU/g Mierocrystalline cellulose behind the sterilization 15min under 121 ℃ of conditions), beta-glucosidase 0.16g(enzyme dosage 8IU/g Mierocrystalline cellulose) and the fermentation basic medium, hierarchy of control solid-liquid weight ratio is 1:10, making total liquid volume is 100g.After stirring, place 40 ℃, SSF 60h in the shaking table of 170r/min.Plasma lactate method such as embodiment 3.
Embodiment 8: simultaneous saccharification and fermentation prepares lactic acid.
Produce waste residue as carbon source simultaneous saccharification and fermentation (SSF) preparation lactic acid take xylo-oligosaccharide, fermentation basic medium such as embodiment 3, take by weighing the heavy 20g of 73.91g(over dry, Mierocrystalline cellulose 14.37g wherein, moisture 53.91g) in the 250mL triangular flask, add 1mol/L citrate buffer solution control pH 5.0 ± 0.5 in each triangular flask, add nutritive salt, distilled water.Nutritive salt (g/L): ammonium sulfate 2; Calcium carbonate 30(sterilizes separately); Potassium primary phosphate 0.30; Magnesium sulfate heptahydrate 0.75; Zinc Sulphate Heptahydrate 0.20; Add calcium carbonate (grinding) behind the fermentation 12h.0.1MPa, add simultaneously cellulase solution 1.80g(enzyme dosage 15FPIU/g Mierocrystalline cellulose behind the sterilization 15min under 121 ℃ of conditions), beta-glucosidase 0.32g(enzyme dosage 8IU/g Mierocrystalline cellulose) and the fermentation basic medium, hierarchy of control solid-liquid weight ratio is 1:5, making total liquid volume is 100g.After stirring, place 40 ℃, SSF 96h in the shaking table of 170r/min.Plasma lactate method such as embodiment 3.
Embodiment 9: carrier-free immobilization.
Carry out the substep diastatic fermentation take xylo-oligosaccharide production waste residue as carbon source, enzymolysis and fermentation step such as embodiment 3, after fermentation one is taken turns, the Rhizopus oryzae particle that filtered through gauze obtains behind the bacterial classification employing 48h, add the enzymolysis solution of xylo-oligosaccharide production waste residue as fresh culture, many wheel preparation lactic acid repeat to ferment.
Embodiment 10: the lactic acid decolouring.
The lactic acid decolouring of substep diastatic fermentation and simultaneous saccharification and fermentation preparation, decolorization condition adds granulated active carbon, Powdered Activated Carbon or laccase and decolours for react 30min under 50 ℃ of 80r/min.The solid-liquid weight ratio is that the decolorizing effect of the system fermentation preparation lactic acid of 1:20 sees 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
Annotate: A is the front absorbancy at 420nm of decolouring; B is rear in the 420nm absorbancy for decolouring; C is the front lactic acid concn of decolouring; D is the rear lactic acid concn of decolouring.
Claims (9)
1. the technique of an xylo-oligosaccharide coproduction lactic acid is characterized in that, prepares the solid slag that produces in the xylo-oligosaccharide process take corn cob and is carbon source, fermentation preparation lactic acid.
2. the technique of xylo-oligosaccharide coproduction lactic acid according to claim 1 is characterized in that the method comprises the steps:
(1) take the pretreated corn cob of process as raw material, take the fungi zytase as initial enzyme preparation xylo-oligosaccharide;
(2) solid slag that produces in step (1) production process again is as carbon source, take fungal cellulase and beta-glucosidase as zymin, through Rhizopus oryzae fermentation preparation lactic acid.
3. the technique of xylo-oligosaccharide coproduction lactic acid according to claim 2, it is characterized in that, step (2), concrete grammar is for to mix the solid slag that produces in step (1) production process with fungal cellulase and beta-glucosidase, add distilled water, being mixed to the solid-liquid weight ratio is 1:5～20, and regulating the pH value is 5.0 ± 0.5, in 50 ± 5 ℃ of lower hydrolysis 48 ~ 96h that stir; Take hydrolyzate as carbon source,, make through decolouring again through the Rhizopus oryzae fermenting lactic acid in 40 ℃ of lower 48 ~ 96h that stir again.
4. the technique of xylo-oligosaccharide coproduction lactic acid according to claim 3, it is characterized in that, described hydrolyzate can be behind the hydrolysis reaction without the liquid of solid-liquid separation and the mixture of solid, also can be that the supernatant liquor that obtains through solid-liquid separation behind the hydrolysis reaction is the enzymolysis glucose solution.
5. the technique of xylo-oligosaccharide coproduction lactic acid according to claim 2, it is characterized in that, step (2), concrete grammar is for to mix the solid slag that produces in step (1) production process with fungal cellulase and beta-glucosidase, add entry and Rhizopus oryzae, being mixed to the solid-liquid weight ratio is 1:5～20, and regulating the pH value is 5.0 ± 0.5, realize simultaneous saccharification and fermentation production lactic acid in 40 ℃ of lower hydrolysis 48 ~ 96h that stir, make through decolouring again.
6. according to claim 2 or the technique of 3 or 5 described xylo-oligosaccharide coproduction lactic acid, it is characterized in that for every g Mierocrystalline cellulose, the consumption of cellulase is 15FPIU/g, the consumption of beta-glucosidase is 8IU/g.
7. the technique of xylo-oligosaccharide coproduction lactic acid according to claim 2, 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.
8. according to claim 2 or the technique of 3 or 5 described xylo-oligosaccharide coproduction lactic acid, it is characterized in that described Rhizopus oryzae can be the particle that carrier-free immobilization forms.
9. according to claim 3 or the technique of 5 described xylo-oligosaccharide coproduction lactic acid, it is characterized in that described lactic acid decolouring can be adopted the activated carbon decolorizing method.
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| CN113718002A (en) * | 2021-08-31 | 2021-11-30 | 江苏菁康生物科技有限公司 | Method for preparing micromolecular galactomannan and galactomannan-oligosaccharide by using alkali extraction residues of corncobs |
| CN113718002B (en) * | 2021-08-31 | 2024-04-12 | 南京林业大学 | Method for preparing micromolecular galactomannan and galactomannan-oligosaccharide by using corncob alkali extraction residues |
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