Method for preparing lactic acid by continuous saccharification and fermentation of straws
Through the continuous saccharification fermentation method of straw, the time difference between the two enzymatic fermentation and fermentation processes is adopted, and the problems of low efficiency and high energy consumption of straw fermentation in the prior art are solved, achieving efficient and economical lactic acid production.
Patent Information
- Application Number
- CN202510748412.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-06
- Publication Date
- 2025-08-19
AI Technical Summary
In the prior art, the method for preparing lactic acid in straw fermentation has the problems of many process steps, insufficient enzymatic substrate, high energy consumption for concentration, and difficulty in competition between substrate and product when enzymatic and fermentation are carried out simultaneously.
The straw continuous saccharification fermentation method is adopted, and the two enzymatic lysis processes and the time difference between the two enzymatic lysis processes and the fermentation process is used to perform enzymatic lysis and fermentation respectively. The enzymatic lysis process and fermentation process are carried out simultaneously. The complex enzyme and enzymatic lysis buffer are used for saccharification, and then fermentation sugar is obtained and fermented using Bacillus coagulis.
The enzymatic decomposition and fermentation process are achieved simultaneously, saving time and equipment costs, improving the saccharification rate and lactic acid production rate, reducing energy consumption, and increasing the lactic acid yield and the target product content of the fermentation broth.
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Figure CN120505375A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological fermentation, and particularly relates to a method for preparing lactic acid by continuous saccharification and fermentation of straw. Background Art
[0002] Currently, through fermentation technology, the cellulose and hemicellulose in straw can be converted into lactic acid. Using straw as a raw material to ferment lactic acid can not only reduce the environmental pollution problems caused by straw burning, but also make full use of renewable resources. Reducing dependence on traditional food resources has significant environmental and economic benefits. However, most existing technologies use methods of enzymatic hydrolysis and concentration followed by fermentation or simultaneous saccharification and fermentation. The former has many process steps, insufficient enzymatic hydrolysis of the substrate, and high energy consumption for concentration. The latter, because enzymatic hydrolysis and fermentation are carried out simultaneously, there is competitive inhibition between the substrate and the product, and subsequent separation and purification are difficult. Therefore, it is necessary to develop a more efficient, economical, and easy-to-operate method for preparing lactic acid using straw fermentation. Summary of the Invention
[0003] In view of the deficiencies in the prior art, the present invention aims to provide a method for preparing lactic acid by continuous saccharification and fermentation of straw, which solves the problems in the prior art.
[0004] The purpose of the present invention can be achieved through the following technical solutions:
[0005] A method for preparing lactic acid by continuous saccharification and fermentation of straw comprises the following steps:
[0006] The straw is dusted, cleaned, cut and then soaked, taken out, water is controlled and mixed with caustic soda solution, and then kneaded and washed to obtain wet heald fiber;
[0007] The wet heald fiber is dried and then immersed in an enzymatic hydrolysis buffer or the wet heald fiber and the enzymatic hydrolysis buffer are evenly mixed, and then a composite enzyme is added, and enzymatic hydrolysis and saccharification are carried out in a primary enzymatic hydrolysis tank and a secondary enzymatic hydrolysis tank. The clear liquid obtained in the primary enzymatic hydrolysis tank is filtered to obtain a mixed sugar liquid; the solids in the lower layer of the primary enzymatic hydrolysis tank are sent to the secondary enzymatic hydrolysis tank for saccharification with the composite enzyme again, and then filtered to obtain a mixed sugar liquid;
[0008] The finished sugar is compounded with the mixed sugar solution to obtain fermentable sugar;
[0009] Using fermentable sugar to ferment bacteria in a fermentation tank to obtain lactic acid fermentation liquid;
[0010] After the saccharification is completed, the first-level enzymatic hydrolysis tank is emptied, and the solids continue to be hydrolyzed in the second-level enzymatic hydrolysis tank; after the first-level enzymatic hydrolysis tank is cleaned and sterilized, it is used as a fermentation tank for lactic acid fermentation; after the fermentation is completed, the first-level enzymatic hydrolysis tank is used again for comprehensive fiber enzymatic hydrolysis; and the second-level enzymatic hydrolysis tank is emptied and cleaned and then a new round of enzymatic hydrolysis is carried out.
[0011] Furthermore, the straw is one or more of rice straw, wheat straw, corn straw, and cotton straw.
[0012] Furthermore, the complex enzyme comprises: cellulase, hemicellulase, xylanase and xylosidase.
[0013] Furthermore, the enzymatic hydrolysis buffer is a combination of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, citric acid, sodium citrate, phosphoric acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate.
[0014] Furthermore, the pH value of the enzymatic hydrolysis buffer is 4.5-6.5.
[0015] Furthermore, the saccharification temperature of the first-level enzymolysis tank and the second-level enzymolysis tank is 40-55°C, and the stirring speed is 120-200r / min; the saccharification time of the first-level enzymolysis tank is 40h, the enzyme addition amount is 15-25FPU / g, and the saccharification time of the second-level enzymolysis tank is 48h, and the enzyme addition amount is 10-25FPU / g.
[0016] Furthermore, the step of compounding the finished sugar with the mixed sugar solution includes: first measuring the concentrations of glucose, xylose, and arabinose in the mixed sugar solution, and then compounding it with finished glucose, finished xylose, and finished arabinose so that the mass concentration ratio is glucose:xylose:arabinose=8:2:0.5, and the total sugar concentration is 100-160g / L.
[0017] Furthermore, the fermentation process includes: using a seed culture medium to cultivate a bacterial fermentation liquid, and then inoculating the bacterial fermentation liquid containing Bacillus coagulans into a fermentation medium at an inoculation rate of 1:10 to ferment, thereby obtaining a fermentation liquid containing lactic acid.
[0018] Furthermore, the seed culture medium includes: 50g / L glucose, 10g / L yeast extract, 0.01g / L sodium chloride, 0.5g / L sodium acetate, 0.2g / L ammonium citrate, 0.2g / L potassium dihydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, 0.05g / L manganese sulfate heptahydrate and 30g / L calcium carbonate.
[0019] Furthermore, the fermentation medium comprises: 100-160 g / L fermentation sugar, 5 g / L yeast extract, 0.03 g / L sodium chloride, 1.5 g / L sodium acetate, 0.6 g / L ammonium citrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, and 0.15 g / L manganese sulfate heptahydrate.
[0020] Beneficial effects of the present invention:
[0021] 1. The present invention achieves continuous saccharification and fermentation of straw, carrying out the enzymatic hydrolysis and fermentation processes simultaneously, significantly saving the time and cost of enzymatic hydrolysis and fermentation. Simultaneously, a single device performs both enzymatic hydrolysis and fermentation, maximizing equipment utilization and saving equipment investment costs.
[0022] 2. The present invention adopts a two-stage enzymatic hydrolysis process to improve the saccharification rate of the comprehensive fiber; at the same time, the dilute sugar obtained by enzymatic hydrolysis does not need to be concentrated like the traditional method, eliminating the energy consumption of the evaporation and concentration step. Instead, the finished sugar can be directly used for compounding to obtain a sugar solution with the target sugar concentration.
[0023] 3. The compound sugar obtained by this method has a good fermentation effect and a fast lactic acid production rate. Under the same fermentation conditions, the initial sugar concentration can be increased, and ultimately the lactic acid yield and the high content of the target product in the fermentation liquid can be increased under the same fermentation time. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.
[0025] Figure 1 The present invention is a flow chart of preparing lactic acid by continuous saccharification and fermentation of straw. DETAILED DESCRIPTION
[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.
[0027] like Figure 1 As shown, a method for preparing lactic acid by continuous saccharification and fermentation of straw comprises the following steps:
[0028] S1, dusting, washing, cutting and soaking the straw in clean water, removing the straw, controlling the water content and mixing it with caustic soda solution, feeding it into a twin-screw extruder for kneading, and subjecting the discharged material to heat preservation treatment and then washing it in the equipment to obtain wet heald fiber;
[0029] S2, drying the wet holofiber and then soaking and softening it in an enzymatic hydrolysis buffer or uniformly mixing the wet holofiber with the enzymatic hydrolysis buffer, then adding a complex enzyme, fully saccharifying it in an enzymatic hydrolysis tank, and centrifuging and filtering it to obtain a mixed sugar solution (mainly pentose and hexose sugars, also known as straw sugar);
[0030] S3, using finished sugar and mixed sugar solution (straw sugar) to compound to achieve fermentable sugar with suitable concentration for fermentation;
[0031] S4, fermenting the strain using fermentable sugar in a fermentation tank at a temperature of 40-47° C. to obtain a lactic acid fermentation liquid.
[0032] In S1, the straw is one or more of rice straw, wheat straw, corn straw, cotton straw or is prepared according to the ratio of the mixed sugar solution components.
[0033] In S2, the complex enzyme includes: cellulase, hemicellulase, xylanase and xylosidase.
[0034] In S2, the pH value of the enzymatic hydrolysis buffer is 4.5-6.5, and it is any combination of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, citric acid, sodium citrate, phosphoric acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate. The mass ratio (solid content) of the holofiber material to water is 10-20%.
[0035] In S2, the enzymolysis tank includes a primary enzymolysis tank and a secondary enzymolysis tank; the clear liquid obtained in the primary enzymolysis tank is directly sent to a centrifugal filter for filtration to obtain a mixed sugar solution; the solids in the lower layer of the primary enzymolysis tank are sent to the secondary enzymolysis tank for saccharification with a complex enzyme again; after completion, the liquid is first coarsely filtered through a plate and frame and then refiltered in a centrifugal filter to obtain a mixed sugar solution.
[0036] In S2, the saccharification temperature of the first and second enzymatic hydrolysis tanks is 40-55°C, and the stirring speed is 120-200r / min; the saccharification time of the first enzymatic hydrolysis tank is 40h, the enzyme addition amount is 15-25FPU / g, and the saccharification time of the second enzymatic hydrolysis tank is 48h, and the compound enzyme addition amount is 10-25FPU / g.
[0037] In S3, the step of compounding the finished sugar and the mixed sugar solution includes: measuring the concentrations of glucose, xylose, and arabinose in the mixed sugar solution, and then compounding it with finished glucose, finished xylose, and finished arabinose so that the mass concentration ratio is glucose:xylose:arabinose = 8:2:0.5, and the total sugar (glucose + xylose + arabinose) concentration is 100-160g / L.
[0038] In S4, the fermentation tank used for fermentation is an empty first-level enzymolysis tank, that is, after the enzymolysis liquid is emptied and disinfected, the mixed sugar liquid is poured into the first-level enzymolysis tank, and nutrients and nitrogen sources are added to prepare the fermentation medium. The present invention cleverly utilizes the time difference between enzymolysis and fermentation, and performs the enzymolysis process and the fermentation process simultaneously, thereby achieving continuous sugar production and fermentation while saving time and equipment investment costs. That is, the first-level enzymolysis tank is emptied after the saccharification is completed, and the solids continue to be enzymolyzed in the second-level enzymolysis tank. After the first-level enzymolysis tank is cleaned and sterilized, it can be used as a fermentation tank for lactic acid fermentation. After the fermentation is completed, it is used as a first-level enzymolysis tank for comprehensive fiber enzymolysis again. After the second-level enzymolysis tank is emptied and cleaned, a new round of enzymolysis is carried out again.
[0039] In S4, the fermentation process includes preparing a seed culture medium to culture a bacterial fermentation broth, and then inoculating the bacterial fermentation broth containing Bacillus coagulans into the fermentation medium at an inoculum ratio of 1:10 to obtain a fermentation broth containing L-lactic acid. The fermentation temperature is 50° C., the rotation speed is 150 rpm, and the fermentation time is 36-40 hours. The seed culture temperature is 45° C., the rotation speed is 120 rpm, and the culture time is 8-10 hours.
[0040] The seed culture medium includes: 50 g / L glucose, 10 g / L yeast extract powder, 0.01 g / L sodium chloride, 0.5 g / L sodium acetate, 0.2 g / L ammonium citrate, 0.2 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate heptahydrate, and 30 g / L calcium carbonate.
[0041] The fermentation medium includes: 100-160 g / L fermentation sugar, 5 g / L yeast extract powder, 0.03 g / L sodium chloride, 1.5 g / L sodium acetate, 0.6 g / L ammonium citrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, and 0.15 g / L manganese sulfate heptahydrate.
[0042] The technical solution of the present invention is described in detail below through the following embodiments:
[0043] Example 1
[0044] S1. Wheat straw is cleaned and chopped into 4-10 cm long segments. The segments are then soaked in clean water, removed, and mixed with a 5% caustic soda solution. The segments are then processed in a twin-screw extruder and placed in an insulated box for 1 hour. The segments are then re-entered the twin-screw extruder, and water is added using a metering pump. Wet fiber is then obtained through multi-stage countercurrent displacement washing. Excess water has been removed from the extruder at this stage, allowing the fibers to be directly used for enzymatic hydrolysis.
[0045] S2, wet heald fiber and PH=5.0 enzymolysis buffer solution, with 10% solid-liquid ratio, are configured into enzymolysis raw material, squeeze into the first-level enzymolysis tank for high temperature sterilization, cool to 50 ℃, open the stirring paddle and stir for 10min to mix, then add complex enzyme (including cellulase, hemicellulase, xylanase, xylosidase), the addition amount of complex enzyme is 20FPU / g. Under 50 ℃, 200rpm conditions, after enzymolysis 40h, keep warm and stand, the supernatant is centrifuged to obtain mixed sugar solution, the lower heavy phase is squeezed into the secondary enzymolysis tank, and the first-level enzymolysis tank is cleaned. Add appropriate amount of enzymolysis buffer solution sterilized in advance in the secondary enzymolysis tank, warm up to 50 ℃ and add complex enzyme (including cellulase, hemicellulase, xylanase, xylosidase), the addition amount is 20FPU / g, continue enzymolysis 48h. After completion, first pass through plate and frame coarse filtration and then enter centrifugal filter refiltration, the filtrate and the sugar solution in the previous step are mixed to obtain mixed sugar solution.
[0046] S3. The concentration of the trisaccharide (glucose + xylose + arabinose) in the obtained mixed sugar solution is measured, and then it is compounded with finished sugars (finished glucose + finished xylose + finished arabinose) to make the mass concentration ratio of glucose: xylose: arabinose = 8:2:0.5, and the total sugar concentration is 100 g / L.
[0047] S4. Use the emptied primary enzymatic hydrolysis tank as a fermentation tank for lactic acid fermentation. Use the fermentation sugar prepared in the previous step as the fermentation carbon source, and add 5 g / L yeast extract powder, 0.03 g / L sodium chloride, 1.5 g / L sodium acetate, 0.6 g / L ammonium citrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, and 0.15 g / L manganese sulfate heptahydrate. Sterilize at 115°C for 20 min until ready for use.
[0048] S5. Inoculate the Bacillus coagulans liquid into the fermentation medium containing the carbon source and ferment to obtain a fermentation liquid containing L-lactic acid. The fermentation temperature is 50° C., the rotation speed is 120 rpm, and the fermentation time is 48 h.
[0049] When the above process conditions are adopted, the content of L-lactic acid is ≥96.2 g / L, the sugar-acid conversion rate is ≥96.2%, and the lactic acid purity is ≥99.8%.
[0050] Example 2
[0051] S1. Wheat straw is cleaned and chopped into 4-10 cm long segments. The segments are then soaked in clean water, removed, and mixed with a 5% caustic soda solution. The segments are then processed in a twin-screw extruder and placed in an insulated box for 1 hour. The segments are then re-entered the twin-screw extruder, and water is added using a metering pump. Wet fiber is then obtained through multi-stage countercurrent displacement washing. Excess water has been removed from the extruder at this stage, allowing the fibers to be directly used for enzymatic hydrolysis.
[0052] S2, the obtained wet fiber and PH = 5.0 enzymolysis buffer solution are configured into enzymolysis raw materials with a solid-liquid ratio of 10%, and are poured into a primary enzymolysis tank for high temperature sterilization, cooled to 50 ° C, turned on the stirring paddle and stirred for 10 min to mix, and then a complex enzyme (including cellulase, hemicellulase, xylanase, xylosidase) is added. The amount of the complex enzyme is 20FPU / g. Under the conditions of 50 ° C and 200 rpm, after enzymolysis for 40 hours, the heat is kept still, the supernatant is centrifuged to obtain a sugar solution, and the lower heavy phase is poured into a secondary enzymolysis tank, and the primary enzymolysis tank is cleaned. An appropriate amount of enzymolysis buffer solution sterilized in advance is added to the secondary enzymolysis tank, and the temperature is raised to 50 ° C and a complex enzyme is added later, with an addition amount of 20FPU / g, and the enzymolysis is continued for 48 hours. After completion, it is first subjected to plate and frame coarse filtration and then enters the centrifugal filter for refiltration. The filtrate is mixed with the sugar solution in the previous step to obtain a mixed sugar solution.
[0053] S3. The concentration of the trisaccharide (glucose + xylose + arabinose) in the obtained mixed sugar solution was measured, and then compounded with finished sugars (finished glucose + finished xylose + finished arabinose) to make the mass concentration ratio of glucose: xylose: arabinose = 8:2:0.5, and the total sugar concentration was 120 g / L.
[0054] S4. Use the emptied primary enzymatic hydrolysis tank as a fermentation tank for lactic acid fermentation. Use the fermentation sugar prepared in the previous step as the fermentation carbon source, and add 5 g / L yeast extract powder, 0.03 g / L sodium chloride, 1.5 g / L sodium acetate, 0.6 g / L ammonium citrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, and 0.15 g / L manganese sulfate heptahydrate. Sterilize at 115°C for 20 min until ready for use.
[0055] S5. Inoculate the Bacillus coagulans liquid into the fermentation medium containing the carbon source and ferment to obtain a fermentation liquid containing L-lactic acid. The fermentation temperature is 50° C., the rotation speed is 120 rpm, and the fermentation time is 48 h.
[0056] When the above process conditions are adopted, the content of L-lactic acid is ≥114.6 g / L, the sugar-acid conversion rate is ≥95.5%, and the lactic acid purity is ≥99.8%.
[0057] Example 3
[0058] S1. Wheat straw is cleaned and chopped into 4-10 cm long segments. The segments are then soaked in clean water, removed, and mixed with a 5% caustic soda solution. The segments are then processed in a twin-screw extruder and placed in an insulated box for 1 hour. The segments are then re-entered the twin-screw extruder, and water is added using a metering pump. Wet fiber is then obtained through multi-stage countercurrent displacement washing. Excess water has been removed from the extruder at this stage, allowing the fibers to be directly used for enzymatic hydrolysis.
[0059] S2, the obtained holofiber and PH=5.0 enzymolysis buffer solution are configured into enzymolysis raw materials with a solid-liquid ratio of 10%, and are poured into a primary enzymolysis tank for high temperature sterilization, cooled to 50 ° C, turned on the stirring paddle and stirred for 10 min to mix, and then a complex enzyme (mainly including cellulase, hemicellulase, xylanase, xylosidase) is added. The addition amount of the complex enzyme is 20FPU / g. Under the conditions of 50 ° C and 200 rpm, after enzymolysis for 40 hours, the supernatant is centrifuged to obtain a sugar solution, and the lower heavy phase is poured into a secondary enzymolysis tank, and the primary enzymolysis tank is cleaned. An appropriate amount of enzymolysis buffer solution sterilized in advance is added to the secondary enzymolysis tank, and the complex enzyme is added after heating to 50 ° C, and the addition amount is 20FPU / g. The enzymolysis is continued for 48 hours. After completion, it is first subjected to plate and frame coarse filtration and then enters the centrifugal filter for refiltration. The filtrate is mixed with the sugar solution in the previous step to obtain a mixed sugar solution.
[0060] S3. The concentration of the trisaccharide (glucose + xylose + arabinose) in the obtained mixed sugar solution was measured, and then compounded with finished sugars (finished glucose + finished xylose + finished arabinose) to make the mass concentration ratio of glucose: xylose: arabinose = 8:2:0.5, and the total sugar concentration was 140 g / L.
[0061] S4. Use the emptied primary enzymatic hydrolysis tank as a fermentation tank for lactic acid fermentation. Use the fermentation sugar prepared in the previous step as the fermentation carbon source, and add 5 g / L yeast extract powder, 0.03 g / L sodium chloride, 1.5 g / L sodium acetate, 0.6 g / L ammonium citrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, and 0.15 g / L manganese sulfate heptahydrate. Sterilize at 115°C for 20 min until ready for use.
[0062] S5. Inoculate the Bacillus coagulans liquid into the fermentation medium containing the carbon source and ferment to obtain a fermentation liquid containing L-lactic acid. The fermentation temperature is 50° C., the rotation speed is 120 rpm, and the fermentation time is 48 h.
[0063] When the above process conditions are adopted, the content of L-lactic acid is ≥132.8 g / L, the sugar-acid conversion rate is ≥94.9%, and the lactic acid purity is ≥99.6%.
[0064] Example 4
[0065] S1. Wheat straw is cleaned and chopped into 4-10 cm long segments. The segments are then soaked in clean water, removed, and mixed with a 5% caustic soda solution. The segments are then processed in a twin-screw extruder and placed in an insulated box for 1 hour. The segments are then re-entered the twin-screw extruder, and water is added using a metering pump. Wet fiber is then obtained through multi-stage countercurrent displacement washing. Excess water has been removed from the extruder at this stage, allowing the fibers to be directly used for enzymatic hydrolysis.
[0066] S2, the obtained holofiber and PH=5.0 enzymolysis buffer solution are configured into enzymolysis raw materials with a solid-liquid ratio of 10%, and are poured into a primary enzymolysis tank for high temperature sterilization, cooled to 50 ° C, turned on the stirring paddle and stirred for 10 min to mix, and then a complex enzyme (mainly including cellulase, hemicellulase, xylanase, xylosidase) is added. The addition amount of the complex enzyme is 20FPU / g. Under the conditions of 50 ° C and 200 rpm, after enzymolysis for 40 hours, the supernatant is centrifuged to obtain a sugar solution, and the lower heavy phase is poured into a secondary enzymolysis tank, and the primary enzymolysis tank is cleaned. An appropriate amount of enzymolysis buffer solution sterilized in advance is added to the secondary enzymolysis tank, and the complex enzyme is added after heating to 50 ° C, and the addition amount is 20FPU / g. The enzymolysis is continued for 48 hours. After completion, it is first subjected to plate and frame coarse filtration and then enters the centrifugal filter for refiltration. The filtrate is mixed with the sugar solution in the previous step to obtain a mixed sugar solution.
[0067] S3. The concentration of the trisaccharide (glucose + xylose + arabinose) in the obtained mixed sugar solution was measured, and then compounded with finished sugars (finished glucose + finished xylose + finished arabinose) to make the mass concentration ratio of glucose: xylose: arabinose = 8:2:0.5, and the total sugar concentration was 160 g / L.
[0068] S4. Use the emptied primary enzymatic hydrolysis tank as a fermentation tank for lactic acid fermentation. Use the fermentation sugar prepared in the previous step as the fermentation carbon source, and add 5 g / L yeast extract powder, 0.03 g / L sodium chloride, 1.5 g / L sodium acetate, 0.6 g / L ammonium citrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, and 0.15 g / L manganese sulfate heptahydrate. Sterilize at 115°C for 20 min until ready for use.
[0069] S5. Inoculate the Bacillus coagulans liquid into the fermentation medium containing the carbon source and ferment to obtain a fermentation liquid containing L-lactic acid. The fermentation temperature is 50° C., the rotation speed is 120 rpm, and the fermentation time is 48 h.
[0070] When the above process conditions are adopted, the content of L-lactic acid is ≥145.7 g / L, the sugar-acid conversion rate is ≥91.1%, and the lactic acid purity is ≥99.6%.
[0071] The results of measuring L-lactic acid content, sugar-acid conversion, and lactic acid purity in Examples 1-4 show that, under the same fermentation conditions, the sugar-acid conversion rate gradually decreases with increasing fermentation sugar concentration. When the fermentation sugar concentration was 100 g / L, the sugar-acid conversion rate was 96.2%. When the sugar concentration increased to 160 g / L, the conversion rate dropped to 91.1%. The optical purity of the lactic acid was ≥99.5%.
[0072] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
[0073] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.
Claims
1. A method for preparing lactic acid by continuous saccharification and fermentation of straw, characterized in that: The following steps are involved: The straw is dusted, cleaned, cut and then soaked, taken out, water is controlled and mixed with caustic soda solution, and then kneaded and washed to obtain wet heald fiber; The wet heald fiber is dried and then immersed in an enzymatic hydrolysis buffer or the wet heald fiber and the enzymatic hydrolysis buffer are evenly mixed, and then a composite enzyme is added, and enzymatic hydrolysis and saccharification are carried out in a primary enzymatic hydrolysis tank and a secondary enzymatic hydrolysis tank. The clear liquid obtained in the primary enzymatic hydrolysis tank is filtered to obtain a mixed sugar liquid; the solids in the lower layer of the primary enzymatic hydrolysis tank are sent to the secondary enzymatic hydrolysis tank for saccharification with the composite enzyme again, and then filtered to obtain a mixed sugar liquid; The finished sugar is compounded with the mixed sugar solution to obtain fermentable sugar; Using fermentable sugar to ferment bacteria in a fermentation tank to obtain lactic acid fermentation liquid; After the saccharification is completed, the first-level enzymatic hydrolysis tank is emptied, and the solids continue to be hydrolyzed in the second-level enzymatic hydrolysis tank; after the first-level enzymatic hydrolysis tank is cleaned and sterilized, it is used as a fermentation tank for lactic acid fermentation; after the fermentation is completed, the first-level enzymatic hydrolysis tank is used again for comprehensive fiber enzymatic hydrolysis; and the second-level enzymatic hydrolysis tank is emptied and cleaned and then a new round of enzymatic hydrolysis is carried out.
2. The method for preparing lactic acid by continuous saccharification and fermentation of straw according to claim 1, characterized in that: The straw is one or more of rice straw, wheat straw, corn straw, and cotton straw.
3. The method for preparing lactic acid by continuous saccharification and fermentation of straw according to claim 1, characterized in that: The complex enzyme comprises cellulase, hemicellulase, xylanase and xylosidase.
4. The method for preparing lactic acid by continuous saccharification and fermentation of straw according to claim 1, characterized in that: The enzymatic hydrolysis buffer is composed of a combination of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, citric acid, sodium citrate, phosphoric acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate.
5. The method for preparing lactic acid by continuous saccharification and fermentation of straw according to claim 1, characterized in that: The pH value of the enzymolysis buffer is 4.5-6.
5.
6. The method for preparing lactic acid by continuous saccharification and fermentation of straw according to claim 1, characterized in that: The saccharification temperature of the first-level enzymolysis tank and the second-level enzymolysis tank is 40-55°C, and the stirring speed is 120-200r / min; the saccharification time of the first-level enzymolysis tank is 40h, the enzyme addition amount is 15-25FPU / g, and the saccharification time of the second-level enzymolysis tank is 48h, and the enzyme addition amount is 10-25FPU / g.
7. The method for preparing lactic acid by continuous saccharification and fermentation of straw according to claim 1, characterized in that: The steps of compounding the finished sugar and the mixed sugar solution include: firstly measuring the concentrations of glucose, xylose and arabinose in the mixed sugar solution, and then compounding the mixed sugar solution with finished glucose, finished xylose and finished arabinose so that the mass concentration ratio is glucose:xylose:arabinose=8:2:0.5, and the total sugar concentration is 100-160g / L.
8. The method for preparing lactic acid by continuous saccharification and fermentation of straw according to claim 1, characterized in that: The fermentation process includes: using a seed culture medium to cultivate a bacterial fermentation liquid, and then inoculating the bacterial fermentation liquid containing Bacillus coagulans into a fermentation medium at an inoculation rate of 1:10 to ferment, thereby obtaining a fermentation liquid containing lactic acid.
9. The method for preparing lactic acid by continuous saccharification and fermentation of straw according to claim 8, characterized in that: The seed culture medium includes: 50 g / L glucose, 10 g / L yeast extract, 0.01 g / L sodium chloride, 0.5 g / L sodium acetate, 0.2 g / L ammonium citrate, 0.2 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate heptahydrate and 30 g / L calcium carbonate.
10. The method for preparing lactic acid by continuous saccharification and fermentation of straw according to claim 8, characterized in that: The fermentation medium comprises: 100-160 g / L fermentation sugar, 5 g / L yeast extract powder, 0.03 g / L sodium chloride, 1.5 g / L sodium acetate, 0.6 g / L ammonium citrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate and 0.15 g / L manganese sulfate heptahydrate.