A method for co-producing ethanol and L-arabinose using corn bran

Abstract

The present invention provides a method for co-producing ethanol and L-arabinose using corn bran, the method comprising pretreatment, enzymolysis, fermentation, alcohol distillation, decolorization, ion exchange, concentration, and crystallization. The present invention obtains alcohol and L-arabinose by processing corn bran, which can further improve the utilization value of corn bran and increase the income. In addition, the obtained alcohol can be used as a clean fuel, which meets the needs of environmental protection, and L-arabinose has a broad market as a health functional sugar; the method of the present invention, relative to the dry basis of corn bran, the alcohol yield reaches 22-23%, and the L-arabinose yield is 15.5-16%.

Description

Technical Field

[0001] The invention relates to a method for co-producing ethanol and L-arabinose by utilizing corn bran, and belongs to the technical field of waste utilization. Background Art

[0002] Corn is one of the three major food crops in the world, and is also an important feed and industrial raw material. my country is a major corn consumer. In my country, more than 10% of corn is used as corn starch and starch sugar production raw materials every year, producing a large amount of by-products - corn bran. Corn bran is the main by-product in corn wet processing. Corn bran contains rich hemicellulose, cellulose, protein, crude fat and a small amount of ash, phenolic compounds and other substances. CN101100685A discloses a method for preparing L-arabinose from corn bran. The invention uses amylase to treat bran to remove starch from the bran, which requires amylase cost and washing water waste. The removed starch concentration is low and difficult to reuse. The subsequent acid hydrolysis sugar concentration is low. Microbial treatment can only remove single glucose, which requires repeated treatment, resulting in high cost, uneconomical, and high product sugar content. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a method for co-producing ethanol and L-arabinose using corn bran, which improves the yield of ethanol and L-arabinose and prepares L-arabinose with higher purity.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] The following are further improvements to the above technical solution:

[0006] 1. Preprocessing

[0007] Water is added to corn bran to prepare a reaction system, wherein the mass proportion of the dry basis of corn bran is 15-20%, and 65-75% (w / v) sulfuric acid solution is added according to 0.3-1.0% (w / w) of the dry basis mass, and the mixture is treated at 105-130° C. for 1-2.5 hours to obtain a pretreated sugar solution.

[0008] The pretreated sugar solution has a glucose content of 1.65-1.7 g / dl, a xylose content of 2.7-2.75 g / dl, a reducing sugar content of 6.5-6.7 g / dl, and an L-arabinose content of 1.3-1.5 g / dl.

[0009] 2. Enzymatic hydrolysis

[0010] The pretreated sugar solution is pumped into the enzymolysis tank, adjusted to pH 4.0-6.0, and Novozymes cellulose and fungal xylanase are added. The dosage of both enzymes is 40-50U / g dry basis. The enzymolysis is carried out at a temperature of 55-65°C for 24-48h to obtain an enzymolysis sugar solution containing glucose, xylose, and L-arabinose.

[0011] The enzymatically hydrolyzed sugar solution has a glucose content of 3.9-4.0 g / dl, a xylose content of 3.4-3.6 g / dl, a reducing sugar content of 9.6-9.8 g / dl, and an L-arabinose content of 1.8-2.0 g / dl.

[0012] 3. Ethanol fermentation

[0013] The strain used for ethanol fermentation is genetically recombinant Saccharomyces cerevisiae, which can produce alcohol by utilizing glucose and xylose in enzymatically hydrolyzed sugar solution.

[0014] The recombinant Saccharomyces cerevisiae was provided by Qilu University of Technology, and the strain accession number is CGMCC No.11331;

[0015] The specific process is as follows:

[0016] A. Flat plate marking

[0017] The gene-recombinant Saccharomyces cerevisiae is streaked and cultured on a yeast activation plate, and a single colony is grown and used for later use;

[0018] The yeast activation plate is a YPX solid culture medium.

[0019] B. Shake flask culture

[0020] A single colony was picked and inoculated into a shake flask for culture. The culture medium was YPX culture medium. The fermentation liquid was used when the OD600nm value after being diluted 10 times was 0.29-0.32.

[0021] C. Seed cultivation

[0022] Inoculate the fermentation broth from the shake flask into the seed tank at an inoculation rate of 0.9-1.1% (volume ratio). When the OD600nm value of the fermentation broth from the seed culture is 0.58-0.62 after being diluted 10 times, it is ready for use.

[0023] The medium for seed culture was YPX medium.

[0024] D. Fermentation

[0025] The fermentation liquid after seed culture is connected to the fermentation tank at an inoculation rate of 8-15% (volume ratio). The fermentation medium is enzymatic sugar liquid. The temperature is 28-35°C and anaerobic fermentation is performed. The brewer's yeast can use glucose and xylose to ferment and produce ethanol. The fermentation takes 22-30 hours and the fermentation is stopped when the glucose content is 0 g / dl and the residual xylose is less than 0.2g / dl.

[0026] In the fermentation broth, the content of glucose is 0 g / dl, the content of xylose is 0.15-0.18 g / dl, the content of reducing sugar is 2-2.5 g / dl, the content of L-arabinose is 1.6-1.8 g / dl, and the content of ethanol is 2.6-3 g / dl.

[0027] 4. Alcohol distillation: The fermented liquid is filtered through a plate and frame filter to remove yeast and impurities, and ethanol is extracted through a distillation device. The remaining liquid enters the next step.

[0028] 5. Decolorization: Filter the sugar solution through a fine filter and concentrate it to a sugar concentration of 18-22% (w / v). Add 0.9-1.2% (w / v) powdered activated carbon for decolorization. The decolorization temperature is 58-63°C and the decolorization time is 0.9-1.1h. The light transmittance of the sugar solution is increased to more than 80%.

[0029] 6. Ion exchange: The decolorized L-arabinose solution is passed through a cation exchange column and an anion exchange column in sequence to remove impurity ions. The feed rate is 1.1-1.3 times the column volume / h at room temperature, and the sugar components are collected.

[0030] The cationic resin is Seplite D001 strong acid macroporous resin, and the anionic resin is Seplite 201*4 strong base gel, which can effectively remove anions and cations in the sugar solution.

[0031] In the sugar solution after ion exchange, the content of glucose is 0g / dl, the content of xylose is 0.1-0.15g / dl, the content of reducing sugar is 2-2.5g / dl, the content of L-arabinose is 1.6-1.8g / dl, the content of sulfate ion is 0mg / dl, the content of sodium ion is 0mg / dl, and the content of chloride ion is 0mg / dl.

[0032] 7. Concentration: Concentrate the sugar solution after ion exchange to a sugar solution mass percentage concentration of 50-60% (w / v) at a concentration temperature of 58-62°C.

[0033] 8. Crystallization:

[0034] A. First Crystallization

[0035] The concentrated L-arabinose solution was cooled and crystallized, and seed crystals (L-arabinose) were added at 1.4-1.6% (w / v) dry matter. The initial temperature was 58-62°C, and the temperature was started to be lowered at 0.8-1.2°C / h to 34-36°C. The temperature was kept at 4.5-5.5h, filtered, and dried to obtain crude L-arabinose crystals.

[0036] B. Recrystallization

[0037] The crude L-arabinose crystals obtained by dissolving in deionized water have a mass percentage concentration of 72-77%, and are subjected to cooling crystallization. Seed crystals (L-arabinose) are added, and the amount of seed crystals added is 0.28-0.32% (w / w) of the dry matter weight of the L-arabinose solution. The initial temperature is 58-62°C. The cooling rate is 1.4-1.6°C / h, the temperature is reduced to 28-31°C, and the temperature is kept for 7.5-8.5h. The crystals are centrifuged and dried to obtain L-arabinose crystals with a crystal mass content of 98%, a moisture content of less than 0.1%, and an ash content of less than 0.05%.

[0038] The yield of L-arabinose calculated on a dry basis of corn bran was 15.5-16%.

[0039] The invention utilizes corn fiber hydrolysis to prepare fiber sugar liquid, utilizes cellulase and xylanase to hydrolyze, obtains sugar liquid containing glucose, xylose and L-arabinose, utilizes recombinant yeast to consume glucose and xylose to produce ethanol, distills the remaining fermentation liquid of ethanol, treats it, purifies it by microfiltration and decolorizes it by adsorption, concentrates it to prepare L-arabinose syrup, and obtains L-arabinose by crystallization or spray drying. The process converts low-value-added corn bran into high-value ethanol and L-arabinose after treatment, thereby improving the utilization value of corn bran.

[0040] The genetically recombinant brewer's yeast of the present invention is classified and named as: Saccharomyces cerevisiae , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 8, 2015, and the deposit number is CGMCC No.11331.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The invention processes corn bran to obtain alcohol and L-arabinose, which can further improve the utilization value of corn bran and increase the income. In addition, the obtained alcohol can be used as clean fuel, which meets the environmental protection requirements, and L-arabinose has a broad market as a health functional sugar.

[0043] According to the method of the present invention, the alcohol yield reaches 22-23% and the L-arabinose yield reaches 15.5-16% relative to the dry basis of corn bran.

[0044] The L-arabinose prepared by the invention has a crystal mass content of 97.5-98.5%, a moisture content of less than 0.1%, and an ash content of less than 0.05%. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0046] Example 1

[0047] (1) Preprocessing

[0048] Take the corn bran squeezed from the workshop, with a moisture content of 60% (w / w) and a dry basis of 40% (w / w). Water is added to the corn bran to prepare the reaction system, in which the mass proportion of the dry basis of the corn bran is 15%. According to 0.5% (w / w) of the dry basis mass, 70% (w / v) sulfuric acid solution is added. The sample is placed in a high-temperature reactor, and the stirring speed is set to 200 rpm and the temperature is 120°C. The reaction is carried out for 2 hours to obtain a pretreated sugar solution.

[0049] In the pretreated sugar solution, the content of glucose was 1.68 g / dl, the content of xylose was 2.72 g / dl, the content of reducing sugar was 6.61 g / dl, and the content of L-arabinose was 1.4 g / dl.

[0050] (2) Enzymatic hydrolysis

[0051] The treated sugar solution was adjusted to pH 5.0 using a saturated sodium hydroxide solution, and cellulase and xylanase were added at 40 U / g dry basis, wherein the cellulase activity unit was 11,000 U / g, the xylanase activity unit was 9,000 U / g, the cellulase addition amount in the reaction system was 3.63 mg / g dry basis bran, and the xylanase addition amount was 4.44 mg / g dry basis bran, and enzymolysis was performed at 60° C. for 24 hours to obtain an enzymolysis sugar solution containing glucose, xylose, and L-arabinose;

[0052] The enzymatically hydrolyzed sugar solution has a glucose content of 3.94 g / dl, a xylose content of 3.5 g / dl, a reducing sugar content of 9.7 g / dl, and an L-arabinose content of 1.9 g / dl.

[0053] (3) Ethanol fermentation

[0054] The strain used for ethanol fermentation is genetically recombinant Saccharomyces cerevisiae, which can produce alcohol by utilizing glucose and xylose in enzymatically hydrolyzed sugar solution.

[0055] The recombinant Saccharomyces cerevisiae was provided by Qilu University of Technology, and the strain accession number is CGMCC No.11331;

[0056] The specific process is as follows:

[0057] A. Flat plate marking

[0058] The yeast activation plate is a YPX solid culture medium, which contains 1g / dl yeast extract, 2g / dl peptone, 2g / dl xylose, pH 6.0, and 2g / dl agar. Use an inoculation loop to inoculate the bacteria in the glycerol tank, streak the plate, and culture it at 30°C for 2 days. After a single colony grows, it is ready for use.

[0059] B. Shake flask culture

[0060] A single colony was picked and inoculated into a shake flask for culture. The culture medium was YPX medium, which contained 1 g / dl yeast extract, 2 g / dl peptone, and 2 g / dl xylose, pH 6.0. The culture was carried out at 30°C and 200 rpm for 24 hours. The fermentation liquid was used when the OD600nm value after being diluted 10 times was 0.3.

[0061] C. Seed cultivation

[0062] The culture medium in the seed tank is YPX culture medium, with the formula: yeast extract powder 1g / dl, peptone 2g / dl, xylose 2g / dl. According to the inoculation amount of 1% (volume ratio), the fermentation liquid cultured in the shake flask is inoculated into the seed tank. The culture conditions are 30°C, 200rpm, pH6.0. When the OD600nm value of the fermentation liquid cultured in the seed tank is 0.6 after being diluted 10 times, it is ready for use.

[0063] D. Fermentation

[0064] At a 10% inoculation rate (volume ratio), the fermentation liquid after seed culture is connected to the fermentation tank, and the fermentation medium is enzymatic sugar liquid.

[0065] The fermentation tank was cultured at a temperature of 30°C and a rotation speed of 200 rpm, and anaerobic fermentation was performed. The fermentation was stopped when the residual xylose content was below 0.2 g / dl.

[0066] The contents of various substances in the fermentation broth were detected as follows: glucose content was 0 g / dl, xylose content was 0.16 g / dl, reducing sugar content was 2.2 g / dl, L-arabinose content was 1.7 g / dl, and ethanol content was 2.69 g / dl.

[0067] After the yeast fermentation liquid is filtered through a plate and frame filter to remove the bacteria and solids, the filtrate is distilled for alcohol at a distillation temperature of 75°C and a cooling tank temperature of 10°C. The resulting alcohol is stored in a collection tank.

[0068] (4) Decolorization

[0069] The sugar solution after alcohol distillation is filtered through a fine filter, and the filtrate is concentrated to a sugar concentration of 20% (w / v) through an MVR evaporator. 1% (w / v) powdered activated carbon is added and decolorized at 60°C for 1h. The light transmittance of the sugar solution is increased to more than 80%.

[0070] (5) Ion exchange

[0071] The decolorized sugar solution was passed through a cation exchange column and an anion exchange column in sequence, fed at room temperature at a feed rate of 1.2 times the column volume / h, and the sugar components were collected.

[0072] The cationic resin is Seplite D001 strong acid macroporous resin, and the anionic resin is Seplite 201*4 strong base gel, which can effectively remove anions and cations in the sugar solution.

[0073] In the sugar solution after ion exchange, the content of glucose was 0 g / dl, the content of xylose was 0.12 g / dl, the content of reducing sugar was 2.1 g / dl, the content of L-arabinose was 1.7 g / dl, the content of sulfate ion was 0 mg / dl, the content of sodium ion was 0 mg / dl, and the content of chloride ion was 0 mg / dl.

[0074] (6) Vacuum concentration

[0075] The sugar solution after ion exchange was vacuum concentrated at 60°C and vacuum degree -0.08Mpa to a sugar content of 55% (w / v).

[0076] (7) Cooling crystallization

[0077] A. First Crystallization

[0078] The concentrated sugar solution was cooled and crystallized, and seed crystals (L-arabinose) were added at 1.5% (w / v). The initial temperature was 60°C, and the temperature was slowly cooled to 35°C at a cooling rate of 1°C / h and maintained for 5 hours. The L-arabinose crystals were filtered and dried to obtain crude L-arabinose crystals.

[0079] B. Recrystallization

[0080] The crude L-arabinose crystals obtained by dissolving in deionized water had a mass percentage concentration of 75%, and were subjected to cooling crystallization. Seed crystals (L-arabinose) were added in an amount of 0.3% (w / w) of the dry matter mass of the L-arabinose solution. The initial temperature was 60°C. The cooling rate was 1.5°C / h, the temperature was reduced to 30°C, and the temperature was kept at this temperature for 8h. The crystals were centrifuged and dried at a drying temperature of 60°C to obtain L-arabinose crystals with a crystal mass content of 98%, a moisture content of less than 0.1%, and an ash content of less than 0.05%.

[0081] The yield of L-arabinose calculated on a dry basis of corn bran was 16%.

[0082] After acid hydrolysis, enzymolysis and yeast fermentation, glucose, xylose and a part of polysaccharides in the fiber hydrolyzed sugar are fermented into alcohol by yeast, and the alcohol yield reaches 23% (relative to the dry basis of bran). After distillation and filtration, a small amount of xylose, polysaccharides and product L-arabinose remain in the filtrate. After ion exchange adsorption, the content of sulfate ions, chloride ions and sodium ions in the filtrate is basically undetectable, the filtrate has high purity and is nearly transparent in color. After crystallization and recrystallization processes, high-purity L-arabinose can be obtained, and the yield can reach 16% of the dry basis of corn bran. Using this process, a higher product value can be obtained, and the use value of bran is improved.

Claims

1. A method for co-producing ethanol and L-arabinose using corn bran, characterized in that: The method includes pretreatment, enzymolysis, fermentation, alcohol distillation, decolorization, ion exchange, concentration, and crystallization; The pretreatment method comprises the following steps: taking squeezed corn bran with a water content of 60% (w / w) and a dry basis of 40% (w / w), adding water to the corn bran to prepare a reaction system, wherein the mass proportion of the dry basis of the corn bran is 15%, adding a 70% (w / v) sulfuric acid solution according to 0.5% (w / w) of the dry basis mass, placing the sample in a high-temperature reactor, setting a stirring speed of 200 rpm and a temperature of 120° C., and treating for 2 hours to obtain a pretreated sugar solution; In the pretreated sugar solution, the content of glucose is 1.68 g / dl, the content of xylose is 2.72 g / dl, the content of reducing sugar is 6.61 g / dl, and the content of L-arabinose is 1.4 g / dl; The enzymatic hydrolysis method comprises the following steps: adjusting the pH of the pretreated sugar solution to 5.0 using a saturated sodium hydroxide solution, adding cellulase and xylanase at 40 U / g dry basis, wherein the cellulase activity unit is 11,000 U / g, the xylanase activity unit is 9,000 U / g, the cellulase addition amount in the reaction system is 3.63 mg / g dry basis bran, the xylanase addition amount is 4.44 mg / g dry basis bran, and enzymatic hydrolysis is performed at 60° C. for 24 hours to obtain an enzymatic hydrolyzed sugar solution containing glucose, xylose, and L-arabinose; The enzymatically hydrolyzed sugar solution has a glucose content of 3.94 g / dl, a xylose content of 3.5 g / dl, a reducing sugar content of 9.7 g / dl, and an L-arabinose content of 1.9 g / dl.