Method for extracting ethanol and oligosaccharide from wheat bran

Abstract

The invention relates to the technical field of fermentation engineering, in particular to a method for extracting ethyl alcohol and oligosaccharide from wheat bran and application thereof.The method for extracting the ethyl alcohol and the oligosaccharide from the wheat bran comprises the preparation steps that 1, the wheat bran is smashed and then pretreated in dilute acid, and a pretreatment substance is obtained; (2) adding a compound enzyme solution into the pretreatment substance, and carrying out primary enzymolysis to obtain a supernate rich in oligosaccharide and residues; (3) uniformly mixing the residues and deionized water, firstly adding feruloyl esterase, performing secondary enzymolysis, then adding cellulase and pectinase, performing final enzymolysis, and after the enzymolysis is finished, performing enzyme deactivation treatment to obtain enzymatic hydrolysate; and (4) inoculating the complex microbial inoculant into the enzymatic hydrolysate, carrying out aerobic and anaerobic two-stage combined fermentation, distilling the fermentation liquor, and collecting fractions to obtain the ethanol. According to the method, high-valued conversion of the wheat bran is realized through resource graded utilization, enzymolysis synergistic interaction and fermentation metabolism complementation, and the yield of oligosaccharide and the yield of ethanol are relatively high.

Description

Technical Field

[0001] The present application relates to the technical field of fermentation engineering, and more specifically, to a method for extracting ethanol and oligosaccharides from wheat bran. Background Art

[0002] Wheat bran is a major byproduct of wheat processing, with an annual production exceeding 20 million tons. Currently, bran is primarily used as feed or a food additive, with relatively low added value. With the advancement of biomass refining technology, extracting high-value chemicals from the abundant cellulose (35-40%) and hemicellulose (25-30%) in bran has become a research hotspot.

[0003] Traditional wheat bran extraction technology mostly focuses on the development of a single component. For example, polysaccharides are extracted through alkaline extraction combined with hypochlorous acid neutralization and decolorization. Although this can improve the extraction efficiency, there are problems such as the decolorant adsorbing polysaccharides leading to loss of target product, complex process flow, and failure to comprehensively utilize cellulose. The preparation of oligosaccharides often relies on acid hydrolysis of hemicellulose, which produces strong acid waste liquid that pollutes the environment and has a yield of only about 30%. In addition, existing ethanol production mainly relies on the fermentation of grain starch, and the utilization rate of the cellulose component in the bran is insufficient, resulting in waste of raw materials and increased costs. In response to the above-mentioned defects, this application proposes a method for extracting ethanol and oligosaccharides from wheat bran. The development of a method that fully utilizes wheat bran to extract ethanol and oligosaccharides at the same time has broad prospects. Summary of the Invention

[0004] In order to solve the technical problems mentioned in the background technology, the present application provides a method for extracting ethanol and oligosaccharides using wheat bran.

[0005] A method for extracting ethanol and oligosaccharides from wheat bran, comprising the following preparation steps: (1) crushing the wheat bran, adding it to dilute acid, pre-treating it at 90-100° C. for 1-2 hours, filtering, and washing to obtain a pre-treated product;

[0006] (2) adding the complex enzyme solution to the pre-treated product, adding deionized water, performing initial enzymatic hydrolysis for 4-6 hours at a pH of 4.5-5.0 and a temperature of 50-60°C, and centrifuging to obtain a supernatant and residue rich in oligosaccharides;

[0007] (3) After the residue and deionized water are evenly mixed, ferulic acid esterase is first added, and secondary enzymolysis is performed for 3-6 hours, and then cellulase and pectinase are added, and final enzymolysis is performed for 6-10 hours. After the enzymolysis is completed, the temperature is raised to 90-95°C, the enzyme is inactivated for 20-30 minutes, and the solution is cooled to room temperature and filtered to obtain an enzymolysis solution;

[0008] (4) The composite bacterial agent is inoculated into the enzymatic hydrolysate and aerobic and anaerobic fermentation is carried out. The first stage is the aerobic fermentation stage. After 10-12 hours of aerobic fermentation, the second stage is anaerobic fermentation. The anaerobic fermentation lasts for 36-48 hours. The fermentation liquid is distilled and the fractions are collected to obtain ethanol.

[0009] Preferably, the complex enzyme solution in step (2) is composed of xylanase, mannanase, arabinofuranosidase and acetate buffer in a mass ratio of 12-15:1-2:1-3:100-150;

[0010] Preferably, the concentration of the acetate buffer in step (2) is 0.1-0.12 mol / L.

[0011] Preferably, in step (2), the mass ratio of the complex enzyme solution, the pre-treated material, and deionized water is 5-10:100-110:550-600.

[0012] Preferably, in step (3), the mass ratio of the residue, deionized water, ferulic acid esterase, cellulase and pectinase is 100-120:800-1000:0.3-0.5:3-4:1-1.2.

[0013] Preferably, the pH of the secondary enzymatic hydrolysis in step (3) is 5-6 and the temperature is 40-45°C; the final enzymatic hydrolysis temperature is 48-52°C.

[0014] Preferably, the composite bacterial agent in step (4) is composed of Saccharomyces cerevisiae, Candida tropicalis and Trichoderma reesei with an effective viable cell count ratio of 10-12:2-4:1-2.

[0015] Preferably, the inoculation amount of the composite bacterial agent in step (4) is 10-15%.

[0016] Preferably, the aerobic fermentation conditions in step (4) are as follows: controlling the pH of the aerobic fermentation to be 5-5.5, the fermentation temperature to be 30-32° C., and the dissolved oxygen concentration in the aerobic fermentation stage to be 5-10%.

[0017] Preferably, the anaerobic fermentation conditions in step (4) are as follows: controlling the anaerobic fermentation temperature to 35-40° C. and adjusting the dissolved oxygen concentration to less than 0.5%.

[0018] In summary, this application has the following beneficial effects:

[0019] This application uses xylanase, mannanase, and arabinofuranosidase as a composite enzyme to enzymatically hydrolyze wheat bran to extract oligosaccharides. Xylanase can act on the β-1,4-glycosidic bonds of the xylan backbone to generate xylooligosaccharides and directly release the main oligosaccharide product, while mannanase removes the arabinose residues of the xylan side chains. Arabinofuranosidase preferentially cuts the arabinose in the xylan side chains, destroying the spatial structure of xylan and making it easier for xylanase to access the backbone. The combination of the two can significantly increase the degradation rate of xylan. Mannanase promotes the diffusion of xylanase and arabinofuranosidase into the deep substrate, improving the overall enzymatic hydrolysis efficiency. The composite enzyme weakens the cell wall structure through synergistic action, releasing more oligosaccharides.

[0020] In the preparation process of the enzymatic hydrolyzate, the present application first adds ferulic acid esterase, and then introduces cellulase and pectinase to enzymatically hydrolyze the residue. Ferulic acid esterase can preferentially decompose the dense lignin-carbohydrate complex formed by cross-linking xylan and lignin through ferulic acid ester bonds, remove the physical wrapping of lignin on cellulose and hemicellulose (such as xylan), and degrade the phenolic acid modification of xylan side chains, reducing steric hindrance and significantly improving the accessibility of subsequent enzymes to substrates. After ferulic acid esterase destroys the cross-linked structure, cellulase and pectinase can efficiently decompose the exposed cellulose backbone, remove residual acetyl groups, completely disintegrate the network structure of the plant cell wall, release more oligosaccharides and fermentable sugars, and provide substrates for subsequent ethanol fermentation.

[0021] This application utilizes Saccharomyces cerevisiae, Candida tropicalis, and Trichoderma reesei as composite bacterial agents to ferment the enzymatic hydrolyzate. Saccharomyces cerevisiae is mainly responsible for efficiently converting monosaccharides (glucose, fructose, etc.) into ethanol and carbon dioxide, while Candida tropicalis can utilize pentoses (such as xylose) and complex polysaccharides to supplement the insufficient utilization of non-hexose sugars by Saccharomyces cerevisiae and improve substrate utilization. Trichoderma reesei can secrete cellulase to further hydrolyze residual cellulose and release more fermentable sugars. The three-way combination can fully utilize various sugars such as glucose, xylose, and cellobiose in the enzymatic hydrolysate, significantly improving the utilization rate of raw materials. In addition, through aerobic and anaerobic fermentation, in the aerobic fermentation stage, tropical yeast and Trichoderma reesei can rapidly proliferate and initially decompose complex substrates through aerobic respiration. In the anaerobic fermentation stage, ethanol fermentation is dominated by Saccharomyces cerevisiae, while tropical yeast and Trichoderma reesei continue to metabolize the remaining sugars. Through a synergistic effect, the cellulase secreted by Trichoderma reesei and the xylose metabolizing enzyme secreted by tropical yeast work together to reduce the accumulation of inhibitory intermediates (such as xylose) and promote the efficient ethanol production of Saccharomyces cerevisiae. By pretreatment, the lignocellulose structure of bran is destroyed, hemicellulose is enzymatically hydrolyzed step by step to produce oligosaccharides, and cellulose is converted into fermentable sugars for ethanol production. At the same time, the bioenzymatic method is used to replace strong acid / strong base treatment to reduce pollution. Ultimately, the cascade utilization of all components of bran is achieved, the oligosaccharide yield is increased, and the ethanol conversion efficiency is high, which is both environmentally friendly and economical. DETAILED DESCRIPTION

[0022] The present application is further described in detail below with reference to the embodiments.

[0023] The wheat bran used in the examples and comparative examples of the present application was purchased from Lingshou County Hengshuo Mineral Products Co., Ltd.; xylanase (enzyme activity: 280,000 CFU / g) was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd.; mannanase (enzyme activity: 50,000 CFU / g) was purchased from Hebei Jiuyu Biotechnology Co., Ltd.; arabinofuranosidase was purchased from Hebei Hongtao Bioengineering Co., Ltd.; feruloyl esterase was purchased from Shanghai Quanyan Biotechnology Co., Ltd.; cellulase (enzyme activity: 100,000 CFU / g) was purchased from Zhejiang Yicun Biotechnology Co., Ltd.; pectinase was purchased from Guangdong Dingsheng Food Ingredients Co., Ltd.; brewer's yeast (product name: Saccharomyces cerevisiae, product number: B98026), tropical Candida (product name: Candida tropicalis, product number: BMZ129285) and Trichoderma reesei (product name: Trichoderma eesei, product number: B89536) were purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0024] Examples 1-3 and Comparative Examples 1-6 provide a method for extracting ethanol and oligosaccharides from wheat bran.

[0025] Example 1

[0026] A method for extracting ethanol and oligosaccharides from wheat bran comprises the following preparation steps:

[0027] (1) After the wheat bran is crushed, it is passed through a 40-mesh sieve. The sieved wheat bran is added to the dilute sulfuric acid with a concentration of 0.5 wt% in a mass ratio of 1:5. The mixture is stirred at 90°C and a speed of 2000 rpm for pretreatment for 1 hour. The mixture is filtered and washed with deionized water for 3 times to obtain a pretreated product.

[0028] (2) adding the complex enzyme solution to the pre-treated material, adding deionized water, performing the initial enzymatic hydrolysis for 4 hours at a pH of 4.5 and a temperature of 50°C, and centrifuging at a centrifugal speed of 5000 rpm for 10 minutes to obtain a supernatant and residue rich in oligosaccharides, wherein the complex enzyme solution is composed of xylanase, mannanase, arabinofuranosidase and acetate buffer in a mass ratio of 12:1:1:100, the concentration of the acetate buffer is 0.1 mol / L, and the mass ratio of the complex enzyme solution, the pre-treated material and deionized water is 5:100:550;

[0029] (3) After the residue and deionized water are evenly mixed, feruloyl esterase is first added, and secondary enzymolysis is carried out at a pH of 5 and a temperature of 40°C for 3 hours. Cellulase and pectinase are then added, and final enzymolysis is carried out at a temperature of 48°C for 6 hours. After the enzymolysis is completed, the temperature is raised to 90°C, the enzyme is inactivated for 20 minutes, and the mixture is cooled to room temperature and filtered to obtain an enzymolysis solution, wherein the mass ratio of the residue, deionized water, feruloyl esterase, cellulase, and pectinase is 100:800:0.3:3:1;

[0030] (4) The composite bacterial agent was inoculated into the enzymatic hydrolysate at an inoculation rate of 10%, and aerobic and anaerobic combined fermentation was carried out. The first stage was the aerobic fermentation stage. The pH of the aerobic fermentation was controlled to be 5, the fermentation temperature was 30°C, and the dissolved oxygen concentration in the aerobic fermentation stage was 5%. After 10 hours of aerobic fermentation, the second stage was anaerobic fermentation. The anaerobic fermentation temperature was 35°C, the dissolved oxygen concentration was 0.3%, and the anaerobic fermentation was carried out for 36 hours. The fermentation liquid was distilled at a distillation temperature of 78°C, and the fractions were collected to obtain ethanol. The composite bacterial agent was composed of Saccharomyces cerevisiae, Candida tropicalis, and Trichoderma reesei with an effective live bacteria ratio of 10:2:1.

[0031] Example 2

[0032] A method for extracting ethanol and oligosaccharides from wheat bran comprises the following preparation steps:

[0033] (1) After the wheat bran is crushed, it is passed through a 50-mesh sieve. The sieved wheat bran is added to the dilute sulfuric acid with a concentration of 1 wt% in a mass ratio of 1:8. The mixture is stirred at 95°C and a speed of 250 rpm for pretreatment for 1.5 h. The mixture is filtered and washed with deionized water for 3 times to obtain a pretreated product.

[0034] (2) adding the complex enzyme solution to the pre-treated material, adding deionized water, performing initial enzymatic hydrolysis for 5 h at a pH of 4.8 and a temperature of 55°C, and centrifuging at a centrifugal speed of 7000 rpm for 15 min to obtain a supernatant and residue rich in oligosaccharides, wherein the complex enzyme solution is composed of xylanase, mannanase, arabinofuranosidase, and acetate buffer in a mass ratio of 13:1.5:2:125, the concentration of the acetate buffer is 0.11 mol / L, and the mass ratio of the complex enzyme solution, the pre-treated material, and deionized water is 8:105:580;

[0035] (3) After the residue and deionized water are evenly mixed, ferulic acid esterase is first added, and secondary enzymolysis is performed at a pH of 5.5 and a temperature of 42°C for 4 hours. Cellulase and pectinase are then added, and final enzymolysis is performed at a temperature of 50°C for 8 hours. After the enzymolysis is completed, the temperature is raised to 92°C, the enzyme is inactivated for 25 minutes, and the mixture is cooled to room temperature and filtered to obtain an enzymolysis solution, wherein the mass ratio of the residue, deionized water, ferulic acid esterase, cellulase, and pectinase is 110:900:0.4:3.5:1.1;

[0036] (4) The composite bacterial agent was inoculated into the enzymatic hydrolysate at an inoculation rate of 12%, and aerobic and anaerobic combined fermentation was carried out. The first stage was the aerobic fermentation stage. The pH of the aerobic fermentation was controlled to be 5.2, the fermentation temperature was 31°C, and the dissolved oxygen concentration in the aerobic fermentation stage was 8%. After 11 hours of aerobic fermentation, the second stage was anaerobic fermentation. The anaerobic fermentation temperature was 38°C, the dissolved oxygen concentration was 0.2%, and the anaerobic fermentation was carried out for 42 hours. The fermentation liquid was distilled at a distillation temperature of 79°C, and the fractions were collected to obtain ethanol. Among them, the composite bacterial agent was composed of Saccharomyces cerevisiae, Candida tropicalis, and Trichoderma reesei with an effective live bacteria ratio of 11:3:1.5.

[0037] Example 3

[0038] A method for extracting ethanol and oligosaccharides from wheat bran comprises the following preparation steps:

[0039] (1) After the wheat bran is crushed, it is passed through a 60-mesh sieve. The sieved wheat bran is added to the dilute sulfuric acid with a concentration of 1.5 wt% in a mass ratio of 1:10. The mixture is stirred at 300 rpm at 100°C for 2 h, filtered, and washed with deionized water three times to obtain a pre-treated product.

[0040] (2) adding the complex enzyme solution to the pre-treated material, adding deionized water, performing the initial enzymatic hydrolysis at a pH of 5 and a temperature of 55°C for 6 hours, centrifuging at a centrifugal speed of 8000 rpm for 20 minutes, and obtaining a supernatant and residue rich in oligosaccharides, wherein the complex enzyme solution is composed of xylanase, mannanase, arabinofuranosidase, and acetate buffer at a mass ratio of 15:2:3:150, the concentration of the acetate buffer is 0.12 mol / L, and the mass ratio of the complex enzyme solution, the pre-treated material, and deionized water is 10:110:600;

[0041] (3) After the residue and deionized water are evenly mixed, ferulic acid esterase is first added, and secondary enzymolysis is carried out at a pH of 6 and a temperature of 45°C for 6 hours. Cellulase and pectinase are then added, and final enzymolysis is carried out at a temperature of 52°C for 10 hours. After the enzymolysis is completed, the temperature is raised to 95°C, the enzyme is inactivated for 30 minutes, and the mixture is cooled to room temperature and filtered to obtain an enzymolysis solution, wherein the mass ratio of the residue, deionized water, ferulic acid esterase, cellulase, and pectinase is 120:1000:0.5:4:1.2;

[0042] (4) The composite bacterial agent was inoculated into the enzymatic hydrolysate at an inoculation rate of 15%, and aerobic and anaerobic combined fermentation was carried out. The first stage was the aerobic fermentation stage. The pH of the aerobic fermentation was controlled to be 5.5, the fermentation temperature was 32°C, and the dissolved oxygen concentration in the aerobic fermentation stage was 10%. After 12 hours of aerobic fermentation, the second stage was anaerobic fermentation. The anaerobic fermentation temperature was 40°C, the dissolved oxygen concentration was 0.1%, and the anaerobic fermentation was carried out for 48 hours. The fermentation liquid was distilled at a distillation temperature of 80°C, and the fractions were collected to obtain ethanol. The composite bacterial agent was composed of Saccharomyces cerevisiae, Candida tropicalis, and Trichoderma reesei with an effective live bacteria ratio of 12:4:2.

[0043] Comparative Example 1

[0044] A method for extracting ethanol and oligosaccharides from wheat bran comprises the following preparation steps:

[0045] (1) After the wheat bran is crushed, it is passed through a 40-mesh sieve. The sieved wheat bran is added to the dilute sulfuric acid with a concentration of 0.5 wt% in a mass ratio of 1:5. The mixture is stirred at 90°C and a speed of 2000 rpm for pretreatment for 1 hour. The mixture is filtered and washed with deionized water for 3 times to obtain a pretreated product.

[0046] (2) adding the complex enzyme solution to the pre-treated material, adding deionized water, performing the initial enzymatic hydrolysis for 4 hours at a pH of 4.5 and a temperature of 50°C, and centrifuging at a centrifugal speed of 5000 rpm for 10 minutes to obtain a supernatant and residue rich in oligosaccharides, wherein the complex enzyme solution is composed of xylanase, arabinofuranosidase, and acetate buffer at a mass ratio of 12:1:100, the concentration of the acetate buffer is 0.1 mol / L, and the mass ratio of the complex enzyme solution, the pre-treated material, and deionized water is 5:100:550;

[0047] (3) After the residue and deionized water are evenly mixed, feruloyl esterase is first added, and secondary enzymolysis is carried out at a pH of 5 and a temperature of 40°C for 3 hours. Cellulase and pectinase are then added, and final enzymolysis is carried out at a temperature of 48°C for 6 hours. After the enzymolysis is completed, the temperature is raised to 90°C, the enzyme is inactivated for 20 minutes, and the mixture is cooled to room temperature and filtered to obtain an enzymolysis solution, wherein the mass ratio of the residue, deionized water, feruloyl esterase, cellulase, and pectinase is 100:800:0.3:3:1;

[0048] (4) The composite bacterial agent was inoculated into the enzymatic hydrolysate at an inoculation rate of 10%, and aerobic and anaerobic combined fermentation was carried out. The first stage was the aerobic fermentation stage. The pH of the aerobic fermentation was controlled to be 5, the fermentation temperature was 30°C, and the dissolved oxygen concentration in the aerobic fermentation stage was 5%. After 10 hours of aerobic fermentation, the second stage was anaerobic fermentation. The anaerobic fermentation temperature was 35°C, the dissolved oxygen concentration was 0.3%, and the anaerobic fermentation was carried out for 36 hours. The fermentation liquid was distilled at a distillation temperature of 78°C, and the fractions were collected to obtain ethanol. The composite bacterial agent was composed of Saccharomyces cerevisiae, Candida tropicalis, and Trichoderma reesei with an effective live bacteria ratio of 10:2:1.

[0049] Comparative Example 2

[0050] A method for extracting ethanol and oligosaccharides from wheat bran comprises the following preparation steps:

[0051] (1) After the wheat bran is crushed, it is passed through a 40-mesh sieve. The sieved wheat bran is added to the dilute sulfuric acid with a concentration of 0.5 wt% in a mass ratio of 1:5. The mixture is stirred at 90°C and a speed of 2000 rpm for pretreatment for 1 hour. The mixture is filtered and washed with deionized water for 3 times to obtain a pretreated product.

[0052] (2) adding the complex enzyme solution to the pre-treated material, adding deionized water, performing the initial enzymatic hydrolysis for 4 hours at a pH of 4.5 and a temperature of 50°C, and centrifuging at a centrifugal speed of 5000 rpm for 10 minutes to obtain a supernatant and residue rich in oligosaccharides, wherein the complex enzyme solution is composed of xylanase, mannanase and acetate buffer at a mass ratio of 12:1:100, the concentration of the acetate buffer is 0.1 mol / L, and the mass ratio of the complex enzyme solution, the pre-treated material and deionized water is 5:100:550;

[0053] (3) After the residue and deionized water are evenly mixed, feruloyl esterase is first added, and secondary enzymolysis is carried out at a pH of 5 and a temperature of 40°C for 3 hours. Cellulase and pectinase are then added, and final enzymolysis is carried out at a temperature of 48°C for 6 hours. After the enzymolysis is completed, the temperature is raised to 90°C, the enzyme is inactivated for 20 minutes, and the mixture is cooled to room temperature and filtered to obtain an enzymolysis solution, wherein the mass ratio of the residue, deionized water, feruloyl esterase, cellulase, and pectinase is 100:800:0.3:3:1;

[0054] (4) The composite bacterial agent was inoculated into the enzymatic hydrolysate at an inoculation rate of 10%, and aerobic and anaerobic combined fermentation was carried out. The first stage was the aerobic fermentation stage. The pH of the aerobic fermentation was controlled to be 5, the fermentation temperature was 30°C, and the dissolved oxygen concentration in the aerobic fermentation stage was 5%. After 10 hours of aerobic fermentation, the second stage was anaerobic fermentation. The anaerobic fermentation temperature was 35°C, the dissolved oxygen concentration was 0.3%, and the anaerobic fermentation was carried out for 36 hours. The fermentation liquid was distilled at a distillation temperature of 78°C, and the fractions were collected to obtain ethanol. The composite bacterial agent was composed of Saccharomyces cerevisiae, Candida tropicalis, and Trichoderma reesei with an effective live bacteria ratio of 10:2:1.

[0055] Comparative Example 3

[0056] A method for extracting ethanol and oligosaccharides from wheat bran comprises the following preparation steps:

[0057] (1) After the wheat bran is crushed, it is passed through a 40-mesh sieve. The sieved wheat bran is added to the dilute sulfuric acid with a concentration of 0.5 wt% in a mass ratio of 1:5. The mixture is stirred at 90°C and a speed of 2000 rpm for pretreatment for 1 hour. The mixture is filtered and washed with deionized water for 3 times to obtain a pretreated product.

[0058] (2) adding the complex enzyme solution to the pre-treated material, adding deionized water, performing the initial enzymatic hydrolysis for 4 hours at a pH of 4.5 and a temperature of 50°C, and centrifuging at a centrifugal speed of 5000 rpm for 10 minutes to obtain a supernatant and residue rich in oligosaccharides, wherein the complex enzyme solution is composed of xylanase, mannanase, arabinofuranosidase and acetate buffer in a mass ratio of 12:1:1:100, the concentration of the acetate buffer is 0.1 mol / L, and the mass ratio of the complex enzyme solution, the pre-treated material and deionized water is 5:100:550;

[0059] (3) After the residue and deionized water are evenly mixed, cellulase and pectinase are added, and finally enzymolysis is carried out at 48°C for 9 hours. After the enzymolysis is completed, the temperature is raised to 90°C, the enzyme is inactivated for 20 minutes, and the mixture is cooled to room temperature and filtered to obtain an enzymolysis solution, wherein the mass ratio of the residue, deionized water, ferulic acid esterase, cellulase and pectinase is 100:800:0.3:3:1;

[0060] (4) The composite bacterial agent was inoculated into the enzymatic hydrolysate at an inoculation rate of 10%, and aerobic and anaerobic combined fermentation was carried out. The first stage was the aerobic fermentation stage. The pH of the aerobic fermentation was controlled to be 5, the fermentation temperature was 30°C, and the dissolved oxygen concentration in the aerobic fermentation stage was 5%. After 10 hours of aerobic fermentation, the second stage was anaerobic fermentation. The anaerobic fermentation temperature was 35°C, the dissolved oxygen concentration was 0.3%, and the anaerobic fermentation was carried out for 36 hours. The fermentation liquid was distilled at a distillation temperature of 78°C, and the fractions were collected to obtain ethanol. The composite bacterial agent was composed of Saccharomyces cerevisiae, Candida tropicalis, and Trichoderma reesei with an effective live bacteria ratio of 10:2:1.

[0061] Comparative Example 4

[0062] A method for extracting ethanol and oligosaccharides from wheat bran comprises the following preparation steps:

[0063] (1) After the wheat bran is crushed, it is passed through a 40-mesh sieve. The sieved wheat bran is added to the dilute sulfuric acid with a concentration of 0.5 wt% in a mass ratio of 1:5. The mixture is stirred at 90°C and a speed of 2000 rpm for pretreatment for 1 hour. The mixture is filtered and washed with deionized water for 3 times to obtain a pretreated product.

[0064] (2) adding the complex enzyme solution to the pre-treated material, adding deionized water, performing the initial enzymatic hydrolysis for 4 hours at a pH of 4.5 and a temperature of 50°C, and centrifuging at a centrifugal speed of 5000 rpm for 10 minutes to obtain a supernatant and residue rich in oligosaccharides, wherein the complex enzyme solution is composed of xylanase, mannanase, arabinofuranosidase and acetate buffer in a mass ratio of 12:1:1:100, the concentration of the acetate buffer is 0.1 mol / L, and the mass ratio of the complex enzyme solution, the pre-treated material and deionized water is 5:100:550;

[0065] (3) After the residue and deionized water are evenly mixed, ferulic acid esterase, cellulase and pectinase are added, and the enzymatic hydrolysis is carried out at a pH of 5 and a temperature of 40°C for 3 hours, and the temperature is raised to 48°C, and the enzymatic hydrolysis is finally carried out for 6 hours. After the enzymatic hydrolysis is completed, the temperature is raised to 90°C, the enzyme is inactivated for 20 minutes, and the enzyme is cooled to room temperature and filtered to obtain an enzymatic hydrolyzate, wherein the mass ratio of the residue, deionized water, ferulic acid esterase, cellulase and pectinase is 100:800:0.3:3:1;

[0066] (4) The composite bacterial agent was inoculated into the enzymatic hydrolysate at an inoculation rate of 10%, and aerobic and anaerobic combined fermentation was carried out. The first stage was the aerobic fermentation stage. The pH of the aerobic fermentation was controlled to be 5, the fermentation temperature was 30°C, and the dissolved oxygen concentration in the aerobic fermentation stage was 5%. After 10 hours of aerobic fermentation, the second stage was anaerobic fermentation. The anaerobic fermentation temperature was 35°C, the dissolved oxygen concentration was 0.3%, and the anaerobic fermentation was carried out for 36 hours. The fermentation liquid was distilled at a distillation temperature of 78°C, and the fractions were collected to obtain ethanol. The composite bacterial agent was composed of Saccharomyces cerevisiae, Candida tropicalis, and Trichoderma reesei with an effective live bacteria ratio of 10:2:1.

[0067] Comparative Example 5

[0068] A method for extracting ethanol and oligosaccharides from wheat bran comprises the following preparation steps:

[0069] (1) After the wheat bran is crushed, it is passed through a 40-mesh sieve. The sieved wheat bran is added to the dilute sulfuric acid with a concentration of 0.5 wt% in a mass ratio of 1:5. The mixture is stirred at 90°C and a speed of 2000 rpm for pretreatment for 1 hour. The mixture is filtered and washed with deionized water for 3 times to obtain a pretreated product.

[0070] (2) adding the complex enzyme solution to the pre-treated material, adding deionized water, performing the initial enzymatic hydrolysis for 4 hours at a pH of 4.5 and a temperature of 50°C, and centrifuging at a centrifugal speed of 5000 rpm for 10 minutes to obtain a supernatant and residue rich in oligosaccharides, wherein the complex enzyme solution is composed of xylanase, mannanase, arabinofuranosidase and acetate buffer in a mass ratio of 12:1:1:100, the concentration of the acetate buffer is 0.1 mol / L, and the mass ratio of the complex enzyme solution, the pre-treated material and deionized water is 5:100:550;

[0071] (3) After the residue and deionized water are evenly mixed, ferulic acid esterase is first added, and secondary enzymolysis is carried out at a pH of 5 and a temperature of 40°C for 3 hours. Cellulase and pectinase are then added, and final enzymolysis is carried out at a temperature of 48°C for 6 hours. After the enzymolysis is completed, the temperature is raised to 90°C, the enzyme is inactivated for 20 minutes, and the solution is filtered after cooling to room temperature to obtain an enzymatic solution, wherein the mass ratio of the residue, deionized water, ferulic acid esterase, cellulase, and pectinase is 100:800:0.3:3:1;

[0072] (4) The composite bacterial agent was inoculated into the enzymatic hydrolysate at an inoculation rate of 10%, and aerobic and anaerobic combined fermentation was carried out. The first stage was the aerobic fermentation stage. The pH of the aerobic fermentation was controlled to be 5, the fermentation temperature was 30°C, and the dissolved oxygen concentration in the aerobic fermentation stage was 5%. After 10 hours of aerobic fermentation, the second stage was anaerobic fermentation. The anaerobic fermentation temperature was 35°C, the dissolved oxygen concentration was 0.3%, and the anaerobic fermentation was carried out for 36 hours. The fermentation liquid was distilled at a distillation temperature of 78°C, and the fractions were collected to obtain ethanol. The composite bacterial agent was composed of Saccharomyces cerevisiae and Trichoderma reesei with an effective live bacteria ratio of 10:1.

[0073] Comparative Example 6

[0074] A method for extracting ethanol and oligosaccharides from wheat bran comprises the following preparation steps:

[0075] (1) After the wheat bran is crushed, it is passed through a 40-mesh sieve. The sieved wheat bran is added to the dilute sulfuric acid with a concentration of 0.5 wt% in a mass ratio of 1:5. The mixture is stirred at 90°C and a speed of 2000 rpm for pretreatment for 1 hour. The mixture is filtered and washed with deionized water for 3 times to obtain a pretreated product.

[0076] (2) adding the complex enzyme solution to the pre-treated material, adding deionized water, performing the initial enzymatic hydrolysis for 4 hours at a pH of 4.5 and a temperature of 50°C, and centrifuging at a centrifugal speed of 5000 rpm for 10 minutes to obtain a supernatant and residue rich in oligosaccharides, wherein the complex enzyme solution is composed of xylanase, mannanase, arabinofuranosidase and acetate buffer in a mass ratio of 12:1:1:100, the concentration of the acetate buffer is 0.1 mol / L, and the mass ratio of the complex enzyme solution, the pre-treated material and deionized water is 5:100:550;

[0077] (3) After the residue and deionized water are evenly mixed, feruloyl esterase is first added, and secondary enzymolysis is carried out at a pH of 5 and a temperature of 40°C for 3 hours. Cellulase and pectinase are then added, and final enzymolysis is carried out at a temperature of 48°C for 6 hours. After the enzymolysis is completed, the temperature is raised to 90°C, the enzyme is inactivated for 20 minutes, and the mixture is cooled to room temperature and filtered to obtain an enzymolysis solution, wherein the mass ratio of the residue, deionized water, feruloyl esterase, cellulase, and pectinase is 100:800:0.3:3:1;

[0078] (4) The composite bacterial agent was inoculated into the enzymatic hydrolysate at an inoculation rate of 10%, and aerobic and anaerobic combined fermentation was carried out. The first stage was the aerobic fermentation stage. The pH of the aerobic fermentation was controlled to be 5, the fermentation temperature was 30°C, and the dissolved oxygen concentration in the aerobic fermentation stage was 5%. After 10 hours of aerobic fermentation, the second stage was anaerobic fermentation. The anaerobic fermentation temperature was 35°C, the dissolved oxygen concentration was 0.3%, and the anaerobic fermentation was carried out for 36 hours. The fermentation liquid was distilled at a distillation temperature of 78°C, and the fractions were collected to obtain ethanol. Among them, the composite bacterial agent was composed of Saccharomyces cerevisiae and Candida tropicalis with an effective live bacteria ratio of 10:2.

[0079] Performance Testing

[0080] The methods for extracting ethanol and oligosaccharides from wheat bran provided in Examples 1-3 and Comparative Examples 1-6 of the present application are as follows:

[0081] Oligosaccharide yield: The oligosaccharide-rich supernatants obtained in Examples 1-3 and Comparative Examples 1-6 were respectively heated to 85°C, inactivated for 15 min, centrifuged for 10 min, and centrifuged at a speed of 5000 rpm. Ultrafiltration was performed using a filter membrane with a molecular weight cutoff of 10 kDa. The pressure was controlled at 0.1 MPa and the temperature was 40°C. The ultrafiltration cycle was continued until the volume was concentrated to 1 / 3 of the original volume. Finally, nanofiltration was performed using a filter membrane with a molecular weight cutoff of 1000 Da at a pressure of 0.3 MPa and a temperature of 35°C. The concentrate was collected and concentrated to a solid content of 25% using a rotary evaporator. The concentrate was then spray-dried. The vacuum degree of the rotary evaporator was -0.09 MPa, the temperature was 45°C, the spray drying air inlet temperature was 160°C, the air outlet temperature was 70°C, and the atomization pressure was 0.2 MPa to obtain oligosaccharides.

[0082] Oligosaccharide yield (%) = oligosaccharide mass (g) / initial wheat bran mass (g) × 100%;

[0083] The average molecular weight of the oligosaccharides was measured using a digital rotational viscometer.

[0084] Ethanol yield: Ethanol yield (%) = ethanol yield (g) / initial wheat bran mass × 100%;

[0085] The test results are shown in Table 1.

[0086] Table 1 Oligosaccharide yield, average molecular weight of oligosaccharides and ethanol yield

[0087] Test items Oligosaccharide yield (%) Average molecular weight of oligosaccharides (Da) Ethanol yield (%) Example 1 18.9 870 14.8 Example 2 18.7 850 14.6 Example 3 19.1 880 14.2 Comparative Example 1 15.4 2690 11.5 Comparative Example 2 14.7 2730 11.1 Comparative Example 3 18.6 890 9.5 Comparative Example 4 18.8 800 10.3 Comparative Example 5 18.9 880 8.8 Comparative Example 6 18.6 900 9.3

[0088] As can be seen from Table 1, the method provided in the present application for extracting ethanol and oligosaccharides from wheat bran achieves high-value conversion of wheat bran through step-by-step enzymatic hydrolysis and two-stage fermentation, through graded resource utilization, synergistic enhancement of enzymatic hydrolysis, and complementary fermentation metabolism. The oligosaccharide yield and ethanol yield are high, and the method is environmentally friendly, economical, and has product diversity.

[0089] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for extracting ethanol and oligosaccharides from wheat bran, characterized in that: The method comprises the following preparation steps: (1) Crush wheat bran, add it to dilute acid, pretreat it at 90-100° C. for 1-2 hours, filter it, and wash it to obtain a pretreated product; (2) adding the complex enzyme solution to the pre-treated product, adding deionized water, performing initial enzymatic hydrolysis for 4-6 hours at a pH of 4.5-5.0 and a temperature of 50-60°C, and centrifuging to obtain a supernatant and residue rich in oligosaccharides; (3) After the residue and deionized water are evenly mixed, ferulic acid esterase is first added, and secondary enzymolysis is performed for 3-6 hours, and then cellulase and pectinase are added, and final enzymolysis is performed for 6-10 hours. After the enzymolysis is completed, the temperature is raised to 90-95°C, the enzyme is inactivated for 20-30 minutes, and the solution is cooled to room temperature and filtered to obtain an enzymolysis solution; (4) The composite bacterial agent is inoculated into the enzymatic hydrolysate and aerobic and anaerobic fermentation is carried out. The first stage is the aerobic fermentation stage. After 10-12 hours of aerobic fermentation, the second stage is anaerobic fermentation. The anaerobic fermentation lasts for 36-48 hours. The fermentation liquid is distilled and the fractions are collected to obtain ethanol.

2. The method for extracting ethanol and oligosaccharides from wheat bran according to claim 1, wherein In the step (2), the complex enzyme solution is composed of xylanase, mannanase, arabinofuranosidase and acetate buffer in a mass ratio of 12-15:1-2:1-3:100-150; the concentration of the acetate buffer is 0.1-0.12 mol / L.

3. The method for extracting ethanol and oligosaccharides from wheat bran according to claim 1, wherein In the step (2), the mass ratio of the complex enzyme solution, the pre-treated material, and the deionized water is 5-10:100-110:550-600.

4. The method for extracting ethanol and oligosaccharides from wheat bran according to claim 1, wherein In the step (3), the mass ratio of the residue, deionized water, ferulic acid esterase, cellulase and pectinase is 100-120:800-1000:0.3-0.5:3-4:1-1.

2.

5. The method for extracting ethanol and oligosaccharides from wheat bran according to claim 1, wherein The pH of the secondary enzymolysis in step (3) is 5-6, and the temperature is 40-45°C; the final enzymolysis temperature is 48-52°C.

6. The method for extracting ethanol and oligosaccharides from wheat bran according to claim 1, wherein The composite bacterial agent in step (4) is composed of saccharomyces cerevisiae, Candida tropicalis and Trichoderma reesei with an effective live bacterial count ratio of 10-12:2-4:1-2.

7. The method for extracting ethanol and oligosaccharides from wheat bran according to claim 1, wherein The inoculation amount of the composite bacterial agent in step (4) is 10-15%.

8. The method for extracting ethanol and oligosaccharides from wheat bran according to claim 1, wherein The aerobic fermentation conditions in step (4) are as follows: controlling the pH of the aerobic fermentation to be 5-5.5, the fermentation temperature to be 30-32° C., and the dissolved oxygen concentration in the aerobic fermentation stage to be 5-10%.

9. The method for extracting ethanol and oligosaccharides from wheat bran according to claim 1, wherein The anaerobic fermentation conditions in step (4) are as follows: controlling the anaerobic fermentation temperature to 35-40° C. and adjusting the dissolved oxygen concentration to less than 0.5%.