Method for preparing cellobiose based on bacterial enzyme synergy of bamboo reed as raw material and application of cellobiose

Abstract

The invention relates to a method for preparing cellobiose based on bacteria and enzyme synergy of a whole bamboo reed as a raw material. The method comprises the following steps: a raw material pretreatment link: carrying out superfine grinding on naturally dried bamboo reed and carrying out thermal swelling; in the enzymolysis link, commodity xylanase is selected, the concentration of cellobiose in the enzymolysis process is kept to be lower than 5 g / L by diluting enzymatic hydrolysate, and inhibition of the cellobiose on enzyme activity is reduced; in the fermentation link, the enzymatic hydrolysate is used as a carbon source, and Escherichia coli str.K-12 is used as a production strain for fermentation; and a fermentation liquid treatment link: filtering the fermentation liquid through a plate frame to obtain clear liquid, filtering the clear liquid through an RO membrane to obtain a cellobiose concentrated solution, and drying to obtain high-purity cellobiose. According to the method, the concentration of cellobiose in the enzymolysis process is controlled, so that the enzyme consumption is reduced, and other non-cellobiose reducing sugars are metabolized through escherichia coli, so that the cellobiose is purified. The method can be used for low-cost biological fermentation preparation of cellobiose of the bamboo reed raw material.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of cellobiose, and in particular to a method for preparing cellobiose in a coordinated manner using a fungus enzyme based on reed as a raw material and an application thereof. Background Art

[0002] Cellobiose is a hydrolysis product of cellulose. It is an oligosaccharide composed of two glucose molecules connected by a β-1,2-glycosidic bond. It has broad application prospects in the fields of food, medicine and chemical industry, so the market demand for cellobiose is large. my country's non-grain biomass raw materials have a large output and low utilization rate. In recent years, with the demand for the utilization of non-grain biomass, the development of biodegradable cellulose has ushered in a huge market demand.

[0003] Phragmites australis is a perennial herbaceous plant of the genus Phragmites in the Poaceae family. It is generally harvested after December, and the closer to the rhizome the better, which is conducive to future management. Phragmites australis stalks can be used to prepare pulp fiber by enzymatic method (CN202410832755.1), and silage can be made into feed when Phragmites australis grows to August (CN202410682981.6). Normally harvested Phragmites australis has a high cellulose content and can be used to prepare cellobiose, with low raw material cost.

[0004] The existing methods for producing cellobiose mainly include acid-catalyzed glucose dehydration, cellulase hydrolysis, and alkali-catalyzed condensation of two glucose molecules. Among them, cellulase hydrolysis has the advantages of environmental protection and high efficiency. Enzymatic hydrolysis mediated by cellulase is a key process for achieving high cellulose sugar yield and improving biorefining production efficiency and economy, but the cost and stability of the enzyme are key factors affecting its industrial application. Chinese invention patent CN201610057567.1 discloses a production process for cellobiose, which breaks the glycosidic bond molecular structure in the cellulose macromolecular chain by high-energy explosion of biomass raw materials, and extracts and separates cellobiose of different contents. This method has the characteristics of low dissolution rate and low purity.

[0005] Chinese invention patent CN201680062329.8 provides a method for producing cello-oligosaccharides with a degree of polymerization of 3-6, establishes a reaction relationship between reaction temperature and reaction time, and optimizes the reaction conditions by using carbon catalysts, etc., but the reaction conditions are complicated and the operation method is not simple. Chinese invention patent CN201610057255.0 provides a method for preparing cello-oligosaccharides by directional two-stage hydrolysis of fibers, which effectively removes the inhibitory effect of hydrolysis products through solid-liquid separation, improves enzyme activity and reaction efficiency, but the yield is still not too high, and it is difficult to meet the requirements for the yield of cellobiose. Chinese invention patent 202310175807.8 provides a method for producing cellobiose based on straw fermentation residue, which breaks the cellulose in the residue and increases enzyme activity through ultrasonic treatment, and determines the calculation method of exoglucanase activity and establishes the calculation method of residue reaction rate by optimizing reaction conditions, but acid is still required in the reaction process, the amount of alcohol is large, the reaction conditions are complicated and the cost is high.

[0006] The inhibition of cellulase activity by cellobiose and glucose increases the amount of enzyme used in cellulose hydrolysis and the cost of enzymes, which may hinder the economic feasibility of the biorefining process. The preparation of cellobiose by enzymatic hydrolysis has the problems of low efficiency and impure products. The hydrolysis of cellulose and hemicellulose produces a large amount of reducing sugars, including cellobiose, glucose, xylose, galactose, arabinose, etc. Wild Escherichia coli can metabolize and utilize glucose, xylose, galactose, and arabinose, but cannot metabolize and utilize cellobiose (Lam, Katie Jing Kay et al. Computational and structuralbiotechnology journal vol. 20 6287-6301. 12 Nov. 2022). Summary of the invention

[0007] In order to realize low-cost production of cellobiose, the present invention provides a fermentation method for efficiently producing cellobiose using reed bamboo as raw material. By using the method of the present invention, the conversion rate of the reed bamboo raw material to cellobiose reaches more than 14%, the generated concentration of cellobiose reaches more than 5 g / L, and the chemical purity of cellobiose is higher than 90%.

[0008] The present invention aims to provide a method for producing cellobiose by enzymatic hydrolysis using reed as a raw material and purifying cellobiose by fermentation with Escherichia coli, and an efficient production technology for preparing reducing sugar by enzymatic hydrolysis using the whole plant of reed as a raw material and purifying cellobiose by directly metabolizing other reducing sugars other than cellobiose by Escherichia coli.

[0009] In order to achieve the above object, the present invention provides the following technical solutions: A method for preparing cellobiose by using Phragmites australis as raw material and using bacterial enzymes in a coordinated manner, characterized in that the method comprises a raw material pretreatment step, an enzymatic hydrolysis step, a fermentation step and a fermentation liquid treatment step, and the specific steps are as follows: S1. The whole plant of Phragmites australis was ultrafinely crushed after natural drying to obtain Phragmites australis powder, and the ultrafine Phragmites australis powder was collected by sieving; S2. 1 to 3% NaOH solution was added to the ultrafine Phragmites australis powder obtained in S1, and the mixture was thermally swollen at a temperature of 50 to 60 ° C to obtain a Phragmites australis hydrolyzate; S3. Add commercial xylanase (the ratio of ultrafine arundinaceae powder to enzyme activity is 1:0.01-0.05) to the arundinaceae hydrolysate obtained in S2, mix well, and react at 30-60℃ for 7-10 h. During the enzymolysis process, keep the cellobiose content in the hydrolysate below 5.0 g / L. After the enzymolysis is completed, sterilize at 115℃ for 20 min to obtain the hydrolysate; S4. The enzymatic hydrolysate obtained in S3 was used as the carbon source for microbial fermentation. The culture was carried out at 35-50℃, ventilation 0-7 vvm, 200-900 r / min, and ammonia was added to maintain the pH at 6.5-7.5. The culture time was 18-30 h, and the bacterial cell volume OD 600 Reach 30~60; S5. The fermentation broth obtained in S4 is subjected to plate and frame filtration to obtain a precipitate and a clear liquid; S6. The supernatant obtained in S5 is concentrated through an RO membrane to obtain a cellobiose concentrate; S7. Drying the cellobiose concentrate obtained in S6 to obtain high-purity cellobiose. The whole plant of Phragmites australis in S1 includes branches, stems and leaves of Phragmites australis. The whole plant of Phragmites australis ultrafine grinding is that the whole plant of Phragmites australis is ground and then passed through a 40-70 mesh sieve; The thermal swelling system described in S2 has a solid-liquid ratio of 1:2~6, a reaction temperature of 50~60°C, and a reaction time of 4~6 h; in S3, the ratio of ultrafine reed powder to enzyme activity is 1:0.01~0.05, and the reaction is carried out at 30~60°C for 7~10 h.

[0010] During the S3 enzymatic hydrolysis process, the cellobiose concentration was kept below 5 g / L by diluting the enzymatic hydrolysate to reduce the inhibition of cellobiose on enzyme activity.

[0011] The microorganism described in S4 is Escherichia coli, including but not limited to Escherichia coli str. K-12, MG1655, W3110, and other E. coli production strains, preferably Escherichia coli Str. K-12.

[0012] The fermentation conditions in S4 were: 35-50℃, ventilation 0-7 vvm, 200-900 r / min, and ammonia was added to maintain the pH at 6.5-7.5; the culture time was 18-30 h, and the bacterial mass reached OD 600 It is 30~60.

[0013] Plate and frame filtration in S5 separates the bacteria and large particle impurities from the fermentation liquid to obtain a clear liquid containing cellobiose.

[0014] The RO membrane in S6 allows water molecules to pass through, and the clear liquid can be passed through the RO membrane to obtain the cellobiose concentrate, and the filtered water can be reused after treatment.

[0015] The chemical purity of cellobiose obtained by S7 drying is over 90%.

[0016] The present invention also discloses the application of the method for preparing cellobiose based on the whole plant of Arundo donax fungus enzyme in improving the conversion rate of Arundo donax. The experimental results show that the conversion rate of Arundo donax raw material to cellobiose reaches more than 14%, the generated concentration of cellobiose reaches more than 5 g / L, and the chemical purity of cellobiose is higher than 90%.

[0017] The method for preparing cellobiose by using Phyllostachys australis as raw material and using bacterial enzymes disclosed in the present invention has the following beneficial effects: The present invention provides a fermentation production method for efficiently producing cellobiose using whole reed as raw material. By using the method of the present invention, the conversion rate of reed raw material to cellobiose reaches more than 14%, the generated concentration of cellobiose reaches more than 5g / L, and the chemical purity of cellobiose is higher than 90%. The method can be used to efficiently produce cellobiose using reed as raw material or raw material containing cellulose, significantly improves the enzyme activity inhibition phenomenon in cellobiose production, reduces the amount of enzyme used, saves the enzyme cost, and realizes low-cost preparation of cellobiose. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The growth of E. coli on M9 plates with different reducing sugars as the sole carbon source; Figure 2 The process of E. coli fermentation and utilization of sugars under enzymatic treatment; Figure 3 The obtained cellobiose (right) was prepared by ultrafine Phragmites australis powder (left) through a 40-70 mesh sieve. DETAILED DESCRIPTION The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available.

[0020] The starting strain used in the present invention Escherichia coli str. K-12 is a known strain and has been deposited in the American Type Culture Collection (ATCC) with the accession number ATCC 25404.

[0021] The commercial cellulase and commercial xylanase used in the present invention are composite enzymes, which are composed of endoglucanase, exoglucanase and beta-glucosidase, and can be obtained by purchasing commercial enzyme preparations.

[0022] Preparation of Arundo donax samples: The mixture of Phragmites australis branches, stems and leaves is crushed in a grinder and passed through a 40-70 mesh sieve to obtain ultrafine Phragmites australis powder.

[0023] Thermal swelling: Ultrafine Phragmites australis powder was mixed with 1-3% NaOH solution at a solid-liquid ratio of 1:2-6, and reacted at 50-60°C for 4-6 h to obtain Phragmites australis hydrolyzate.

[0024] The enzymatic hydrolysis method of the present invention is that the ratio of ultrafine reed powder to enzyme activity is 1:0.01-0.05, and the reaction is carried out at 30-60°C for 7-10 hours. During the enzymatic hydrolysis process, the concentration of cellobiose is kept below 5 g / L by diluting the enzymatic hydrolyzate to reduce the inhibition of cellobiose on enzyme activity. After the enzymatic hydrolysis is completed, the reed is immediately placed in a sterilizing pot for sterilization at 115°C for 20 minutes.

[0025] The method for producing cellobiose by microbial method of the present invention comprises the following steps: in the initial stage of fermentation, 100 g of arundinaceus enzymatic hydrolyzate in the form of ultrafine arundinaceus powder is added to a basic fermentation medium, the culture is carried out at 30-50°C, ventilation is 0-7 vvm, and 200-900 r / min, and ammonia water is added to maintain the pH at 7.0; the culture time is 18-30 h, and the bacterial cell volume reaches OD 600 It is 30~60.

[0026] The main implementation methods adopted by the present invention are as follows: 1. Exoglucanase activity assay Enzyme activity definition: Under the specific conditions of 50℃ and pH 5.0, the reaction time is 30 min, and 1 mL of enzyme solution hydrolyzes CMC-Na to release 1 µmol of cellobiose per unit time, which is one unit of enzyme activity (U).

[0027] Preheat 50 µL of the diluted enzyme solution at 50°C for 5 min. First, add 350 µL of 1% CMC-Na solution to the experimental group, react for 30 min, and then add 600 µL of DNS. Add 600 µL of DNS to the control group, then add 350 µL of 1% CMC-Na solution. After the reaction, boil for 7 min, cool to room temperature, add 2 mL of water, and measure the absorbance at 540 nm.

[0028] 2. Determination of endoglucanase activity Definition of enzyme activity: Under the specific conditions of 50℃ and pH 5.0, the reaction time is 30 min, and 1 mL of enzyme solution hydrolyzes 1 mg of CMC-Na per unit time, which is one unit of enzyme activity (U).

[0029] Take 30 mL of 1% CMC-Na solution and preheat at 50℃ for 5 min. Add 500 μL of diluted enzyme solution to the experimental group; add 500 μL of supernatant of E. coli fermentation broth to the control group, react at 50℃ for 30 min, boil for 10 min to terminate the reaction, cool to room temperature, and measure the viscosity of the reaction solution several times with NDJ-5S rotational viscometer.

[0030] 3. β-Glucosidase Activity Assay Enzyme activity definition: At 50°C and pH 5.0, the amount of enzyme required to hydrolyze cellobiose to produce 1 μmol of glucose within 1 min is 1 enzyme activity unit (U).

[0031] Using 10 mmol / L cellobiose as substrate, the reaction was carried out at 50℃ and pH 5.0 for 10 min, and the amount of glucose released was measured by a biosensor.

[0032] 4. Determination of the degree of crushing of the whole plant The whole superfinely crushed Phragmites australis was passed through 20, 30, 40, 50, 60, and 70 mesh sieves respectively, and mixed at a mass ratio of superfine Phragmites australis powder: water = 1:10 to observe whether the superfine Phragmites australis powder can be completely suspended in water. The results are shown in Table 1. The selected Phragmites australis was superfinely crushed and then passed through a 40-70 mesh sieve to obtain superfine Phragmites australis powder.

[0033] Table 1 Whether ultrafine Phragmites australis powder can be completely suspended in water with a certain mass ratio Screen mesh number 20 30 40 50 60 70 Can it be completely suspended in water? no no able able able able 5. Determination of thermal swelling system The ultrafine reed powder and 1~3% NaOH solution were mixed at a mass ratio of 1:2, 4, 6, 8, and 10, and the time required for the ultrafine reed powder to settle was observed. The results are shown in Table 2. It was found that when the mass ratio of ultrafine reed powder: 1~3% NaOH solution = 1:2~6 was mixed, the swelling effect was good. The ultrafine reed powder: 1~3% NaOH solution = 1:2~6 was mixed at a mass ratio of 1:2~6, and the reaction was carried out at 30, 40, 50, 60, and 70℃ for 1 h. The ultrafine reed powder was observed at a mass ratio of 1:2~6. The results are shown in Table 3. It was found that when the reaction temperature was 50~60℃, the swelling effect was good. The ultrafine reed powder: 1~3% NaOH solution = 1:2~6 was mixed at a mass ratio of 1:2~6, and the reaction was carried out at 50~60℃ for 2, 4, 6, 8, and 10 h, and the ultrafine reed powder was observed at a mass ratio of 1:2~6. The reaction was carried out at 50~60℃ for 2, 4, 6, 8, and 10 h, and the ultrafine reed powder was observed at a mass ratio of 1:2~6. The results are shown in Table 4. It was found that the swelling effect was good when the reaction time was 4-6 h. The optimal process for thermal swelling was the mass ratio of ultrafine Phragmites australis powder to 1-3% NaOH solution = 1:2-6, and the reaction was carried out at 50-60℃ for 4-6 h.

[0034] Table 2 Effect of water addition on swelling of ultrafine Phragmites australis powder Ultrafine Phragmites australis powder: 1~3% NaOH solution mass ratio 1：2 1：4 1：6 1：8 1：10 Settling time (min) 8.1 5.3 3.4 1.2 0.7 Table 3 Effect of reaction temperature on swelling of ultrafine Phragmites australis powder Reaction temperature (℃) 30 40 50 60 70 Settling time (min) 3.2 5.0 9.3 9.2 8.4 Table 4 Effect of reaction time on swelling of superfine Phragmites australis powder Reaction time (h) 2 4 6 8 10 Settling time (min) 10.2 12.4 12.1 10.9 9.6 6. Compounding of commercial enzymes 1 g of ultrafine Phragmites australis powder was enzymolyzed using commercial cellulase, commercial xylanase, and a composite enzyme with a mass ratio of commercial cellulase to commercial xylanase of 1. The results are shown in Table 5. When the enzyme activity ratio of ultrafine reed powder to commercial cellulase is 1:0.1, the hydrolysis rates of cellulose and hemicellulose can reach 91.31% (calculated in glucose) and 96.16% (calculated in xylose), respectively, and the enzyme cost is 5.4 yuan / g ultrafine reed powder; when the enzyme activity ratio of ultrafine reed powder to commercial xylanase is 1:0.09, the hydrolysis rates of cellulose and hemicellulose can reach 93.62% (calculated in glucose) and 96.83% (calculated in xylose), respectively, and the enzyme cost is 1.4 yuan / g ultrafine reed powder; when the activity ratio of ultrafine reed powder to compound commercial enzyme is 1:0.1, the hydrolysis rates of cellulose and hemicellulose can reach 94.08% (calculated in glucose) and 97.11% (calculated in xylose), respectively, and the enzyme cost is 3.4 yuan / g ultrafine reed powder; at this time, the cellulose and hemicellulose in the reed are almost completely hydrolyzed. It can be seen that, without considering the cost of enzyme use, both the existing commercial cellulase and commercial xylanase can effectively hydrolyze cellulose and hemicellulose in Phragmites australis. Considering the cost of use, the present invention uses commercial xylanase to hydrolyze ultrafine Phragmites australis powder.

[0035] Table 5 Enzyme hydrolysis results of Phragmites australis

[0036] 7. Determination of Final Cellobiose Concentration High concentration of cellobiose content can inhibit exoglucanase and β-glucosidase, increasing the amount of enzyme. So on the basis of the above research, by using CMC-Na as substrate, changing the amount of cellobiose added, measuring the exoglucanase and β-glucosidase enzyme activities in the enzymolysis solution, the results are shown in Table 6. It is found that when the cellobiose concentration is below 5 g / L, the exoglucanase enzyme activity inhibition rate is less than 40%, and the β-glucosidase enzyme activity inhibition rate is less than 55%. Since the content of β-glucosidase is less in the commercial xylanase and the purpose of the present invention is to prepare cellobiose, the final concentration of cellobiose enzymolysis is selected to be 5 g / L.

[0037] Table 6 Effect of cellobiose concentration on enzyme activity

[0038] By adding deionized water to keep the cellobiose content in the lysate of Phragmites australis below 5 g / L, the amount of commercial xylanase added was gradually reduced, and the conversion rate of Phragmites australis raw materials to cellobiose and the cellulose hydrolysis rate in the enzymatic hydrolysis solution were determined. The results are shown in Table 7. It was found that when the ratio of ultrafine Phragmites australis powder to enzyme activity was 1:0.01-0.05, the cellobiose conversion rate was high and the enzyme cost was low.

[0039] Table 7 Effect of dilution multiple of superfine Phragmites australis lysate on enzyme addition amount

[0040] 8. Shake flask fermentation experiment Streak the fermentation strain from the glycerol storage tube onto LB (Zhuge Jian and Wang Zhengxiang Industrial Microbiology Experimental Technology Manual, Beijing: China Light Industry Press, 1994) solid plate, and culture at 30-50℃ for 12 hours; take 3-5 single colonies of E. coli, inoculate them into 50 mL LB liquid medium, and culture them at 30-50℃ and 100-250 r / min for 5-15 hours as the first-level seed liquid. Inoculate 5% of the first-level seed liquid into 50 mL LB liquid medium, and culture them at 30-50℃ and 200 r / min for about 10 hours as the second-level seed liquid.

[0041] The Phyllostachys australis liquid after enzymatic hydrolysis was used as the fermentation medium. The strain seed liquid was inoculated into the fermentation medium at an inoculation rate of 5-10%, and cultured at 30-50°C and a rotation speed of 100-250 r / min for 12 h.

[0042] Phragmites australis liquid after enzymatic hydrolysis: In the pretreatment of Phragmites australis, ultrafine Phragmites australis powder that has been sieved through a 40-70 mesh sieve is thermally swollen with 1-3% NaOH solution at a material-liquid ratio of 1:2-6, a reaction temperature of 50-60°C, and a reaction time of 4-6 h. In the Phragmites australis enzymatic hydrolysis step, the ratio of ultrafine Phragmites australis powder to enzyme activity is 1:0.01-0.05, and the reaction is carried out at 30-60°C for 7-10 h. During the enzymatic hydrolysis process, the concentration of cellobiose is kept below 5 g / L by diluting the enzymatic hydrolyzate to reduce the inhibition of cellobiose on enzyme activity. After the enzymatic hydrolysis is completed, it is immediately placed in a sterilizer and sterilized at 115°C for 20 min. The fermentation medium is obtained.

[0043] 9. Fermentation and purification of cellobiose in a fermenter Streak the fermentation strain from the glycerol storage tube onto an LB solid plate and culture at 30-50℃ for 12 h; select 3-5 single colonies and inoculate them into LB liquid culture medium and culture at 37℃ and 200 r / min for 9-12 h as the primary seed solution. Take 6 1 mL primary seed solutions and resuspend them twice in M9 liquid culture medium with glucose as the carbon source (Tian Kangming et al., Journal of Biotechnology, 29: 111-114, 2013; Tian Kangming et al., Journal of Biotechnology, 29: 1-10, 2013) and add them to 6 50 mL M9 liquid culture medium with glucose as the carbon source. Culture at 37℃ and 200 r / min for 10 h as the secondary seed solution (i.e., seed solution). The secondary seed solution was added according to the starting OD 600The inoculum with a value of 0.1-1.0 was inoculated into the fermentation medium in a 5 L fermenter, and fermentation was started at 30-50°C, ventilation 0-7 vvm, and 200-900 r / min. Ammonia water was added to maintain the pH at 7.0. The culture time was 18-24 h, and the bacterial volume reached OD 600 It is 30~60.

[0044] 10. Fermentation process analysis Sample preparation: Take 5 mL of fermentation broth and measure OD 600 , pH value. Take 1 mL into a 1.5 mL centrifuge tube and centrifuge at 12000r / min for 5 min, and take the supernatant for relevant component determination and analysis.

[0045] (1) Glucose concentration determination: After the sample is appropriately diluted, the glucose concentration is determined using an SBA-40C biosensor, and the average of three parallel data is taken.

[0046] (2) Determination of cellobiose concentration: The concentration of cellobiose in the sample was determined by liquid phase method.

[0047] Example 1 Purification of cellobiose by shake flask fermentation with Escherichia coli Escherichia coli Escherichia coli str. K-12 is the production strain. Escherichia coli str. 3 to 5 single colonies of K-12 were inoculated into 50 mL of LB liquid medium and cultured at 37°C and 200 r / min for 12 h as the primary seed liquid. 600 The second seed solution was inoculated with 0.05 in 50 mL LB liquid medium in a 250 mL Erlenmeyer flask at 37°C, 200 r / min, and cultured at constant temperature for 12 h as the secondary seed solution. The secondary seed solution was transferred to the fermentation medium (2.0 g of superfine reed powder and 1.5% NaOH solution were mixed at a mass ratio of 1:2.5 and kept at a constant temperature of 55°C for 4 h; the ratio of superfine reed powder to commercial xylanase activity was 1:0.011, and the reaction was carried out at 50°C for 8 h. The cellobiose concentration was kept below 5 g / L by diluting the enzymatic hydrolysate to reduce the inhibition of cellobiose on enzyme activity. After the enzymatic hydrolysis, the enzyme was steam sterilized at 115°C for 20 min), the fermentation temperature was 37°C, and the culture was continued for 24 h. The sugar content in the fermentation broth was sampled and determined, and it was found that the content of non-cellobiose reducing sugar in the fermentation broth was close to 0. Example

[0048] Purification of cellobiose by fermentation of E. coli in a fermenter Take E. coli Escherichia coli str.3 to 5 single colonies of K-12 were inoculated into 50 mL of LB liquid medium and cultured at 37°C and 200 r / min for 12 h as the primary seed liquid. The primary seed liquid was resuspended in M9 medium and then heated according to the initial OD 600 0.05 was inoculated into 50 mL M9 liquid medium in a 250 mL Erlenmeyer flask and cultured at 30-50°C, 200 r / min, for 12 h as the secondary seed liquid. 600 The inoculum with a value of 0.3 was inoculated into the fermentation medium in a 5 L fermenter (M9 medium components, magnesium sulfate 1.2 g / L, superfine reed powder 100.0 g and 1.5% NaOH solution were mixed at a mass ratio of 1:2.5 and kept at 55°C for 4 h; the ratio of superfine reed powder to commercial xylanase activity was 1:0.011, and the reaction was carried out at 50°C for 8 h. The cellobiose concentration was kept below 5 g / L by diluting the enzymatic hydrolysate to reduce the inhibition of cellobiose on enzyme activity. The enzymatic hydrolysate was concentrated to 2.75 L, and the components were mixed with the concentrated fermentation liquid and placed in the fermenter for steam sterilization at 115°C for 20 min). The initial volume of the fermenter after inoculation was 60% of the working volume. During the fermentation stage, the temperature was controlled at 37°C, the pH was maintained at about 7.0, the ventilation was 0-7.0 vvm, the rotation speed was 200-900 r / min, and the sugar content and OD in the fermentation liquid were measured every 2 h. 600 , until the fermentation is completed. Example

[0049] Cellobiose purification The fermentation liquid in the upper tank was filtered by plate and frame, and the precipitate was preserved intact for subsequent animal feed. The clear liquid was filtered by RO membrane to obtain cellobiose concentrate. The cellobiose concentrate was freeze-dried in a vacuum freeze dryer for 24 hours to obtain cellobiose solid, and the purity of cellobiose was measured by liquid chromatography.

[0050] Liquid chromatography conditions Chromatographic column: Shodex Asahipak NH2P-50 4E 250 (4.6 mm ID x 250 mm).

[0051] Mobile phase: CH3CN / H2O=75 / 25.

[0052] Column temperature: 30℃.

[0053] Detector: Refractive Index Detector (RID).

[0054] Flow rate: 1.0 mL / min.

[0055] Injection volume: 10 μL.

[0056] Liquid chromatography has shown that the purity of cellobiose can reach 90%, which makes it possible to produce cellobiose from Phragmites australis at a low cost. The produced cellobiose can be used in the fields of food, medicine and chemical industry. The bacterial cells filtered from the fermentation liquid can be used to prepare animal feed, etc. This has realized the resource utilization of Phragmites australis.

Claims

1. A method for preparing cellobiose by using Arundo donax as raw material and using bacterial enzymes, characterized in that: The method includes raw material pretreatment, enzymatic hydrolysis, fermentation and fermentation liquid treatment, and the specific steps are as follows: S1. The naturally dried Phragmites australis is ultrafinely crushed to obtain Phragmites australis powder, and the ultrafine Phragmites australis powder is collected by sieving; the Phragmites australis includes: branches, leaves, and stems; S2. Add 1 to 3% NaOH solution to the ultrafine Phragmites australis powder obtained in S1, and heat swell at a temperature of 50 to 60 ° C to obtain Phragmites australis hydrolyzate; S3. Add commercial xylanase to the Phyllostachys australis hydrolyzate obtained in S2, mix well, and react at 30-60°C for 7-10 hours. During the enzymolysis process, the cellobiose content in the hydrolyzate is kept below 5.0 g / L. After the enzymolysis is completed, sterilize at 115°C for 20 min to obtain the hydrolyzate; S4. The enzymatic hydrolysate obtained in S3 was used as the carbon source for microbial fermentation. The culture was carried out at 35-50℃, ventilation 0-7 vvm, 200-900 r / min, and ammonia was added to maintain the pH at 6.5-7.

5. The culture time was 18-30 h, and the bacterial cell volume OD 600 Reach 30~60; S5. The fermentation broth obtained in S4 is subjected to plate and frame filtration to obtain a precipitate and a clear liquid; S6. The supernatant obtained in S5 is concentrated through an RO membrane to obtain a cellobiose concentrate; S7. Drying the cellobiose concentrate obtained in S6 to obtain high-purity cellobiose.

2. The method for producing cellobiose based on whole-plant Arundo donax microbial method according to claim 1, characterized in that: The whole plant of Phragmites australis ultrafine grinding in S1 is that the whole plant of Phragmites australis is ground and then passed through a 40-70 mesh sieve; The thermal swelling system described in S2 has a solid-liquid ratio of 1:2~6, a reaction temperature of 50~60°C, and a reaction time of 4~6 h; in S3, the ratio of ultrafine reed powder to enzyme activity is 1:0.01~0.05, and the reaction is carried out at 30~60°C for 7~10 h.

3. The method for producing cellobiose based on the whole plant of Phragmites australis microbial method according to claim 1, characterized in that: During the S3 enzymatic hydrolysis process, the cellobiose concentration was kept below 5 g / L by diluting the enzymatic hydrolysate to reduce the inhibition of cellobiose on enzyme activity.

4. The method for preparing cellobiose based on the enzyme of whole plant Arundo donax according to claim 1, characterized in that: The microorganism described in S4 is Escherichia coli, including but not limited to Escherichia coli K-12, MG1655, W3110, and other E. coli production strains, specific E. coli Escherichia coli str. K-12.

5. The method for preparing cellobiose based on the whole-plant Arundo donax enzyme according to claim 1, characterized in that: The fermentation conditions in S4 were: 35-50℃, ventilation 0-7 vvm, 200-900 r / min, and ammonia was added to maintain the pH at 6.5-7.5; the culture time was 18-30 h, and the bacterial mass reached OD 600 It is 30~60.

6. The method for producing cellobiose based on the whole plant of Phragmites australis microbial method according to claim 1, characterized in that: Plate and frame filtration in S5 separates the bacteria and large particle impurities from the fermentation liquid to obtain a clear liquid containing cellobiose.

7. The method for preparing cellobiose based on the whole-plant Arundo donax enzyme according to claim 1, characterized in that: The RO membrane in S6 allows water molecules to pass through, and the clear liquid can be passed through the RO membrane to obtain the cellobiose concentrate, and the filtered water can be reused after treatment.

8. The method for preparing cellobiose based on the whole-plant Arundo donax enzyme according to claim 1, characterized in that: The chemical purity of cellobiose obtained by S7 drying is over 90%.

9. Use of the method for preparing cellobiose by bacterial enzyme synergistically based on Phragmites australis as raw material as claimed in claim 1 in improving the conversion rate of Phragmites australis.

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