Method for preparing saccharified liquid from crop straws after micro-storage

Abstract

The invention relates to a method for preparing saccharified liquid by utilizing crop straw after micro-storage, which sequentially comprises a crop straw inoculation micro-storage stage and an alkaline hydrolysis stage, the inoculation strain is bacillus subtilis, the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.25552, and the inoculation amount of the bacillus subtilis is 5-15% of the total amount of the crop straw. The bacillus subtilis CGMCC NO.25552 has the advantages that the bacillus subtilis CGMCC NO.25552 is inoculated into the straw for microbiological storage, the decomposability of the crop straw can be effectively promoted, and then the conversion rate of the straw and the concentration of reducing sugar are increased.

Description

Technical Field

[0001] The invention relates to the technical field of straw processing, and in particular to a method for preparing saccharification liquid by micro-storing crop straw. Background Art

[0002] As a structural carbohydrate, lignocellulose in straw is a renewable resource with huge quantity in nature. In my country, the annual output of crop straw is as high as more than 700 million tons. The irrational use of crop straw resources has caused huge waste of resources and environmental pollution.

[0003] With the rapid development of animal husbandry, animal husbandry will account for 50% of the total grain production in the future. It is very important to develop new carbohydrate raw materials to replace traditional energy raw materials. At present, studying the digestion, metabolism and utilization rules of carbohydrates with different structures (such as non-starch polysaccharides) and rational processing to replace traditional energy raw materials is one of the hot spots in the development of animal feed resources. Effectively degrading straw lignocellulose into glucose and easily digestible low-molecular carbohydrates and then converting them into energy feed and protein feed is the entry point for preparing and producing animal feed. This will help solve the problem of straw resource waste and feed shortage in animal husbandry production. The application of enzymatic hydrolysis technology in the development of straw feed is the most effective and safest method, and it is also an indispensable step to improve the nutritional value of straw feed. Its main function is to use cellulase and xylanase to decompose cellulose and hemicellulose and convert them into glucose and xylose. It is difficult to achieve the ideal effect by enzymatic treatment alone.

[0004] For example, Chinese patent CN117187318A discloses a method for preparing fermentable sugar solution by enzymatic hydrolysis of corn straw. The method introduces excess ferric chloride molecules while pretreating the corn straw by adding ferric chloride to strengthen the ethanol solvent. The addition of ferric chloride increases the content of chloride ions and iron ions in the saccharification solution, making it easy for the ferric chloride to form ionic bonds with pigment molecules, making it difficult to remove a large amount of pigment substances in the straw. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing saccharification liquid by micro-storing crop straw. Bacillus subtilis CGMCC NO.25552 is inoculated into the straw and then micro-stored, which can effectively promote the decomposition of the crop straw, thereby improving the conversion rate and reducing sugar concentration of the straw.

[0006] The present invention is realized by the following technical scheme: on the one hand, a method for preparing saccharification liquid by using crop straw micro-storage is provided, which comprises a crop straw inoculation micro-storage stage and an alkaline hydrolysis stage in sequence, the inoculation strain is Bacillus subtilis, the preservation number is CGMCC NO.25552, and the inoculation amount of Bacillus subtilis is 5%-15% of the total amount of the crop straw.

[0007] Through the above technical scheme, appropriate physical and chemical pretreatment is necessary before enzymatic hydrolysis, thereby removing lignin, destroying the crystallinity and polymerization degree of cellulose, breaking long-chain cellulose into small molecular chains, increasing the contact area between enzymes and cellulose and hemicellulose, and accelerating the enzymatic hydrolysis rate. The corn straw is first micro-stored, and the use of microorganisms to pretreat the straw is mainly fungi that produce various hydrolytic enzymes, thereby degrading the hemicellulose or cellulose of lignocellulose. In particular, white, brown and soft rot fungi have been widely used to degrade hemicellulose and lignin in lignocellulosic biomass. White rot fungi are considered to be the most effective microbial candidates for pretreating lignocellulosic biomass. Compared with the unpretreated control samples, the enzymatic saccharification rate of reducing sugars released by lignocellulosic biomass (wheat straw, rice straw and corn straw) after biological pretreatment with white rot fungi increased by 50-65%. The energy demand of microbial pretreatment is also very low, so eco-friendly biological pretreatment using microorganisms is considered to be a candidate to replace harsh chemicals and thermochemical pretreatment.

[0008] Based on the combination of the above-mentioned crop straw inoculation micro-storage stage and alkaline hydrolysis stage, Bacillus subtilis CGMCC NO.25552 can effectively inhibit the growth of harmful bacteria and maintain the nutritional content and freshness of corn straw; Bacillus subtilis CGMCC NO.25552 is used to hydrolyze the lignocellulose in the straw, and then the alkali solution is used to destroy the lignin structure, increase the contact area between the enzyme preparation and the lignocellulose, improve its saccharification efficiency, and remove a large amount of pigment substances in the straw, thereby reducing the chromaticity of the sugar solution.

[0009] Furthermore, the crop straw inoculation micro-storage stage comprises the following steps:

[0010] Step 1) pretreatment of crop straw;

[0011] Step 2) Activating the strains;

[0012] Step 3) inoculating micro-storage;

[0013] Step 4) Drying and sealing.

[0014] Through the above technical scheme, crop straw is pretreated to become fine particles, which is not only conducive to the contact between microorganisms and straw during subsequent micro-storage, but also preliminarily destroys the microstructure of the straw. Compared with straw without pretreatment, the powder state is more conducive to the experiment. First, if the straw is not crushed, the straw is long and difficult to compact and seal, which has higher requirements for the experimental device. Secondly, during micro-storage, it needs to be stirred regularly. The straw in powder state is easier to stir evenly, and the moisture of the straw powder is adjusted before micro-storage. Under this moisture condition, the powder is not easy to fly, which is more conducive to the fermentation of microorganisms, thereby achieving the effect of decomposing the straw. When activating the strain, weigh 10 grams of brown sugar and add it to 1L of sterile water, weigh 1.00g of powdered Bacillus subtilis CGMCC NO.25552 and add it to the brown sugar culture medium, place it in a 37℃ shaker for 12h to activate it, and the number of live bacteria after activation is 1*10 9 CFU / ml, the strain is highly active and has strong decomposition ability for straw. With the extension of micro-storage time, the color of the straw gradually deepens and the texture becomes softer. Under microscopic conditions, it can be seen that the cellulose and hemicellulose in the straw are partially degraded, and the composite structure between cellulose and lignin becomes loose. Spraying is used during inoculation, and stirring is done while spraying to ensure that the strain is evenly inoculated in the straw powder. The inoculation amount determines the fermentation state of the microorganism to a certain extent. If the inoculation amount is too small, the fermentation cycle will be extended. If the inoculation amount is too large, the mycelium will grow too fast, and it is easy to produce viscous substances due to insufficient dissolved oxygen, which makes it difficult to decompose the straw.

[0015] Furthermore, in step 1), the air-dried crop straw is cut into segments and crushed into ultrafine powder with a particle size of 20-40 mesh by a crusher. In traditional micro-storage, the straw is directly collected, bundled, and piled. The straw is not crushed, which increases the floor space. If it is not compacted, it is difficult to create an anaerobic environment, which is not conducive to the fermentation of microorganisms. The straw is bundled and piled, which is inconvenient to turn it over. The straw generates heat under the action of microorganisms during the micro-storage process. The bundled and piled straw is not conducive to heat dissipation and is easy to cause fire. The straw in a powder state increases the contact area between the microorganism and its internal structure, thereby improving the decomposition efficiency of the microorganism.

[0016] Further, in step 2), under a sterile environment, brown sugar is added to sterile water, and powdered Bacillus subtilis CGMCC NO.25552 is added to the brown sugar culture medium, and the mixture is placed in a shaker at 37°C for 12 hours for activation to obtain an activated strain; the Bacillus subtilis (Bacillus subtilis) has a preservation number of CGMCC NO.25552; ​​the preservation time is August 19, 2022; the preservation unit is the General Microbiology Center of the China Culture Collection Administration (CGMCC); the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0017] The specific process of activating the strains through the above technical scheme is as follows: in a sterile environment, 10 grams of brown sugar is weighed and added to 1L of sterile water, 1.00g of powdered Bacillus subtilis CGMCC NO.25552 is weighed and added to the brown sugar culture medium, and then placed in a shaker at 37°C for 12 hours to activate the strains; after the activation, the number of live bacteria in the strains is 1*10 9 CFU / ml, the number of live bacteria is sufficient, and preparing it into a bacterial liquid makes it easier to evenly inoculate the bacteria.

[0018] Further, in step 3), water is added to the straw powder obtained in step 1), the water content is adjusted to 40%-60% of the total amount of the straw, 5%-15% of the total amount of the straw is added with activated bacteria, the powder is evenly stirred and put into a 50-80°C constant temperature incubator, water is sprayed and stirred every 1-4 hours, and the powder is taken out after micro-storage. After micro-storage, the texture of the straw becomes soft, the color deepens, and it has a sweet smell. Under microscopic conditions, it can be observed that the cellulose and hemicellulose in the straw are partially degraded, and the composite structure between cellulose and lignin is obviously loosened.

[0019] Furthermore, in step 4), the drying temperature is 40-70° C. When the docking rods are dried within this temperature range, the straws are not easy to clump and the drying efficiency is high. If the temperature is low, the drying rate is low, the drying time is long, and the straws are prone to mold growth. If the drying temperature is too high, the water is lost quickly and some nutrients are lost, affecting the subsequent saccharification effect.

[0020] Furthermore, in the alkaline hydrolysis stage, the alkali-solid ratio of the micro-stored straw during alkaline hydrolysis is 4-9:1, the alkaline hydrolysis temperature is 70-90°C, and the alkaline hydrolysis time is 0.5-3h. Under this alkali-solid ratio, the micro-stored straw is fully in contact with the alkali solution, and under stirring or shaking conditions, the alkali-solid ratio has low requirements for the stirring power consumption of the equipment, and the alkaline hydrolysis is carried out under heating conditions, which accelerates the reaction and shortens the alkaline hydrolysis time, but the temperature should not be too high and the time should not be too long, otherwise it will cause the evaporation of the alkali solution. The straw after alkaline hydrolysis is in a paste state, and the granular straw is greatly reduced. Under microscopic conditions, it can be seen that the surface pore size of the straw cellulose after alkaline hydrolysis increases, and the fiber bundle structure becomes looser, which is more conducive to enzymatic hydrolysis.

[0021] Furthermore, the alkali solution in the alkaline hydrolysis stage is one of NaOH and KOH, and the concentration of the alkali solution is 1-5%. Alkaline solution treatment of straw is beneficial to the destruction of molecular bonds in the straw, and under certain conditions, it can effectively remove lignin, increase the exposure area of ​​cellulose, and improve the grafting efficiency. KOH and NaOH are both strong alkalis, which are more destructive to their molecular bonds. Using a lower concentration of alkali solution to treat it can not only change its internal scaling state, but also reduce the workload of subsequent washing steps, reduce the application of neutralizers before enzymatic hydrolysis, and reduce production costs.

[0022] Furthermore, it also includes a saccharification stage, and the saccharification stage specifically includes the following steps:

[0023] Step S1: filtration, filtering the mixed solution in the alkaline hydrolysis stage with a vacuum pump and washing the filter residue, wherein the washing water is 3-8 times the amount of the alkaline solution;

[0024] Step S2: adjusting the pH, adding pure water to the filter residue to 4-9 times the total weight of the straw, and adjusting the pH thereof with sulfuric acid / citric acid;

[0025] Step S3: Enzymatic hydrolysis: adding an appropriate amount of enzyme preparation to the pH-adjusted mixed solution and placing the solution in a water bath for enzymatic hydrolysis;

[0026] Step S4: Filtration: Filter the enzymatically hydrolyzed mixed solution to obtain a saccharified solution.

[0027] Through the above technical scheme, in the traditional saccharification step, the pretreated straw is washed and then dried, and then a buffer is added, and the pH is adjusted before adding an enzyme preparation for enzymatic hydrolysis. In the present invention, a proper amount of water is directly added to the straw after washing, and the pH is adjusted before enzymatic hydrolysis, which reduces the drying step and the use of a buffer, saves energy, and the enzyme can still maintain a high activity in the aqueous solution.

[0028] Furthermore, the amount of enzyme preparation added is 0.25%-1.5% of the total amount of micro-storage straw, the enzymolysis temperature is 40-60°C, and the enzymolysis time is 24h-72h. The existing technology is to obtain high-concentration and high-conversion saccharification liquid, continuously increase the amount of enzyme preparation added and extend the enzymolysis time, the enzyme addition amount is mostly 2-5%, and the enzymolysis time is 72-144h. Enzyme preparation is an important cost indicator in the preparation process. The increase in the amount of enzyme preparation added significantly increases the preparation cost. As the enzymolysis time increases, the enzyme hydrolysis efficiency increases, but it is also easy to produce inhibitors to inhibit the enzymolysis. After micro-storage and alkaline hydrolysis pretreatment, this project can significantly reduce the amount of enzyme added and shorten the enzymolysis time to obtain a higher enzymolysis efficiency.

[0029] The beneficial effects of the present invention are:

[0030] 1) The present invention adds a micro-storage step on the basis of alkaline hydrolysis, firstly using microorganisms to hydrolyze the lignocellulose in the straw, and then using alkaline solution to destroy the lignin structure, thereby increasing the contact area between the enzyme preparation and the lignocellulose and improving its saccharification efficiency.

[0031] 2) The present invention washes the straw after alkaline hydrolysis, which can not only reduce the alkalinity caused by alkaline hydrolysis and reduce the amount of neutralizer used, but also remove a large amount of pigment substances in the straw and reduce the chromaticity of the sugar solution. Compared with the unwashed straw, the chromaticity of the sugar solution is reduced by more than 80%.

[0032] 3) The prior art mostly uses acid hydrolysis, alkaline hydrolysis, steam explosion and other methods for pretreatment and then enzymatic hydrolysis, but all of them have the problems of high preparation cost, high requirements for equipment corrosion resistance, high energy consumption cost, etc., and the prepared saccharification liquid has low concentration and low conversion rate. The present invention provides a method for enzymatic hydrolysis of straw after micro-storage + alkaline hydrolysis composite pretreatment. The structure of the treated straw is separated, the cellulose structure is exposed, the crystallinity is reduced, the enzymatic hydrolysis efficiency is improved, the amount of enzyme preparation used is reduced by 1-4%, the enzymatic hydrolysis time is shortened by 50%, the sugar solution concentration is increased by about 2%, and the conversion rate is increased by 10%. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the embodiment of the invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions or parts are by weight. The reagents and raw materials used in the embodiments of the present invention and the comparative examples, if not otherwise specified, can be obtained through commercial channels.

[0035] The strain deposit information involved in the following examples is as follows:

[0036] The deposit number of Bacillus subtilis is CGMCC NO.25552; ​​the deposit date is August 19, 2022; the deposit unit is the General Microbiology Center of China Culture Collection Administration (CGMCC); the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; this strain belongs to a published strain, and the specific source can be found in the Chinese invention patent with publication number CN115851498B.

[0037] Lactobacillus plantarum CGMCC27595, yeast ATCC26497, and Enterococcus faecalis CI CC 20419 are all commercially available and are publicly available strains.

[0038] Example 1

[0039] A method for preparing saccharification liquid by using crop straw micro-storage, which comprises a crop straw inoculation micro-storage stage, an alkaline hydrolysis stage, and a saccharification stage in sequence; wherein the crop straw inoculation micro-storage stage comprises the following steps:

[0040] 1) Pretreatment: Cut the air-dried corn stalks into 4 cm segments and then grind them into ultra-fine powder with an average particle size of 20 mesh using a grinder;

[0041] 2) Activation of bacterial strains: Weigh 10 g of brown sugar and add it into 1 L of sterile water, weigh 1.00 g of powdered Bacillus subtilis CGMCCNO.25552 and add it into the brown sugar culture medium, and place it in a shaker at 37° C. for 12 h to activate it.

[0042] 3) Inoculation micro-storage: Add appropriate amount of water to the straw powder to adjust its moisture content to 40%, then inoculate 10% of the straw weight of activated bacteria, stir evenly and place in a 60°C constant temperature incubator, spray water and stir every 1 hour, and take out after one week;

[0043] 4) Drying: The micro-storage straw taken out from the incubator is placed in a 70° C. oven for drying, and the dried micro-storage straw powder is bagged and sealed.

[0044] Alkaline hydrolysis stage: mix the micro-storage straw powder with 1% NaOH, adjust the alkali-solid ratio to 1:5, and put it into a water bath at 80℃ for alkaline hydrolysis for 1 hour;

[0045] Saccharification stage:

[0046] Filtration: Filter the mixed solution after the reaction with a vacuum pump and wash the residue with water 3 times the mass of the alkali solution;

[0047] Adjust pH: add pure water to the residue to 5 times the mass of the straw, and adjust the pH to 4.56 with 13% sulfuric acid;

[0048] Enzymatic hydrolysis: add 0.25% enzyme preparation by weight of straw to the mixed solution with adjusted pH, and place in a 50℃ water bath for enzymatic hydrolysis for 48h;

[0049] Filtration: The mixed solution after enzymatic hydrolysis was filtered to obtain saccharified liquid. The reducing sugar concentration of the obtained saccharified liquid was 6.63%, the conversion rate was 31.82%, and the chromaticity of the sugar liquid was 530.

[0050] Example 2

[0051] A method for preparing saccharification liquid by using crop straw micro-storage, which comprises a crop straw inoculation micro-storage stage, an alkaline hydrolysis stage, and a saccharification stage in sequence; wherein the crop straw inoculation micro-storage stage comprises the following steps:

[0052] 1) Pretreatment: Cut the air-dried corn stalks into 4 cm segments and then grind them into ultrafine powder with an average particle size of 40 mesh using a grinder;

[0053] 2) Preparation of bacterial strains: Weigh 10 g of brown sugar and add it to 1 L of sterile water, weigh 1.00 g of powdered Bacillus subtilis CGMCCNO.25552 and add it to the brown sugar culture medium, place it in a shaker at 37°C for 12 h to activate it;

[0054] 3) Inoculation micro-storage: add appropriate amount of water to the straw powder to adjust its moisture content to 50%, then inoculate 5% of the straw weight of activated bacteria, stir evenly and place in a 50°C constant temperature incubator, spray water and stir every 2 hours, and take out after one week;

[0055] 4) Drying: The micro-storage straw taken out from the incubator is placed in a 70°C oven for drying, and the dried micro-storage straw powder is bagged and sealed;

[0056] Alkaline hydrolysis stage:

[0057] Alkaline hydrolysis: Mix the micro-storage straw powder with 2% NaOH, adjust the alkali-solid ratio to 1:6, and place it in a water bath at 70℃ for 2h;

[0058] Saccharification stage:

[0059] Filtration: Filter the reaction mixture with a vacuum pump and wash the residue, using 4 times the mass of the alkali solution;

[0060] Adjust pH: add pure water to the residue to 6 times the mass of the straw, and adjust the pH to 4.78 with 14% sulfuric acid;

[0061] Enzymatic hydrolysis: add 0.5% enzyme preparation by weight of straw to the mixed solution with adjusted pH, and place in a 50℃ water bath for enzymatic hydrolysis for 48h;

[0062] Filtration: The enzymatically hydrolyzed mixed solution is filtered to obtain a sugar solution having a reducing sugar concentration of 6.72%, a conversion rate of 38.98%, and a chromaticity of 410.

[0063] Example 3

[0064] A method for preparing saccharification liquid by using crop straw micro-storage, which comprises a crop straw inoculation micro-storage stage, an alkaline hydrolysis stage, and a saccharification stage in sequence; wherein the crop straw inoculation micro-storage stage comprises the following steps:

[0065] 1) Pretreatment: Cut the air-dried corn stalks into 5 cm segments and then grind them into ultrafine powder with an average particle size of 40 mesh using a grinder;

[0066] 2) Preparation of bacterial strains: Weigh 10 g of brown sugar and add it to 1 L of sterile water, weigh 1.00 g of powdered Bacillus subtilis CGMCCNO.25552 and add it to the brown sugar culture medium, place it in a shaker at 37°C for 12 h to activate it;

[0067] 3) Inoculation micro-storage: add appropriate amount of water to the straw powder to adjust its moisture content to 60%, then inoculate 10% of the straw weight of activated bacteria, stir evenly and place in a 60°C constant temperature incubator, spray water and stir every 3 hours, and take out after one week;

[0068] 4) Drying: The micro-storage straw taken out from the incubator is placed in a 60°C oven for drying, and the dried micro-storage straw powder is bagged and sealed;

[0069] Alkaline hydrolysis stage:

[0070] Alkaline hydrolysis: Mix the micro-storage straw powder with 3% NaOH, adjust the alkali-solid ratio to 1:7, and place it in a water bath at 70℃ for 1 hour;

[0071] Saccharification stage:

[0072] Filtration: Filter the reaction mixture with a vacuum pump and wash the residue, using 5 times the mass of the alkali solution;

[0073] Adjust pH: add pure water to the residue to 7 times the mass of the straw, and adjust the pH to 4.82 with 13% sulfuric acid;

[0074] Enzymatic hydrolysis: add 1% enzyme preparation of straw weight to the mixed solution with adjusted pH, and place it in a 55℃ water bath for enzymatic hydrolysis for 72h;

[0075] Filtration: The mixed solution after enzymatic hydrolysis is filtered to obtain a sugar solution with a reducing sugar concentration of 6.88%, a conversion rate of 46.78%, and a chromaticity of 360.

[0076] Example 4

[0077] A method for preparing saccharification liquid by using crop straw micro-storage, which comprises a crop straw inoculation micro-storage stage, an alkaline hydrolysis stage, and a saccharification stage in sequence; wherein the crop straw inoculation micro-storage stage comprises the following steps:

[0078] 1) Pretreatment: Cut the air-dried corn stalks into 4 cm segments and then grind them into ultrafine powder with an average particle size of 40 mesh using a grinder;

[0079] 2) Preparation of bacterial strains: Weigh 10 g of brown sugar and add it to 1 L of sterile water, weigh 1.00 g of powdered Bacillus subtilis CGMCCNO.25552 and add it to the brown sugar culture medium, place it in a shaker at 37°C for 12 h to activate it;

[0080] 3) Inoculation micro-storage: Add appropriate amount of water to the straw powder to adjust its moisture content to 70%, then inoculate 10% of the straw weight of activated bacteria, stir evenly and place in a 70°C constant temperature incubator, spray water and stir every 4 hours, and take out after one week;

[0081] 4) Drying: The micro-storage straw taken out from the incubator is placed in a 70°C oven for drying, and the dried micro-storage straw powder is bagged and sealed;

[0082] Alkaline hydrolysis stage:

[0083] Alkaline hydrolysis: Mix the micro-storage straw powder with 2% NaOH, adjust the alkali-solid ratio to 1:8, and place it in a water bath at 80°C for 2 hours;

[0084] Saccharification stage:

[0085] Filtration: Filter the reaction mixture with a vacuum pump and wash the residue, using 6 times the mass of the alkali solution;

[0086] Adjust pH: add pure water to the residue to 8 times the mass of the straw, and adjust the pH to 4.97 with 15% sulfuric acid;

[0087] Enzymatic hydrolysis: add 1.25% enzyme preparation of the straw weight to the mixed solution with adjusted pH, and place it in a 45℃ water bath for enzymatic hydrolysis for 48h;

[0088] Filtration: The mixed solution after enzymatic hydrolysis is filtered to obtain a sugar solution with a reducing sugar concentration of 6.69%, a conversion rate of 52.18%, and a chromaticity of 320.

[0089] Example 5

[0090] A method for preparing saccharification liquid by using crop straw micro-storage, which comprises a crop straw inoculation micro-storage stage, an alkaline hydrolysis stage, and a saccharification stage in sequence; wherein the crop straw inoculation micro-storage stage comprises the following steps:

[0091] 1) Pretreatment: Cut the air-dried corn stalks into 5 cm segments and then grind them into ultra-fine powder with an average particle size of 20 mesh using a grinder;

[0092] 2) Preparation of bacterial strains: Weigh 10 g of brown sugar and add it to 1 L of sterile water, weigh 1.00 g of powdered Bacillus subtilis CGMCCNO.25552 and add it to the brown sugar culture medium, place it in a shaker at 37°C for 12 h to activate it;

[0093] 3) Inoculation micro-storage: Add appropriate amount of water to the straw powder to adjust its moisture content to 60%, then inoculate 15% of the straw weight of activated bacteria, stir evenly and place in a 50°C constant temperature incubator, spray water and stir every 2 hours, and take out after one week;

[0094] 4) Drying: The micro-storage straw taken out from the incubator is placed in a 60°C oven for drying, and the dried micro-storage straw powder is bagged and sealed;

[0095] Alkaline hydrolysis stage:

[0096] Alkaline hydrolysis: Mix the micro-storage straw powder with 1% NaOH, adjust the alkali-solid ratio to 1:9, and place it in a water bath at 80°C for 1 hour;

[0097] Saccharification stage:

[0098] Filtration: Filter the reaction mixture with a vacuum pump and wash the residue, using 7 times the mass of the alkali solution;

[0099] Adjust pH: add pure water to the residue to 9 times the mass of the straw, and adjust the pH to 4.32 with 15% sulfuric acid;

[0100] Enzymatic hydrolysis: add 1.5% enzyme preparation of straw weight to the mixed solution with adjusted pH value, and place it in a 40℃ water bath for enzymatic hydrolysis for 48h;

[0101] Filtration: The mixed solution after enzymatic hydrolysis is filtered to obtain a sugar solution with a reducing sugar concentration of 6.11%, a conversion rate of 57.64%, and a chromaticity of 280.

[0102] Comparative Example 1

[0103] A method for preparing saccharification liquid by using crop straw micro-storage, which comprises a crop straw inoculation micro-storage stage, an alkaline hydrolysis stage, and a saccharification stage in sequence; wherein the crop straw inoculation micro-storage stage comprises the following steps:

[0104] 1) Pretreatment: Cut the air-dried corn stalks into 4 cm segments and then grind them into ultra-fine powder with an average particle size of 20 mesh using a grinder;

[0105] 2) Preparation of bacterial strains: Weigh 10 g of brown sugar and add it to 1 L of sterile water, weigh 1.00 g of powdered Lactobacillus plantarum CGMCC27595 and add it to the brown sugar culture medium, place it in a shaker at 37° C. for 12 h to activate it;

[0106] 3) Inoculation micro-storage: Add appropriate amount of water to the straw powder to adjust its moisture content to 40%, then inoculate 10% of the straw weight of activated bacteria, stir evenly and place in a 60°C constant temperature incubator, spray water and stir every 1 hour, and take out after one week;

[0107] 4) Drying: The micro-storage straw taken out from the incubator is placed in a 70°C oven for drying, and the dried micro-storage straw powder is bagged and sealed;

[0108] Alkaline hydrolysis stage:

[0109] Alkaline hydrolysis: Mix the micro-storage straw powder with 1% NaOH, adjust the alkali-solid ratio to 1:5, and place it in a water bath at 80°C for 1 hour;

[0110] Saccharification stage:

[0111] Filtration: Filter the reaction mixture with a vacuum pump and wash the residue, using 3 times the mass of the alkali solution;

[0112] Adjust pH: add pure water to the residue to 5 times the mass of the straw, and adjust the pH to 4.56 with 13% sulfuric acid;

[0113] Enzymatic hydrolysis: add 0.25% enzyme preparation of the mass of straw to the mixed solution with adjusted pH value, and place it in a 50℃ water bath for enzymatic hydrolysis for 48h;

[0114] Filtration: The enzymatically hydrolyzed mixed solution is filtered to obtain a sugar solution having a reducing sugar concentration of 5.82%, a conversion rate of 27.94%, and a chromaticity of 580.

[0115] Comparative Example 2

[0116] A method for preparing saccharification liquid by using crop straw micro-storage, which comprises a crop straw inoculation micro-storage stage, an alkaline hydrolysis stage, and a saccharification stage in sequence; wherein the crop straw inoculation micro-storage stage comprises the following steps:

[0117] 1) Pretreatment: Cut the air-dried corn stalks into 4 cm segments and then grind them into ultrafine powder with an average particle size of 40 mesh using a grinder;

[0118] 2) Preparation of bacterial strains: Weigh 10 g of brown sugar and add it to 1 L of sterile water, weigh 1.00 g of powdered ATCC26497 yeast and add it to the brown sugar culture medium, place it in a shaker at 37°C for 12 h to activate it;

[0119] 3) Inoculation micro-storage: add appropriate amount of water to the straw powder to adjust its moisture content to 50%, then inoculate 5% of the straw weight of activated bacteria, stir evenly and place in a 50°C constant temperature incubator, spray water and stir every 2 hours, and take out after one week;

[0120] 4) Drying: The micro-storage straw taken out from the incubator is placed in a 70°C oven for drying, and the dried micro-storage straw powder is bagged and sealed;

[0121] Alkaline hydrolysis stage:

[0122] Alkaline hydrolysis: Mix the micro-storage straw powder with 2% NaOH, adjust the alkali-solid ratio to 1:6, and place it in a water bath at 70℃ for 2h;

[0123] Saccharification stage:

[0124] Filtration: Filter the reaction mixture with a vacuum pump and wash the residue, using 4 times the mass of the alkali solution;

[0125] Adjust pH: add pure water to the residue to 6 times the mass of the straw, and adjust the pH to 4.78 with 14% sulfuric acid;

[0126] Enzymatic hydrolysis: add 0.5% enzyme preparation by weight of straw to the mixed solution with adjusted pH, and place in a 50℃ water bath for enzymatic hydrolysis for 48h;

[0127] Filtration: The enzymatically hydrolyzed mixed solution is filtered to obtain a sugar solution having a reducing sugar concentration of 5.96%, a conversion rate of 34.57%, and a chromaticity of 525.

[0128] Comparative Example 3

[0129] A method for preparing saccharification liquid by using crop straw micro-storage, which comprises a crop straw inoculation micro-storage stage, an alkaline hydrolysis stage, and a saccharification stage in sequence; wherein the crop straw inoculation micro-storage stage comprises the following steps:

[0130] 1) Pretreatment: Cut the air-dried corn stalks into 5 cm segments and then grind them into ultra-fine powder with an average particle size of 20 mesh using a grinder;

[0131] 2) Preparation of bacterial strains: Weigh 10 g of brown sugar and add it to 1 L of sterile water, weigh 1.00 g of powdered CI CC 20419 Enterococcus faecalis and add it to the brown sugar culture medium, place it in a shaker at 37°C for 12 h to activate it;

[0132] 3) Inoculation micro-storage: Add appropriate amount of water to the straw powder to adjust its moisture content to 60%, then inoculate 15% of the straw weight of activated bacteria, stir evenly and place in a 50°C constant temperature incubator, spray water and stir every 2 hours, and take out after one week;

[0133] 4) Drying: The micro-storage straw taken out from the incubator is placed in a 60°C oven for drying, and the dried micro-storage straw powder is bagged and sealed;

[0134] Alkaline hydrolysis stage:

[0135] Alkaline hydrolysis: Mix the micro-storage straw powder with 1% NaOH, adjust the alkali-solid ratio to 1:9, and place it in a water bath at 80°C for 1 hour;

[0136] Saccharification stage:

[0137] Filtration: Filter the reaction mixture with a vacuum pump and wash the residue, using 7 times the mass of the alkali solution;

[0138] Adjust pH: add pure water to the residue to 9 times the mass of the straw, and adjust the pH to 4.32 with 15% sulfuric acid;

[0139] Enzymatic hydrolysis: add 1.5% enzyme preparation by weight of straw to the mixed solution with adjusted pH, and place in a 40℃ water bath for enzymatic hydrolysis for 48h;

[0140] Filtration: The enzymatically hydrolyzed mixed solution is filtered to obtain a sugar solution having a reducing sugar concentration of 5.21%, a conversion rate of 35.43%, and a chromaticity of 385.

[0141] Effect Example

[0142] The sugar solutions prepared in Examples 1-5 were measured and calculated to obtain the reducing sugar content, conversion rate, and color of the sugar solutions. The results are shown in Table 1 below.

[0143] Table 1

[0144]

[0145]

[0146] Table 2

[0147] Glucose ratio Xylose content Arabinose ratio Cellobiose ratio Example 1 76.98％ 19.22％ 2.08％ 0.91％ Example 2 77.72％ 19.17％ 2.10％ 1.00％ Example 3 77.94％ 19.13％ 2.03％ 0.90％ Example 4 77.56％ 19.14％ 2.12％ 0.95％ Example 5 77.83％ 19.12％ 2.06％ 0.99％

[0148] It can be seen from Table 1 that the saccharification liquid obtained by the present invention has a higher concentration than the straw without micro-storage, and the conversion rate of the straw has also been improved. After liquid phase detection, it can be seen from Table 2 that the concentration of glucose contained in the sugar solution accounts for a large proportion, and the proportion of glucose compared to the straw without micro-storage increases, and the proportion of cellobiose is low. The present invention adds a micro-storage step compared to traditional sugar production. First, the straw is preliminarily decomposed by microorganisms to degrade part of the cellulose and hemicellulose, and then lignin is removed by alkaline decomposition, the molecular bonds between the three elements are opened, the exposed area of ​​cellulose is increased, the crystallinity of cellulose is reduced, and the enzymatic hydrolysis efficiency is significantly improved. The sugar concentration is increased by 0.5-1%, the conversion rate can be increased to 15%, the chromaticity is reduced by 60-80%, and the enzyme addition amount of the present invention is reduced compared with the traditional sugar production method, which reduces the production cost. The present invention is better than the original technology in terms of straw conversion rate, and the equipment requirements for sugar production by alkaline hydrolysis are relatively low, and the increase in sugar concentration can reduce the cost of concentration.

[0149] It can be seen from Comparative Examples 1-3 that under the same preparation conditions, the number of viable bacteria of Bacillus subtilis after activation is 1*10 9 CFU / ml, the number of viable Lactobacillus plantarum after activation is 1*10 8 CFU / ml, the number of live yeast cells after activation is 2*10 8 CFU / ml, the number of viable Enterococcus faecalis after activation is 1*10 9CFU / ml, among which Bacillus subtilis and Enterococcus faecalis have more live bacteria after activation, and have stronger decomposition ability on straw, but Bacillus subtilis has stronger stress resistance, high temperature resistance, easy storage, and the ability to maintain anaerobic environment than Enterococcus faecalis. Under the same preparation conditions, the reducing sugar concentration of straw after micro-storage with Bacillus subtilis is 0.9% higher than that of Enterococcus faecalis, and the conversion rate is 22.21% higher. It can be seen that Bacillus subtilis has better decomposition effect on straw than Lactobacillus plantarum, yeast, and Enterococcus faecalis. The decomposition ability of the straw structure after pretreatment with it is strong, which is conducive to the subsequent alkaline hydrolysis and enzymatic hydrolysis, thereby effectively improving the reducing sugar concentration and conversion rate.

[0150] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing saccharified liquid by micro-storage of crop straw, which comprises a crop straw inoculation micro-storage stage and an alkaline hydrolysis stage, wherein the inoculated strain is Bacillus subtilis, with a deposit number of CGMCCNO.25552, and the inoculation amount of Bacillus subtilis is 5%-15% of the total amount of the crop straw.

2. The method for preparing saccharified liquid by micro-storage of crop straw according to claim 1, characterized in that: The crop straw inoculation micro-storage stage comprises the following steps: Step 1) pretreatment of crop straw; Step 2) Activating the strains; Step 3) inoculating micro-storage; Step 4) Drying and sealing.

3. The method for preparing saccharified liquid by micro-storage of crop straw according to claim 2, characterized in that: In step 1), the air-dried crop straw is cut into segments and crushed into ultrafine powder with a particle size of 20-40 mesh using a crusher.

4. The method for preparing saccharified liquid by micro-storage of crop straw according to claim 3, characterized in that: In step 2), under a sterile environment, brown sugar is added to sterile water, and powdered Bacillus subtilis CGMCC NO.25552 is added to the brown sugar culture medium, and the mixture is placed in a shaker at 37°C for 12 hours for activation to obtain an activated strain; the Bacillus subtilis (Bacillus subtilis) has a preservation number of CGMCC NO.25552; ​​the preservation time is August 19, 2022; the preservation unit is the General Microbiology Center of China National Microbiological Culture Collection Administration (CGMCC); the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

5. The method for preparing saccharified liquid by micro-storage of crop straw according to claim 4, characterized in that: In step 3), water is added to the straw powder obtained in step 1) to adjust the moisture content to 40%-60% of the total amount of straw, and activated bacteria of 5%-15% of the total amount of straw are added. After being stirred evenly, the powder is placed in a constant temperature incubator of 50-80°C, sprayed with water and stirred every 1-4 hours, and taken out after being slightly stored.

6. The method for preparing saccharified liquid by using crop straw micro-storage according to claim 4, characterized in that: In step 4), the drying temperature is 40-70°C.

7. The method for preparing saccharified liquid by using crop straw micro-storage according to claim 1, characterized in that: In the alkaline hydrolysis stage, the alkali-solid ratio of the micro-storage straw during alkaline hydrolysis is 4-9:1, the alkaline hydrolysis temperature is 70-90° C., and the alkaline hydrolysis time is 0.5-3 h.

8. The method for preparing saccharified liquid by using crop straw micro-storage according to claim 7, characterized in that: The alkali solution in the alkaline hydrolysis stage is one of NaOH and KOH, and the concentration of the alkali solution is 1-5%.

9. The method for preparing saccharified liquid by using crop straw micro-storage according to claim 1, characterized in that: The saccharification stage is also included, and the saccharification stage specifically includes the following steps: Step S1: filtration, filtering the mixed solution in the alkaline hydrolysis stage with a vacuum pump and washing the filter residue, wherein the washing water is 3-8 times the amount of the alkaline solution; Step S2: adjusting the pH, adding pure water to the filter residue to 4-9 times the total weight of the straw, and adjusting the pH thereof with sulfuric acid / citric acid; Step S3: Enzymatic hydrolysis: adding an appropriate amount of enzyme preparation to the pH-adjusted mixed solution and placing the solution in a water bath for enzymatic hydrolysis; Step S4: Filtration: Filter the enzymatically hydrolyzed mixed solution to obtain a saccharified solution.

10. The method for preparing saccharified liquid by using crop straw micro-storage according to claim 9, characterized in that: The added amount of the enzyme preparation is 0.25%-1.5% of the total amount of the micro-storage straw, the enzymolysis temperature is 40-60°C, and the enzymolysis time is 24h-72h.

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