A method for preparing glucose

Abstract

The invention provides a method for preparing glucose, belonging to the technical field of glucose; the method comprises the steps of preparing starch milk, liquefaction, saccharification, filtering and post-processing; the filtering step comprises filtering saccharification liquid through a 0.5 mm screen, collecting primary filtrate of saccharification liquid, then adding an impurity remover, filtering, collecting secondary filtrate of saccharification liquid, and performing nanofiltration treatment to obtain saccharification filtrate; the method for preparing the impurity remover comprises placing activated carbon in a nitric acid solution, stirring at a high temperature, washing after filtering, putting into deionized water, adding a catalyst, stirring and catalyzing at room temperature for 28-32 minutes, then adding dodecylamine and glutamic acid, stirring and reacting at room temperature, filtering and washing, putting into a chitosan solution, stirring at 34-36°C for 38-42 minutes, filtering, washing and drying to obtain the impurity remover; by adopting the preparation method of the invention, the glucose yield and purity are high, the stability of functional saccharifying enzyme is good, and the energy consumption is low.

Description

Technical Field

[0001] The invention belongs to the technical field of glucose, and in particular relates to a method for preparing glucose. Background Art

[0002] Glucose, also known as corn glucose, corn sugar, or glucose for short, has a chemical name of 2,3,4,5,6-pentahydroxyhexanal. It is the most widely distributed and important sugar in nature. Pure glucose is a colorless crystal, sweet but not as sweet as sucrose. It is easily soluble in water, slightly soluble in ethanol, and insoluble in ether. In aqueous solution, glucose has rightward optical rotation, so it is also called "dextrose".

[0003] Glucose is an indispensable nutrient for metabolism in living organisms. The heat released by its oxidation reaction is an important source of energy required for human life activities and plays an important role in the biological field. Glucose can be used to produce fructose after being treated with isomerase and has become an important product of the current sugar industry. Glucose can also be used as a reducing agent in the printing and dyeing leather industry. It is often used as a reducing agent in the mirror industry and the silver plating process of thermos flasks. Glucose is also used as a raw material in the industry to synthesize vitamin C.

[0004] The preparation process of glucose includes acid hydrolysis, acid enzyme method and double enzyme method; the acid hydrolysis method is to hydrolyze α-1,4-glycosidic bonds with a high concentration of acid solution, and finally generate sweet glucose syrup, but the acid hydrolysis method has the defects of poor sugar conversion rate and easy generation of bitter impurities, and it also requires pressure and acid resistant equipment, which increases the cost; the acid enzyme method is to hydrolyze starch under high pressure and high acid conditions, and then use enzymes to saccharify the hydrolyzate, but it still produces bitter miscellaneous sugars and is darker in color;

[0005] The dual enzyme method is to first gelatinize the starch, then use α-amylase to hydrolyze the gelatinized starch into short-chain maltodextrin and oligosaccharides, and then use saccharifying enzyme to hydrolyze the α-1,4-glycosidic bond from the non-reducing end to produce glucose, and finally completely convert the dextrin oligosaccharides in the saccharification liquid into glucose. The dual enzyme method for preparing glucose does not require pressure-resistant and acid-resistant equipment, and has the advantages of mild reaction conditions, making it a preparation process for large-scale production of glucose.

[0006] However, in the process of preparing glucose by the double enzyme method, there are the following defects:

[0007] First, the DE value of the liquefied liquid is very important, as it directly affects the quality of the product. If the DE value is high, some sugars will be further decomposed into other small molecular compounds, and the saccharifying enzyme cannot fully act, which reduces the quality of the product. If the DE value is low, it means that the degree of starch hydrolysis is insufficient, and some starch still maintains a larger molecular structure, resulting in high viscosity of the liquefied liquid, which is not conducive to subsequent saccharification. In addition, the number of substrates at the exposed non-reducing end is limited, and the saccharifying enzyme does not have enough substrates to act on, which affects the saccharification efficiency and ultimately the yield of the product.

[0008] Second, the stability of glucoamylase is poor and it is greatly affected by the environment. The enzyme activity is unstable, which leads to incomplete starch hydrolysis. The presence of large molecular dextrin makes crystallization and centrifugal separation very difficult or even impossible, affecting the purity of glucose.

[0009] Third, when separating and extracting glucose from the saccharification liquid, plate and frame filtration is usually used to remove suspended matter and residual starch, and then a large amount of anion and cation exchange resins are used to remove salt in the saccharification liquid, and finally the product is crystallized; however, this process produces more wastewater, and the resin is easily destroyed, resulting in a large amount of wastewater, high energy consumption, and low work efficiency. Summary of the invention

[0010] In order to solve the technical problems existing in the prior art, the present invention provides a method for preparing glucose, which reasonably controls the degree of starch hydrolysis, ensures sufficient and complete saccharification, has high and stable enzyme activity, consumes less energy, and has high glucose yield and purity.

[0011] In view of the above technical problems, the present invention adopts the following technical solutions:

[0012] A method for preparing glucose, comprising the steps of preparing starch milk, liquefaction, saccharification, filtration and post-processing, which are specifically as follows:

[0013] 1. Preparation of starch milk

[0014] Add corn starch to deionized water for slurry adjustment, control the mass concentration of the slurry after slurry adjustment to be 16-20%, then add decyl glucoside, continue to stir evenly, add 9.6-10.3wt% hydrochloric acid solution to adjust the pH to 4.4-4.6, increase the temperature to 43-47°C, keep stirring for 0.8-1.2h, and obtain starch milk;

[0015] The mass of the decyl glucoside is 0.6-0.8% of the mass of corn starch.

[0016] 2. Liquefaction

[0017] Add high temperature resistant α-amylase to starch milk, the addition amount of high temperature resistant α-amylase is 0.30-0.34kg / t starch dry basis, the starch milk is sprayed and liquefied by a liquefaction ejector, the liquefaction temperature is 106-110°C, the liquefaction time is 40-44min, the pH of the liquefied liquid is controlled to be 5.4-5.6, the DE value is 12-14%, and the liquefied liquid is obtained by centrifugal separation.

[0018] 3. Saccharification

[0019] The temperature of the liquefied liquid is controlled at 58-62°C, the pH is adjusted to 4.5-4.7, and a functional saccharifying enzyme is added to carry out a saccharification reaction. The reaction time is 46.0-50.0 hours, the DE value is ≥98.0%, and the DX value is ≥96.5%, to obtain a saccharified liquid;

[0020] The mass ratio of the liquefied liquid to the functional saccharifying enzyme is 1000:1.5-2.0;

[0021] The method for preparing the functional saccharifying enzyme comprises the steps of preparing an enzyme carrier and loading the enzyme;

[0022] The steps of preparing the enzyme carrier are as follows: placing diatomaceous earth in a sodium hydroxide solution, raising the temperature to 58-62° C., keeping warm and stirring for 2.8-3.2 hours, filtering, washing, and drying after the stirring is completed, and then placing the solution in a muffle furnace for calcination, calcining at 215-225° C. for 0.8-1.2 hours, and calcining at 357-363° C. for 3.0-3.4 hours, and obtaining porous diatomaceous earth after the calcination is completed; placing the porous diatomaceous earth in an ethanol solution, adding kH550, kH602, and triethanolamine, raising the temperature to 61-65° C., keeping warm and stirring for 3.8-4.2 hours, filtering, washing, and drying after the reaction is completed to obtain aminodiatomaceous earth; placing the aminodiatomaceous earth in acetonitrile, and then adding ethylene glycol diglycidyl ether, raising the temperature to 63-67° C., stirring and reacting for 5.8-6.2 hours, filtering out and washing after the reaction is completed to obtain a primary carrier;

[0023] The particle size of the diatomaceous earth is 310-330nm;

[0024] The mass ratio of the diatomaceous earth to the sodium hydroxide solution is 9.8-10.2:67-73;

[0025] The mass concentration of the sodium hydroxide solution is 21-24%;

[0026] The mass ratio of the porous diatomaceous earth, ethanol solution, kH550, kH602, and triethanolamine is 9.0-9.5:97-105:0.5-0.8:0.4-0.6:0.8-1.2;

[0027] The mass concentration of the ethanol solution is 34-37%;

[0028] The mass ratio of aminodiatomite, acetonitrile and ethylene glycol diglycidyl ether is 8.3-8.7:95-105:2.0-2.2;

[0029] The enzyme loading step comprises: adding pullulanase into deionized water, adding Tween 80, stirring evenly, adding a primary carrier, then adding a chitosan solution and continuing to stir evenly, then adding a citral solution, stirring and reacting at 32-35° C. for 2.8-3.2 hours, adding sodium alginate after the stirring reaction is completed, continuing to stir for 0.8-1.2 hours, filtering and drying, and obtaining a functional saccharifying enzyme;

[0030] The mass ratio of the pullulanase, deionized water, Tween 80, primary carrier, chitosan solution, citral solution, and sodium alginate is 3.7-4.2:96-105:0.30-0.34:12.3-12.7:8.5-8.8:6.2-6.6:2.6-3.0;

[0031] The chitosan solution is an acetic acid solution of chitosan, the volume concentration of the acetic acid solution is 1.8-2.2%, and the mass concentration of the chitosan solution is 2.4-2.6%;

[0032] The mass concentration of the citral solution is 2.1-2.5%.

[0033] 4. Filter

[0034] The saccharification liquid is filtered through a 0.5 mm mesh, the primary filtrate of the saccharification liquid is collected, and then an impurity remover is added, and the mixture is stirred at 240-260 rpm for 9.0-12.0 min. After the stirring is completed, the mixture is allowed to stand for 1.4-1.6 h. After the standing is completed, the secondary filtrate of the saccharification liquid is collected by filtration, and the temperature of the secondary filtrate of the saccharification liquid is increased to 38-42° C., and the secondary filtrate is subjected to nanofiltration treatment through a nanofiltration membrane, and the feed pressure is controlled to be 1.8-2.2 MPa, and the retention amount is 270-300 MW, to obtain a saccharification filtrate;

[0035] The mass ratio of the primary filtrate of the saccharified liquid to the impurity remover is 100:0.32-0.36;

[0036] The preparation method of the impurity remover comprises placing activated carbon in a nitric acid solution, raising the temperature to 66-70° C., keeping the temperature and stirring for 4.8-5.2 hours, filtering out and washing after the end of the keeping temperature and stirring, putting it into deionized water, adding a catalyst, stirring and catalyzing at room temperature for 28-32 minutes, then adding dodecylamine and glutamic acid, stirring and reacting at room temperature for 3.2-3.7 hours, filtering and washing, putting it into a chitosan solution, stirring at 34-36° C. for 38-42 minutes, filtering, washing, and drying to obtain the impurity remover;

[0037] The particle size of the activated carbon is 10-14 μm;

[0038] The mass concentration of the nitric acid solution is 73-77%;

[0039] The catalyst is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2.2-2.6:1.5-1.8;

[0040] The chitosan solution is a mixture of chitosan and 5.0wt% acetic acid solution, and the mass ratio of the chitosan to the 5.0wt% acetic acid solution is 1:12.2-12.5;

[0041] The mass ratio of the activated carbon, nitric acid solution, deionized water, catalyst, dodecylamine, glutamic acid and chitosan solution is 9.0-9.4:82-88:490-510:4.0-4.2:3.6-4.0:2.1-2.3:46-53.

[0042] 5. Post-processing

[0043] The saccharification filtrate is heated and concentrated to 50% of the original volume, and then crystallized and dried to obtain the glucose product.

[0044] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0045] 1. The present invention uses corn starch to prepare glucose, controls a suitable DE value in the liquefaction step, ensures sufficient hydrolysis of starch, and does not cause excessive hydrolysis, so that the saccharification reaction can be fully carried out. In the saccharification step, a carrier is used to solidify the enzyme, specifically, diatomaceous earth is modified by amino groups, and then the amino groups on the surface of the diatomaceous earth are reacted by epoxy groups, and then the enzyme is adsorbed, chitosan solution is added, and aldehyde groups are introduced for cross-linking to form a stable cross-linking structure, which effectively avoids the loss of the enzyme, ensures the activity of the enzyme, and improves the stability of the functionalized enzyme, so that the enzyme can be reused and the saccharification reaction can be promoted. When removing impurities, the protein has a large volume and structure, and its surface contains polar groups, and the glucose Glucose has fewer surface charges and polar groups, and activated carbon is used as a porous adsorption material to effectively remove pigments from the saccharification liquid. The activated carbon is specifically treated, and then amino groups are introduced for amidation reaction. The long-chain alkyl groups of dodecylamine are well combined with the hydrophobic groups of proteins, effectively repelling small molecules such as glucose. In addition, the amide groups and chitosan can enhance the interaction with proteins and improve the adsorption stability. Ultimately, it can effectively adsorb proteins in the saccharification liquid, while repelling molecules such as glucose, thereby effectively removing impurities from the saccharification liquid. After steps such as concentration, crystallization, and drying, high-quality glucose products are obtained with high yield and purity, good stability of functional saccharifying enzymes, and less energy consumption in the preparation method.

[0046] 2. The glucose product prepared by the method of the present invention has a glucose content of 99.56-99.68% and a yield of 90.18-90.25%;

[0047] 2. After the saccharification step of the present invention is completed, the functional saccharifying enzyme is recovered and reused. The recovery method is to centrifuge the saccharification liquid, wash it with deionized water, and dry it at 60°C to obtain regenerated functional saccharifying enzyme; the number of recovery and reuse is 20 times, and the recovery treatment is carried out after each saccharification step. At the 20th application, the content of the tested glucose product is 96.17-97.09%, and the yield is 87.56-88.44%. DETAILED DESCRIPTION

[0048] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0049] Example 1

[0050] 1. Preparation of starch milk

[0051] The corn starch was added to deionized water for slurry preparation, and the mass concentration of the slurry after slurry preparation was controlled to be 18%, and then decyl glucoside was added, and the mixture was stirred evenly, and a 10 wt % hydrochloric acid solution was added to adjust the pH to 4.5, and the temperature was increased to 45° C., and the mixture was stirred for 1.0 h to obtain starch milk;

[0052] The mass of the decyl glucoside is 0.7% of the mass of corn starch.

[0053] 2. Liquefaction

[0054] Add high temperature resistant α-amylase to starch emulsion, the addition amount of high temperature resistant α-amylase is 0.32kg / t starch dry basis, the starch emulsion is sprayed and liquefied by a liquefaction ejector, the liquefaction temperature is 108°C, the liquefaction time is 42min, the pH of the liquefied liquid is controlled to be 5.5, the DE value is 13%, and the liquefied liquid is obtained by centrifugal separation.

[0055] 3. Saccharification

[0056] The temperature of the liquefied liquid was controlled at 60°C, the pH was adjusted to 4.6, and a functional saccharifying enzyme was added to carry out a saccharification reaction. The reaction time was 48.0 hours, the DE value was ≥98.0%, and the DX value was ≥96.5%, to obtain a saccharified liquid;

[0057] The mass ratio of the liquefied liquid to the functional saccharifying enzyme is 1000:1.7;

[0058] The method for preparing the functional saccharifying enzyme comprises the steps of preparing an enzyme carrier and loading the enzyme;

[0059] The steps of preparing the enzyme carrier are as follows: placing 10.0 g of diatomaceous earth in 70 g of 23 wt% sodium hydroxide solution, raising the temperature to 60° C., keeping warm and stirring for 3.0 h, filtering, washing, drying after the stirring is completed, and putting it into a muffle furnace for calcination, calcining at 220° C. for 1.0 h, and calcining at 360° C. for 3.2 h, and obtaining porous diatomaceous earth after the calcination is completed; placing 9.2 g of porous diatomaceous earth in 100 g of 36 wt% ethanol solution, adding 0.6 g of kH550, 0.5 g of kH602 and 1.0 g of triethanolamine, raising the temperature to 63° C., keeping warm and stirring for 4.0 h, filtering, washing, and drying after the reaction is completed to obtain aminodiatomaceous earth; placing 8.5 g of aminodiatomaceous earth in 100 g of acetonitrile, then adding 2.1 g of ethylene glycol diglycidyl ether, raising the temperature to 65° C., stirring for reaction for 6.0 h, filtering out and washing after the reaction is completed to obtain a primary carrier;

[0060] The particle size of the diatomaceous earth is 320 nm;

[0061] The enzyme loading step comprises: adding 4.0 g of pullulanase into 100 g of deionized water, adding 0.32 g of Tween 80, stirring evenly, adding 12.5 g of primary carrier, then adding 8.7 g of chitosan solution and continuing to stir evenly, then adding 6.4 g of citral solution, stirring and reacting at 33° C. for 3.0 h, adding 2.8 g of sodium alginate after the stirring reaction is completed, continuing to stir for 1.0 h, filtering and drying, and obtaining a functional saccharifying enzyme;

[0062] The chitosan solution is an acetic acid solution of chitosan, the volume concentration of the acetic acid solution is 2.0%, and the mass concentration of the chitosan solution is 2.5%;

[0063] The mass concentration of the citral solution is 2.3%.

[0064] 4. Filter

[0065] The saccharification liquid was filtered through a 0.5 mm mesh, the primary filtrate of the saccharification liquid was collected, and then the impurity remover was added, and stirred at 250 rpm for 10.0 min. After the stirring was completed, it was allowed to stand for 1.5 h. After the standing was completed, the secondary filtrate of the saccharification liquid was collected by filtration, and the temperature of the secondary filtrate of the saccharification liquid was increased to 40° C., and nanofiltration treatment was performed through a nanofiltration membrane, and the feed pressure was controlled to be 2.0 MPa, and the retention amount was 280 MW, to obtain the saccharification filtrate;

[0066] The mass ratio of the primary filtrate of the saccharified liquid to the impurity remover is 100:0.34;

[0067] The preparation method of the impurity remover is as follows: 9.2g of activated carbon is placed in 85g of 75wt% nitric acid solution, the temperature is increased to 68°C, the mixture is kept warm and stirred for 5.0h, after the end of the keeping warm and stirring, the mixture is filtered out and washed, and then put into 500g of deionized water, 4.1g of catalyst is added, and the mixture is stirred and catalyzed at room temperature for 30min, and then 3.8g of dodecylamine and 2.2g of glutamic acid are added, and the mixture is stirred and reacted at room temperature for 3.5h, and after filtering and washing, the mixture is put into 50g of chitosan solution, stirred at 35°C for 40min, filtered, washed, and dried to obtain the impurity remover;

[0068] The particle size of the activated carbon is 12 μm;

[0069] The catalyst is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2.4:1.7;

[0070] The chitosan solution is a mixture of chitosan and 5.0 wt % acetic acid solution, and the mass ratio of the chitosan to the 5.0 wt % acetic acid solution is 1:12.4.

[0071] 5. Post-processing

[0072] The saccharification filtrate is heated and concentrated to 50% of the original volume, and then crystallized and dried to obtain the glucose product.

[0073] The glucose product was prepared by the method of Example 1, with a glucose content of 99.68% and a yield of 90.25%;

[0074] After the saccharification step of Example 1 is completed, the functional saccharifying enzyme is recovered and reused. The recovery method is to centrifuge the saccharification liquid, wash it with deionized water, and dry it at 60°C to obtain regenerated functional saccharifying enzyme; the number of recovery and reuse is 20 times, and the recovery treatment is carried out after each saccharification step. At the 20th application, the content of the tested glucose product is 97.09%, and the yield is 88.44%.

[0075] Example 2

[0076] 1. Preparation of starch milk

[0077] The corn starch was added to deionized water for slurry preparation, and the mass concentration of the slurry after slurry preparation was controlled to be 16%, and then decyl glucoside was added, and the mixture was stirred evenly, and a 9.6wt% hydrochloric acid solution was added to adjust the pH to 4.4, and the temperature was increased to 43°C, and the mixture was stirred for 0.8h to obtain starch milk;

[0078] The mass of the decyl glucoside is 0.6% of the mass of corn starch.

[0079] 2. Liquefaction

[0080] Add high temperature resistant α-amylase to starch emulsion, the addition amount of high temperature resistant α-amylase is 0.30kg / t starch dry basis, the starch emulsion is sprayed and liquefied by a liquefaction ejector, the liquefaction temperature is 106°C, the liquefaction time is 40min, the pH of the liquefied liquid is controlled to be 5.4, the DE value is 12%, and the liquefied liquid is obtained by centrifugal separation.

[0081] 3. Saccharification

[0082] The temperature of the liquefied liquid was controlled at 58°C, the pH was adjusted to 4.5, and a functional saccharifying enzyme was added to carry out a saccharification reaction. The reaction time was 46.0 hours, the DE value was ≥98.0%, and the DX value was ≥96.5%, to obtain a saccharified liquid;

[0083] The mass ratio of the liquefied liquid to the functional saccharifying enzyme is 1000:1.5;

[0084] The method for preparing the functional saccharifying enzyme comprises the steps of preparing an enzyme carrier and loading the enzyme;

[0085] The steps of preparing the enzyme carrier are as follows: placing 9.8g of diatomaceous earth in 67g of 21wt% sodium hydroxide solution, raising the temperature to 58°C, keeping warm and stirring for 2.8h, filtering, washing and drying after the stirring is completed, and then putting it into a muffle furnace for calcination, calcining at 215°C for 1.2h, and calcining at 357°C for 3.0h, and obtaining porous diatomaceous earth after the calcination is completed; placing 9.0g of porous diatomaceous earth in 97g of 34wt% ethanol solution, adding 0.5g kH550, 0.4g kH602 and 0.8g triethanolamine, raising the temperature to 61°C, keeping warm and stirring for 3.8h, filtering, washing and drying after the reaction is completed, and obtaining aminodiatomaceous earth; placing 8.3g of aminodiatomaceous earth in 95g of acetonitrile, then adding 2.0g of ethylene glycol diglycidyl ether, raising the temperature to 63°C, stirring and reacting for 5.8h, filtering out and washing after the reaction is completed, and obtaining a primary carrier;

[0086] The particle size of the diatomaceous earth is 310 nm;

[0087] The enzyme loading step comprises: adding 3.7 g of pullulanase into 96 g of deionized water, adding 0.30 g of Tween 80, stirring evenly, adding 12.3 g of primary carrier, then adding 8.5 g of chitosan solution and continuing to stir evenly, then adding 6.2 g of citral solution, stirring and reacting at 32° C. for 3.2 h, after the stirring reaction is completed, adding 2.6 g of sodium alginate, continuing to stir for 0.8 h, filtering and drying, and obtaining a functional saccharifying enzyme;

[0088] The chitosan solution is an acetic acid solution of chitosan, the volume concentration of the acetic acid solution is 1.8%, and the mass concentration of the chitosan solution is 2.4%;

[0089] The mass concentration of the citral solution is 2.1%.

[0090] 4. Filter

[0091] The saccharification liquid was filtered through a 0.5 mm mesh, the primary filtrate of the saccharification liquid was collected, and then the impurity remover was added, and stirred at 240 rpm for 9.0 min. After the stirring was completed, it was allowed to stand for 1.4 h. After the standing was completed, the secondary filtrate of the saccharification liquid was collected by filtration, and the temperature of the secondary filtrate of the saccharification liquid was increased to 38° C., and nanofiltration treatment was performed through a nanofiltration membrane, and the feed pressure was controlled to be 1.8 MPa, and the retention amount was 270 MW, to obtain the saccharification filtrate;

[0092] The mass ratio of the primary filtrate of the saccharified liquid to the impurity remover is 100:0.32;

[0093] The preparation method of the impurity remover is as follows: 9.0g of activated carbon is placed in 82g of 73wt% nitric acid solution, the temperature is increased to 66°C, the mixture is kept warm and stirred for 4.8h, after the end of the keeping warm and stirring, the mixture is filtered out and washed, and then put into 490g of deionized water, 4.0g of catalyst is added, the mixture is stirred and catalyzed at room temperature for 28min, and then 3.6g of dodecylamine and 2.1g of glutamic acid are added, the mixture is stirred and reacted at room temperature for 3.2h, filtered and washed, and then put into 46g of chitosan solution, stirred at 34°C for 38min, filtered, washed, and dried to obtain the impurity remover;

[0094] The particle size of the activated carbon is 10 μm;

[0095] The catalyst is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2.2:1.5;

[0096] The chitosan solution is a mixture of chitosan and 5.0wt% acetic acid solution, and the mass ratio of the chitosan to the 5.0wt% acetic acid solution is 1:12.2.

[0097] 5. Post-processing

[0098] The saccharification filtrate is heated and concentrated to 50% of the original volume, and then crystallized and dried to obtain the glucose product.

[0099] The glucose product was prepared by the method of Example 2, with a glucose content of 99.56% and a yield of 90.18%;

[0100] After the saccharification step of Example 2 is completed, the functional saccharifying enzyme is recovered and reused. The recovery method is to centrifuge the saccharification liquid, wash it with deionized water, and dry it at 60°C to obtain regenerated functional saccharifying enzyme; the number of recovery and reuse is 20 times, and the recovery treatment is carried out after each saccharification step. At the 20th application, the content of the tested glucose product is 96.17%, and the yield is 87.56%.

[0101] Example 3

[0102] 1. Preparation of starch milk

[0103] The corn starch was added to deionized water for slurry preparation, and the mass concentration of the slurry after slurry preparation was controlled to be 20%, and then decyl glucoside was added, and the mixture was stirred evenly, and a 10.3 wt % hydrochloric acid solution was added to adjust the pH to 4.6, and the temperature was increased to 47° C., and the mixture was stirred for 1.2 h to obtain starch milk;

[0104] The mass of the decyl glucoside is 0.8% of the mass of corn starch.

[0105] 2. Liquefaction

[0106] Add high temperature resistant α-amylase to starch emulsion, the addition amount of high temperature resistant α-amylase is 0.34kg / t starch dry basis, the starch emulsion is sprayed and liquefied by a liquefaction injector, the liquefaction temperature is 110°C, the liquefaction time is 44min, the pH of the liquefied liquid is controlled to be 5.6, the DE value is 14%, and the liquefied liquid is obtained by centrifugal separation.

[0107] 3. Saccharification

[0108] The temperature of the liquefied liquid was controlled at 62°C, the pH was adjusted to 4.75, and a functional saccharifying enzyme was added to carry out a saccharification reaction. The reaction time was 50.0 h, the DE value was ≥98.0%, and the DX value was ≥96.5%, to obtain a saccharified liquid;

[0109] The mass ratio of the liquefied liquid to the functional saccharifying enzyme is 1000:2.05;

[0110] The method for preparing the functional saccharifying enzyme comprises the steps of preparing an enzyme carrier and loading the enzyme;

[0111] The steps of preparing the enzyme carrier are as follows: placing 10.2g of diatomaceous earth in 73g of 24wt% sodium hydroxide solution, raising the temperature to 62°C, keeping warm and stirring for 3.2h, filtering, washing and drying after the stirring is completed, and then putting it into a muffle furnace for calcination, calcining at 225°C for 0.8h, and calcining at 363°C for 3.4h, and obtaining porous diatomaceous earth after the calcination is completed; placing 9.5g of porous diatomaceous earth in 105g of 37wt% ethanol solution, adding 0.8g kH550, 0.6g kH602 and 1.2g of triethanolamine, raising the temperature to 651°C, keeping warm and stirring for 4.2h, filtering, washing and drying after the reaction is completed, and obtaining aminodiatomaceous earth; placing 8.7g of aminodiatomaceous earth in 105g of acetonitrile, and then adding 2.2g of ethylene glycol diglycidyl ether, raising the temperature to 67°C, stirring and reacting for 6.2h, filtering out and washing after the reaction is completed, and obtaining a primary carrier;

[0112] The particle size of the diatomaceous earth is 330 nm;

[0113] The enzyme loading step comprises: adding 4.2 g of pullulanase into 105 g of deionized water, adding 0.34 g of Tween 80, stirring evenly, adding 12.7 g of primary carrier, then adding 8.8 g of chitosan solution and continuing to stir evenly, then adding 6.6 g of citral solution, stirring and reacting at 35° C. for 2.8 h, adding 3.0 g of sodium alginate after the stirring reaction is completed, continuing to stir for 1.2 h, filtering and drying, and obtaining a functional saccharifying enzyme;

[0114] The chitosan solution is an acetic acid solution of chitosan, the volume concentration of the acetic acid solution is 2.2%, and the mass concentration of the chitosan solution is 2.6%;

[0115] The mass concentration of the citral solution is 2.5%.

[0116] 4. Filter

[0117] The saccharification liquid was filtered through a 0.5 mm mesh, and the primary filtrate of the saccharification liquid was collected. Then, an impurity remover was added, and the mixture was stirred at 260 rpm for 12.0 min. After the stirring was completed, the mixture was allowed to stand for 1.6 h. After the standing was completed, the secondary filtrate of the saccharification liquid was collected by filtration. The temperature of the secondary filtrate of the saccharification liquid was raised to 42° C., and the filtrate was treated by nanofiltration through a nanofiltration membrane. The feed pressure was controlled to be 2.2 MPa, and the retention amount was 300 MW to obtain the saccharification filtrate.

[0118] The mass ratio of the primary filtrate of the saccharified liquid to the impurity remover is 100:0.36;

[0119] The preparation method of the impurity remover is as follows: 9.4g of activated carbon is placed in 88g of 77wt% nitric acid solution, the temperature is increased to 70°C, the mixture is kept warm and stirred for 5.2h, after the end of the keeping warm and stirring, the mixture is filtered out and washed, and then put into 5100g of deionized water, 4.2g of catalyst is added, and the mixture is stirred and catalyzed at room temperature for 32min, and then 4.0g of dodecylamine and 2.3g of glutamic acid are added, and the mixture is stirred and reacted at room temperature for 3.7h, and after filtering and washing, the mixture is put into 53g of chitosan solution, stirred at 36°C for 42min, filtered, washed, and dried to obtain the impurity remover;

[0120] The particle size of the activated carbon is 14 μm;

[0121] The catalyst is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2.6:1.8;

[0122] The chitosan solution is a mixture of chitosan and 5.0 wt % acetic acid solution, and the mass ratio of the chitosan to the 5.0 wt % acetic acid solution is 1:12.5.

[0123] 5. Post-processing

[0124] The saccharification filtrate is heated and concentrated to 50% of the original volume, and then crystallized and dried to obtain the glucose product.

[0125] The glucose product was prepared by the method of Example 3, with a glucose content of 99.63% and a yield of 90.22%;

[0126] After the saccharification step of Example 3 is completed, the functional saccharifying enzyme is recovered and reused. The recovery method is to centrifuge the saccharification liquid, wash it with deionized water, and dry it at 60°C to obtain regenerated functional saccharifying enzyme; the number of recovery and reuse is 20 times, and the recovery treatment is carried out after each saccharification step. At the 20th application, the content of the tested glucose product is 96.64%, and the yield is 87.96%.

[0127] Comparative Example 1

[0128] Based on Example 1, the changes are as follows:

[0129] In the process of preparing functional saccharifying enzyme, the step of preparing the enzyme carrier is as follows: 10.0g of diatomaceous earth is placed in 70g of 23wt% sodium hydroxide solution, the temperature is raised to 60°C, and the solution is stirred for 3.0h at the temperature. After the stirring is completed, the solution is filtered, washed, and dried, and then placed in a muffle furnace for calcination. The solution is calcined at 220°C for 1.0h and at 360°C for 3.2h. After the calcination is completed, porous diatomaceous earth is obtained, which is the primary carrier; the particle size of the diatomaceous earth is 320nm;

[0130] During the filtration process, the preparation method of the impurity remover is as follows: 9.2g of activated carbon is placed in 85g of 75wt% nitric acid solution, the temperature is raised to 68°C, and the mixture is stirred for 5.0h. After the stirring is completed, the mixture is filtered out, washed, and dried to obtain the impurity remover; the particle size of the activated carbon is 12μm;

[0131] The rest of the operations are the same.

[0132] The glucose product was prepared by the method of Comparative Example 1, with a glucose content of 89.65% and a yield of 80.14%;

[0133] After the saccharification step of Comparative Example 1, the functional saccharifying enzyme was recovered and reused. The recovery method was to centrifuge the saccharification liquid, wash it with deionized water, and dry it at 60°C to obtain regenerated functional saccharifying enzyme. The number of recovery and reuse was 20 times, and the recovery treatment was carried out after each saccharification step. At the 20th application, the content of the tested glucose product was 73.60%, and the yield was 66.52%.

[0134] Comparative Example 2

[0135] Based on Example 1, the changes are as follows:

[0136] In the process of preparing the functional saccharifying enzyme, the enzyme loading step is to add 4.0g of pullulanase into 100g of deionized water, add 0.32g of Tween 80, stir evenly, add 12.5g of the primary carrier, filter and dry to obtain the functional saccharifying enzyme;

[0137] During the filtration process, the preparation method of the impurity remover is to add activated carbon into 200g chitosan solution, stir at 35°C for 40min, filter, wash and dry to obtain the impurity remover; the particle size of the activated carbon is 12μm;

[0138] The rest of the operations are the same.

[0139] The glucose product was prepared by the method of Comparative Example 2, with a glucose content of 92.47% and a yield of 82.35%;

[0140] After the saccharification step of Comparative Example 2, the functional saccharifying enzyme was recovered and reused. The recovery method was to centrifuge the saccharification liquid, wash it with deionized water, and dry it at 60°C to obtain regenerated functional saccharifying enzyme. The number of recovery and reuse was 20 times, and the recovery treatment was carried out after each saccharification step. At the 20th application, the content of the tested glucose product was 76.94%, and the yield was 69.50%.

[0141] The invention adopts corn starch to prepare glucose, controls a suitable DE value in the liquefaction step, ensures sufficient hydrolysis of starch without causing excessive hydrolysis, and thus enables a saccharification reaction to be fully carried out; in the saccharification step, adopts a carrier to solidify the enzyme, specifically adopts an amino group to modify diatomite, then adopts an epoxy group to react with the amino group on the surface of the diatomite, and then adsorbs the enzyme, adds a chitosan solution, introduces an aldehyde group for cross-linking, forms a stable cross-linking structure, effectively avoids the loss of the enzyme, ensures the activity of the enzyme, improves the stability of the functionalized enzyme, thereby realizing the repeated use of the enzyme, and promoting the saccharification reaction; when removing impurities, the protein has a large volume and structure, and its surface contains polar groups, and the glucose Due to the characteristics of sugar surface charge and less polar groups, activated carbon is used as a porous adsorption material, which can effectively remove pigments in the saccharification liquid. The activated carbon is subjected to specific treatment, and then amino groups are introduced for amidation reaction. The long-chain alkyl groups of dodecylamine are well combined with the hydrophobic groups of proteins, effectively repelling small molecules such as glucose. In addition, the amide groups and chitosan can enhance the interaction with proteins and improve the adsorption stability. Ultimately, it can achieve effective adsorption of proteins in the saccharification liquid, while having a repelling effect on molecules such as glucose, thereby effectively removing impurities from the saccharification liquid. After steps such as concentration, crystallization, and drying, high-quality glucose products are obtained with high yield and purity, good stability of functional saccharifying enzymes, and less energy consumption in the preparation method.

[0142] In the step of preparing the enzyme carrier of Comparative Example 1, diatomaceous earth is treated with sodium hydroxide solution and then calcined to obtain a primary carrier. The primary carrier is used to load the enzyme, and then chitosan and citral are added for cross-linking, and then treated with sodium alginate. The diatomaceous earth is easy to agglomerate and cannot effectively load the enzyme. In addition, the cross-linking of chitosan and citral is not complete, which affects the activity and recovery performance of the functional enzyme. In the process of removing impurities, it is only activated carbon treated with nitric acid, and its adsorption of proteins and pigments is incomplete. Finally, the saccharification reaction of Comparative Example 1 is insufficient, and the impurity removal is incomplete, which affects the yield and purity of glucose, and the recovery of the functional saccharifying enzyme is poor, and there are many impurities.

[0143] In Comparative Example 2, a primary carrier is used to load the enzyme to obtain a functional saccharifying enzyme, which has not been cross-linked, and the enzyme immobilization is unstable, which affects the recovery performance of the enzyme. In addition, in the process of preparing the impurity remover, activated carbon is added to the chitosan solution, so that the protein molecules are adsorbed on the surface of the activated carbon, but the adsorption of the protein is incomplete, and the adsorption of glucose cannot be avoided, which will ultimately reduce the yield and purity of the product.

[0144] Unless otherwise specified, all ratios and percentages described in the present invention are by mass ratios and percentages are by mass percentages.

[0145] 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 is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing glucose, characterized in that: It includes the steps of preparing starch milk, liquefaction, saccharification, filtration and post-processing; The liquefaction step comprises adding a thermostable α-amylase to the starch milk, wherein the amount of the thermostable α-amylase added is 0.30-0.34 kg / t starch dry basis, spraying and liquefying the starch milk through a liquefaction ejector, wherein the liquefaction temperature is 106-110° C., the liquefaction time is 40-44 min, the pH of the liquefied liquid is controlled to be 5.4-5.6, the DE value is 12-14%, and the liquefied liquid is obtained by centrifugation; The saccharification step comprises controlling the temperature of the liquefied liquid to 58-62° C., adjusting the pH to 4.5-4.7, adding a functional saccharifying enzyme to carry out a saccharification reaction, the reaction time is 46.0-50.0 h, the DE value is ≥98.0%, and the DX value is ≥96.5%, to obtain a saccharified liquid; The method for preparing the functional saccharifying enzyme comprises the steps of preparing an enzyme carrier and loading the enzyme; The steps of preparing the enzyme carrier are as follows: placing porous diatomaceous earth in an ethanol solution, adding kH550, kH602 and triethanolamine, raising the temperature to 61-65° C., keeping warm and stirring for 3.8-4.2 hours, filtering, washing and drying after the reaction is completed to obtain aminodiatomaceous earth; placing the aminodiatomaceous earth in acetonitrile, then adding ethylene glycol diglycidyl ether, raising the temperature to 63-67° C., stirring and reacting for 5.8-6.2 hours, filtering and washing after the reaction is completed to obtain a primary carrier; The enzyme loading step comprises: adding pullulanase into deionized water, adding Tween 80, stirring evenly, adding a primary carrier, then adding a chitosan solution and continuing to stir evenly, then adding a citral solution, stirring and reacting at 32-35° C. for 2.8-3.2 hours, adding sodium alginate after the stirring reaction is completed, continuing to stir for 0.8-1.2 hours, filtering and drying, and obtaining a functional saccharifying enzyme; The filtering step comprises filtering the saccharification liquid through a 0.5 mm mesh, collecting the primary filtrate of the saccharification liquid, adding an impurity remover, filtering, collecting the secondary filtrate of the saccharification liquid, and performing nanofiltration treatment to obtain the saccharification filtrate; The preparation method of the impurity remover comprises the following steps: placing activated carbon in a nitric acid solution, raising the temperature to 66-70°C, stirring at the temperature for 4.8-5.2 hours, filtering out and washing after the stirring, placing the activated carbon in deionized water, adding a catalyst, stirring and catalyzing at room temperature for 28-32 minutes, then adding dodecylamine and glutamic acid, stirring and reacting at room temperature for 3.2-3.7 hours, filtering and washing, placing the activated carbon in a chitosan solution, stirring at 34-36°C for 38-42 minutes, filtering, washing, and drying to obtain the impurity remover.

2. The method for preparing glucose according to claim 1, characterized in that: In the saccharification step, the mass ratio of the liquefied liquid to the functional saccharifying enzyme is 1000:1.5-2.

0.

3. The method for preparing glucose according to claim 1, characterized in that: In the step of preparing the enzyme carrier, the porous diatomaceous earth is prepared by placing the diatomaceous earth in a sodium hydroxide solution, raising the temperature to 58-62° C., keeping the temperature and stirring for 2.8-3.2 hours, filtering, washing and drying after the stirring, and putting the diatomaceous earth into a muffle furnace for calcination at 215-225° C. for 0.8-1.2 hours, and at 357-363° C. for 3.0-3.4 hours. After the calcination, the porous diatomaceous earth is obtained; the particle size of the diatomaceous earth is 310-330 nm; The mass ratio of the diatomaceous earth to the sodium hydroxide solution is 9.8-10.2:67-73; The mass concentration of the sodium hydroxide solution is 21-24%.

4. The method for preparing glucose according to claim 1, characterized in that: In the step of preparing the enzyme carrier, the mass ratio of the porous diatomaceous earth, ethanol solution, kH550, kH602, and triethanolamine is 9.0-9.5:97-105:0.5-0.8:0.4-0.6:0.8-1.2; The mass concentration of the ethanol solution is 34-37%; The mass ratio of aminodiatomite, acetonitrile and ethylene glycol diglycidyl ether is 8.3-8.7:95-105:2.0-2.2; In the enzyme loading step, the mass ratio of the pullulanase, deionized water, Tween 80, primary carrier, chitosan solution, citral solution, and sodium alginate is 3.7-4.2:96-105:0.30-0.34:12.3-12.7:8.5-8.8:6.2-6.6:2.6-3.0; The chitosan solution is an acetic acid solution of chitosan, the volume concentration of the acetic acid solution is 1.8-2.2%, and the mass concentration of the chitosan solution is 2.4-2.6%; The mass concentration of the citral solution is 2.1-2.5%.

5. The method for preparing glucose according to claim 1, characterized in that: The filtering step comprises filtering the saccharification liquid through a 0.5 mm mesh, collecting the primary filtrate of the saccharification liquid, adding an impurity remover, stirring at 240-260 rpm for 9.0-12.0 min, standing for 1.4-1.6 h after the stirring, collecting the secondary filtrate of the saccharification liquid by filtering, raising the temperature of the secondary filtrate of the saccharification liquid to 38-42° C., performing nanofiltration treatment through a nanofiltration membrane, controlling the feed pressure to 1.8-2.2 MPa, and the retention amount to 270-300 MW, to obtain the saccharification filtrate; The mass ratio of the primary filtrate of the saccharified liquid to the impurity remover is 100:0.32-0.

36.

6. The method for preparing glucose according to claim 1, characterized in that: In the preparation method of the impurity remover, the particle size of the activated carbon is 10-14 μm; The mass concentration of the nitric acid solution is 73-77%; The catalyst is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2.2-2.6:1.5-1.8; The chitosan solution is a mixture of chitosan and 5.0wt% acetic acid solution, and the mass ratio of the chitosan to the 5.0wt% acetic acid solution is 1:12.2-12.5; The mass ratio of the activated carbon, nitric acid solution, deionized water, catalyst, dodecylamine, glutamic acid and chitosan solution is 9.0-9.4:82-88:490-510:4.0-4.2:3.6-4.0:2.1-2.3:46-53.

7. The method for preparing glucose according to claim 1, characterized in that: The step of preparing starch milk is to add corn starch to deionized water for slurry adjustment, control the mass concentration of the slurry after slurry adjustment to be 16-20%, then add decyl glucoside, continue to stir evenly, add 9.6-10.3wt% hydrochloric acid solution to adjust the pH to 4.4-4.6, increase the temperature to 43-47°C, keep warm and stir for 0.8-1.2h, and obtain starch milk; The mass of the decyl glucoside is 0.6-0.8% of the mass of corn starch.

8. The method for preparing glucose according to claim 1, characterized in that: The post-treatment step is to heat and concentrate the saccharification filtrate to 50% of the original volume, and then crystallize and dry it to obtain a glucose product.