A method for preparing high-concentration isomaltooligosaccharide using cyclodextrin waste mother liquor
By adding β-amylase and transglucosidase to the discarded mother liquor of β-cyclodextrin crystals, high concentration of oligoisomaltose is prepared, solving the problems of waste of resources and high production costs, and achieving efficient recovery of β-cyclodextrin and efficient preparation of oligoisomaltose.
Patent Information
- Application Number
- CN202411901965.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-04-08
AI Technical Summary
In the existing β-cyclodextrin production process, the discarded mother liquor of β-cyclodextrin crystals cannot be effectively utilized, resulting in waste of resources and increased production costs.
High concentration of oligoisomaltose is prepared by adding β-amylase and transglucosidase to the discarded mother liquor of β-cyclodextrin crystallization, performing acid/base regulation and heat treatment, and further shearing and saccharification of oligosaccharides.
The preparation of high concentration of oligoisomal isomaltose is achieved, which improves the recovery rate of β-cyclodextrin, reduces production costs, and increases corporate profits.
Smart Images
Figure BDA0005203431320000041 
Figure BDA0005203431320000051
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biochemical engineering, and in particular to a method for preparing high-concentration isomaltooligosaccharide by utilizing cyclodextrin waste mother liquor. Background Art
[0002] Isomaltooligosaccharide (IMO) is also known as isomaltooligosaccharide, isomaltooligosaccharide, branched oligosaccharide, etc. Its main components are isomaltose, panose, isomaltotriose and oligosaccharides above tetrasaccharide, which are bound by α-1,6 glycosidic bonds between glucose molecules. Because it can promote the significant proliferation of bifidobacteria in the human body, has the function of water-soluble dietary fiber, low calorific value, and anti-caries, it is a widely used functional oligosaccharide. According to the definition in "GB / T 20881-2017 Isomaltooligosaccharide", the total content of isomaltooligosaccharide>50%, the total content of isomaltose, panose, and isomaltotriose>35% is IM0-50 type products, and the total content of isomaltooligosaccharide>90%, the total content of isomaltose, panose, and isomaltotriose>45% is IM0-90 type products.
[0003] β-amylase, also known as starch β-1,4-maltosidase, is a type of amylase, which is widely found in higher plants such as barley, wheat, sweet potato, soybean, and microorganisms such as Bacillus. It is the main saccharifying agent for beer brewing and maltose (maltose syrup) production. By using β-amylase produced by microorganisms such as Bacillus polymyxa and Bacillus megaterium to saccharify starch raw materials that have been acidified or liquefied by α-amylase, high maltose syrup with a maltose content of 60%-70% can be produced.
[0004] Transglucosidase is widely present in animals, plants and microorganisms. It can hydrolyze the α-1,4-glucosidic bonds of oligosaccharides and polysaccharides from the non-reducing end, and can also act on the α-1,6-glycosidic bonds. It can catalyze the transglycosidation reaction in a high glucosidase receptor environment, that is, transfer the glucose group to substrate molecules such as maltose and glucose to obtain oligomaltose. Currently, the only host allowed to produce transglucosidase in "GB2760-2016" is Aspergillus niger.
[0005] The waste mother liquor of β-cyclodextrin crystallization is that some cyclodextrin will remain in the process of β-cyclodextrin crystallization and purification. In addition, 45%-60% of starch that has not generated β-cyclodextrin is cut into oligosaccharides by β-cyclodextrin glucosyltransferase and high temperature resistant a-amylase / saccharifying enzyme and remains in the mother liquor.
[0006] At present, β-cyclodextrin is mainly produced by enzymatic method or fermentation method. The enzymatic production process mainly includes: liquefaction, cyclization, organic solvent recovery, secondary liquefaction, activated carbon impurity removal, crystallization, drying, crushing and other processes. The fermentation method is essentially an enzymatic method, but after the fermentation of the enzyme liquid is completed, the fermentation liquid is directly added to the starch milk for subsequent operation without the refining step. The fermentation method will introduce a large amount of culture medium and bacterial impurities. At present, in large-scale production, different starch raw materials are used to prepare β-cyclodextrin, and the reaction yield is between 40% and 55%. Due to the high impurity content, the mother liquor of the first crystallization is generally directly discarded for sewage treatment or used as a fermentation raw material. In addition, because the discarded mother liquor also contains 3-10g / L of β-cyclodextrin, direct disposal will cause economic losses. Therefore, the existing mother liquor treatment method will not only cause a huge waste of resources, but also increase the production cost of enterprises due to sewage treatment. Summary of the invention
[0007] The invention solves the problems in the prior art and provides a method for preparing high-concentration isomaltooligosaccharide by utilizing waste mother liquor of beta-cyclodextrin crystallization.
[0008] The preparation method of the high-concentration isomaltooligosaccharide comprises the following steps:
[0009] (1) adding acid / base to the waste mother liquor of β-cyclodextrin crystallization with a mass concentration of 10-20% to adjust the pH to 4-8;
[0010] (2) heating the slurry obtained in step (1) to 40-65° C., adding β-amylase and transglucosidase thereto, and reacting for 20-48 hours;
[0011] (3) adding 2-8% activated carbon to the reaction solution obtained in step (2) and heating to 95° C. for adsorption for 1 h, filtering to remove the activated carbon, then evaporating and concentrating at 95-100° C. to 1 / 3-2 / 3 of the original weight, and then cooling and crystallizing;
[0012] (4) The crystals obtained in step (3) are filtered to separate the solids, washed to remove impurities, and dried to obtain the precipitate to obtain β-cyclodextrin. The washing water is added to the mother liquor to simultaneously perform chromatographic separation to remove reducing sugars, and then concentrated until the dry matter content is not less than 75%.
[0013] Further preferably, the optimum pH in step (1) is 4.5-6.
[0014] In step (2), the optimum reaction temperature is 55-60° C., the β-amylase and the transglucosidase are any one of liquid, powder or immobilized enzyme, the immobilized enzyme is a macroporous adsorption resin immobilized enzyme, the addition amount of the β-amylase is 200-1000 U / g dry matter, and the addition amount of the transglucosidase is 5-30 U / g dry matter.
[0015] The most suitable amount of activated carbon added in step (3) is 4-6%.
[0016] The filtration method used in step (4) is any one of tubular centrifugation, drum centrifugation, suction filtration, and plate and frame filtration, and the concentration method used is any one of evaporation, freeze drying, and membrane separation.
[0017] The present invention provides a method for preparing oligomeric isomaltosose using the waste mother liquor of β-cyclodextrin crystallization, wherein β-amylase is added to the waste mother liquor to further shear the long-chain oligosaccharides in the mother liquor into maltose, and transglucosidase is added to link the glucose groups in the mother liquor to substrate molecules such as maltose and glucose in the form of α-1,6 glycosidic bonds to obtain oligomeric isomaltosose. The method is conducive to increasing the yield of β-cyclodextrin, and also obtains high-concentration oligomeric isomaltosose, which reduces production costs while increasing corporate profits, and has broad application prospects. DETAILED DESCRIPTION
[0018] The present invention is further described in conjunction with specific implementation cases, and those skilled in the art can better understand and grasp the present invention by this, rather than limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.
[0019] The β-amylase and transglucosidase used were both from Weifang Kangdian Biotechnology Co., Ltd., with enzyme activities of 70 W U / mL for β-amylase and 30 W U / mL for transglucosidase, respectively. The macroporous adsorption resin used was from Tianjin Nankai Hecheng Technology Co., Ltd., model NKA.
[0020] The implementation steps of the control group are as follows:
[0021] Weigh 800g of β-cyclodextrin crystallization mother liquor (15% DS) and place it in a 1000ml four-necked flask, adjust the pH to 6.0, add transglucosidase at a dry matter ratio of 30U / g; after stirring evenly, maintain 300rpm and start heating to 60°C, and catalyze the reaction for 48h.
[0022] Add 4% activated carbon to the slurry according to the dry matter ratio, heat to 95°C for 1h, filter to remove the activated carbon, then evaporate and concentrate 260ml of water at 95-100°C, then cool and crystallize. The solids are washed, dried and weighed, and 4.5g of precipitate is harvested. The precipitate contains 4.3% water and 96.5% purity after testing, and the recovery rate of β-cyclodextrin is 3.45% (based on dry matter).
[0023] The washing water is added to the mother liquor to simultaneously perform chromatographic separation to remove reducing sugars, and then evaporated and concentrated until the dry matter content is 75%. Samples are taken and the content of oligomeric isomaltosaccharides in the slurry is detected by HPLC.
[0024] Example 1
[0025] Weigh 800g of β-cyclodextrin crystallization mother liquor (18% DS) and place it in a 1000ml four-necked flask, adjust the pH to 6.0, add β-amylase and transglucosidase at a dry matter ratio of 1000U / g and 5U / g respectively; stir evenly and maintain 300rpm, start heating to 60℃, and catalyze the reaction for 48h.
[0026] Add 6% activated carbon to the slurry according to the dry matter ratio, heat to 95°C for 1h, filter to remove the activated carbon, then evaporate and concentrate 260ml of water at 95-100°C, then cool and crystallize. The solids are washed, dried and weighed, and 6g of precipitate is harvested. The precipitate contains 3% water and 98.6% purity after testing, and the recovery rate of β-cyclodextrin is 3.985% (based on dry matter).
[0027] The washing water is added to the mother liquor to simultaneously perform chromatographic separation to remove reducing sugars, and then evaporated and concentrated until the dry matter content is 75%. Samples are taken and the content of oligomeric isomaltosaccharides in the slurry is detected by HPLC.
[0028] Example 2
[0029] Weigh 4 kg of β-cyclodextrin crystallization mother liquor (10% DS) and place it in a 5L fermentation tank, adjust the pH to 5.0, add β-amylase and transglucosidase at a dry matter ratio of 500 U / g and 15 U / g respectively; stir evenly and maintain 300 rpm, start heating to 55°C, and catalyze the reaction for 36 hours.
[0030] Add 4% activated carbon to the slurry according to the dry matter ratio, heat to 95℃ and keep adsorbing for 1h, filter to remove the activated carbon, then evaporate and concentrate 2.7L water at 95-100℃, then cool and crystallize. The solids are washed, dried and weighed, and 16.8g of precipitate is harvested. The precipitate contains 5% water and 97.9% purity after testing, and the recovery rate of β-cyclodextrin is 3.91% (based on dry matter).
[0031] The washing water is added to the mother liquor to simultaneously perform chromatographic separation to remove reducing sugars, and then evaporated and concentrated until the dry matter content is 75%. Samples are taken and the content of oligomeric isomaltosaccharides in the slurry is detected by HPLC.
[0032] Example 3
[0033] Weigh 4 kg of β-cyclodextrin crystallization mother liquor (15% DS) and place it in a 5L four-necked flask, adjust the pH to 5.5, add β-amylase and transglucosidase at a dry matter ratio of 200 U / g and 30 U / g respectively; stir evenly and maintain 300 rpm, start heating to 50°C, and catalyze the reaction for 20 hours.
[0034] Add 5% activated carbon to the slurry according to the dry matter ratio, heat to 95°C for 1h, filter to remove the activated carbon, then evaporate and concentrate 1.3L of water at 95-100°C, and then cool and crystallize. The solids were washed, dried and weighed, and 15.3g of precipitate was harvested. The precipitate was tested to have a water content of 6.7%, a purity of 96.8%, and a recovery rate of β-cyclodextrin of 3.455% (based on dry matter).
[0035] The washing water is added to the mother liquor to simultaneously perform chromatographic separation to remove reducing sugars, and then evaporated and concentrated until the dry matter content is 75%. Samples are taken and the content of oligomeric isomaltosaccharides in the slurry is detected by HPLC.
[0036] Example 4
[0037] Weigh 4kg of β-cyclodextrin crystallization mother liquor (20% DS) and place it in a 5L fermenter, adjust the pH to 4.5, add immobilized β-amylase and transglucosidase at a dry matter ratio of 1000U / g and 30U / g respectively; stir evenly and maintain 300rpm, start heating to 55℃, and catalyze the reaction for 36h. After the reaction is completed, filter out the immobilized enzyme, and determine that the residual enzyme activity of the immobilized β-amylase is 99.2%, and the residual enzyme activity of the immobilized transglucosidase is 98.9%.
[0038] Add 6% activated carbon to the slurry according to the dry matter ratio, heat to 95°C for 1h, filter to remove the activated carbon, then evaporate and concentrate 2L of water at 95-100°C, and then cool and crystallize. The solids were washed, dried and weighed, and 14.7g of precipitate was harvested. The precipitate was tested to contain 3.5% water, 97.9% purity, and 2.89% recovery of β-cyclodextrin (based on dry matter).
[0039] The washing water is added to the mother liquor to simultaneously perform chromatographic separation to remove reducing sugars, and then evaporated and concentrated until the dry matter content is 75%. Samples are taken and the content of oligomeric isomaltosaccharides in the slurry is detected by HPLC.
[0040] Table 1. Content of oligosaccharide in control group and examples
[0041]
[0042]
[0043] It can be seen from the above experimental results that the method for preparing high-concentration isomaltooligosaccharide provided by the present invention can make full use of the waste crystallization mother liquor of β-cyclodextrin to prepare IMO-90 high-concentration isomaltooligosaccharide with an isomaltooligosaccharide content of >90, and at the same time increase the recovery rate of β-cyclodextrin by 2.89%, achieving unexpected technical effects.
[0044] The present invention provides a method for preparing oligomeric isomaltosose using the waste mother liquor of β-cyclodextrin crystallization, wherein β-amylase is added to the waste mother liquor to further shear the long-chain oligosaccharides in the mother liquor into maltose, and transglucosidase is added to link the glucose groups in the mother liquor to substrate molecules such as maltose and glucose in the form of α-1,6 glycosidic bonds to obtain oligomeric isomaltosose. The method is conducive to increasing the yield of β-cyclodextrin, and also obtains high-concentration oligomeric isomaltosose, which reduces production costs while increasing corporate profits, and has broad application prospects.
Claims
1. A method for preparing high-concentration isomaltooligosaccharide using cyclodextrin waste mother liquor, characterized in that: The method comprises the following steps: (1) adding acid / base to the waste mother liquor of β-cyclodextrin crystallization with a mass concentration of 10-20% to adjust the pH to 4-8; (2) heating the slurry obtained in step (1) to 40-65° C., adding β-amylase and transglucosidase thereto, and reacting for 20-48 hours before the reaction is completed; (3) adding 2-8% activated carbon to the reaction solution obtained in step (2) and heating it to 95°C for adsorption for 1 hour, filtering to remove the activated carbon, then evaporating and concentrating at 95-100°C to 1 / 3-2 / 3 of the original weight, and then cooling and crystallizing; (4) The crystals obtained in step (3) are filtered to separate the solids, washed to remove impurities, and dried to obtain the precipitate to obtain β-cyclodextrin. The washing water is added to the mother liquor to simultaneously perform chromatographic separation to remove reducing sugars such as glucose, maltose, and maltotriose, and then concentrated until the dry matter content is greater than 75%.
2. The method according to claim 1, characterized in that The optimum pH in step (1) is 4.5-6.
3. The method according to claim 1 or 2, characterized in that The optimum reaction temperature in step (2) is 55-60°C.
4. The method according to any one of claims 1 to 3, characterized in that: In step (2), the β-amylase and transglucosidase are any one of liquid, powder or immobilized enzyme, wherein the immobilized enzyme is a macroporous adsorption resin immobilized enzyme.
5. The method according to any one of claims 1 to 4, characterized in that: In step (2), the amount of β-amylase added is 200-1000 U / g dry matter.
6. The method according to claims 1 to 5, characterized in that In step (2), the amount of transglucosidase added is 5-30 U / g dry matter.
7. The method according to claims 1 to 6, characterized in that The most suitable amount of activated carbon added in step (3) is 4-6%.
8. The method according to claims 1 to 7, characterized in that The filtration method used in step (4) is any one of tubular centrifugation, drum centrifugation, suction filtration, and plate and frame filtration.
9. The method according to claims 1 to 8, characterized in that The concentration method used in step (4) is any one of evaporation, freeze drying and membrane separation.