CN111454305A - Method for preparing, separating and purifying lactulose by directionally assisting lactose isomerization through phenylboronic acid-based carrier - Google Patents
Method for preparing, separating and purifying lactulose by directionally assisting lactose isomerization through phenylboronic acid-based carrier Download PDFInfo
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- JCQLYHFGKNRPGE-FCVZTGTOSA-N lactulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-FCVZTGTOSA-N 0.000 title claims abstract description 126
- 229960000511 lactulose Drugs 0.000 title claims abstract description 126
- PFCRQPBOOFTZGQ-UHFFFAOYSA-N lactulose keto form Natural products OCC(=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O PFCRQPBOOFTZGQ-UHFFFAOYSA-N 0.000 title claims abstract description 126
- HXITXNWTGFUOAU-UHFFFAOYSA-N phenylboronic acid Chemical compound OB(O)C1=CC=CC=C1 HXITXNWTGFUOAU-UHFFFAOYSA-N 0.000 title claims abstract description 125
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 title claims abstract description 76
- 239000008101 lactose Substances 0.000 title claims abstract description 76
- 238000006317 isomerization reaction Methods 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- 238000001179 sorption measurement Methods 0.000 claims abstract description 23
- 238000003795 desorption Methods 0.000 claims abstract description 19
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- 238000000746 purification Methods 0.000 claims description 14
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- 229920006395 saturated elastomer Polymers 0.000 claims description 5
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- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
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- 239000010452 phosphate Substances 0.000 claims description 3
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims 2
- 239000003929 acidic solution Substances 0.000 claims 2
- 230000001476 alcoholic effect Effects 0.000 claims 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims 2
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims 1
- 239000000920 calcium hydroxide Substances 0.000 claims 1
- 229910001861 calcium hydroxide Inorganic materials 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000001035 drying Methods 0.000 claims 1
- -1 etc. Chemical class 0.000 claims 1
- 239000004310 lactic acid Substances 0.000 claims 1
- 235000014655 lactic acid Nutrition 0.000 claims 1
- 239000002122 magnetic nanoparticle Substances 0.000 claims 1
- 150000007522 mineralic acids Chemical class 0.000 claims 1
- 239000011259 mixed solution Substances 0.000 claims 1
- 150000007524 organic acids Chemical class 0.000 claims 1
- 241000041834 Orthocis Species 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 3
- 239000007788 liquid Substances 0.000 description 13
- 238000002360 preparation method Methods 0.000 description 13
- 238000004519 manufacturing process Methods 0.000 description 9
- 239000000126 substance Substances 0.000 description 8
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000006227 byproduct Substances 0.000 description 5
- 150000001720 carbohydrates Chemical class 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000005715 Fructose Substances 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
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- 229910021641 deionized water Inorganic materials 0.000 description 3
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- 239000000843 powder Substances 0.000 description 3
- 238000000967 suction filtration Methods 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 125000005619 boric acid group Chemical group 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 235000013402 health food Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000011403 purification operation Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 108030002154 Cellobiose epimerases Proteins 0.000 description 1
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-fructofuranose Chemical group OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 1
- 201000010538 Lactose Intolerance Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 239000000706 filtrate Substances 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
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- 239000012982 microporous membrane Substances 0.000 description 1
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- 150000002772 monosaccharides Chemical class 0.000 description 1
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- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229910001388 sodium aluminate Inorganic materials 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
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- 239000007921 spray Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/04—Disaccharides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses a method for preparing lactulose by directionally assisting lactose isomerization by using a phenylboronic acid-based polymer carrier and simultaneously separating and purifying the product lactulose. According to the invention, firstly, the specific affinity adsorption effect between the phenylboronic acid functional group loaded by the phenylboronic acid-based polymer carrier and the lactulose containing an ortho-cis hydroxyl structure is utilized to realize the directional selective adsorption of the lactulose in a lactose isomerization system, so that the isomerization reaction is promoted to be carried out in the direction of generating the lactulose, the conversion rate of a target product can be greatly improved, and finally, the yield of the lactulose can reach more than 65%. Meanwhile, the phenylboronic acid-based polymer carrier for directionally and selectively adsorbing lactulose can obtain a high-purity lactulose desorption solution through desorption operation, and the purity of the desorption solution can reach more than 95%.
Description
Technical Field
The invention relates to a method for preparing lactulose by directionally assisting lactose isomerization by using phenylboronic acid-based carrier and simultaneously separating and purifying the product lactulose, belonging to the technical field of preparation, separation and purification of carbohydrate (especially functional oligosaccharide).
Background
Lactulose (C)12H22O11，4-O-β-DGalactopyranosyl-DFructose) is a non-digestible disaccharide (non-digestible oligosaccharide), has the advantages of low calorie, high safety, good stability, wide application range and the like, accords with the new health consumption concept of broad consumers, and is widely applied to the fields of clinical medicine industry, health food and animal feed.
The preparation technology of lactulose mainly comprises a chemical isomerization method and an enzyme method, wherein the preparation technology of lactulose by the biological enzyme method has the advantages of mild reaction conditions, few byproducts, high safety and the like, and is a research hotspot at present. However, the conventional enzyme method for preparing lactulose not only needs a large amount of fructose as a coaction substrate, but also has the conversion rate of only 10-20%, and meanwhile, the product also contains a large amount of other reaction byproducts such as monosaccharide or lactose and the like, which is far lower than the requirement of commercial lactulose syrup. The novel enzymatic method taking cellobiose epimerase as a representative to prepare lactulose can improve the conversion rate of lactulose to 50-65%, only single lactose is used as a substrate, and the method has a good commercial prospect, but has the problems of poor substrate specificity, low enzyme activity, poor stability and the like, and is still in the laboratory research stage at present.
The chemical isomerization process catalyzes lactose to produce lactulose is the only form of lactulose production currently commercialized. The chemical isomerization process for preparing lactulose includes three different stages, from using NaOH, KOH or other strong alkali reagent or tertiary amine or other strong organic alkali to MgO and NaHSO3Use of composite catalyst, then NaAlO2、H3BO3NaOH and other catalysts with complexing property are used, and the conversion rate of lactulose is also about 10 percentThe yield is improved to about 75 percent. Although the chemical method, especially the complex catalyst represented by boric acid and sodium metaaluminate can greatly improve the conversion rate of lactulose, the complex catalyst is complex to remove, has the risks of environmental pollution, edible safety and the like, has large catalyst dosage, more reaction byproducts, high energy consumption and complex separation and purification of final products, and seriously restricts the further development of preparing lactulose by a chemical isomerization method.
The separation and purification of high-purity lactulose is a great problem in the field of lactulose preparation. On the one hand, both the substrate lactose and the product lactose are isomers of lactulose, which have structural similarities and are therefore difficult to separate by conventional methods. Considering that lactulose contains a unique ortho-cis hydroxyl structure (fructofuranose group), research reports that the method for separating and purifying lactulose adopts a borate affinity adsorption method (see Mingming Wang et al J. Agric. FoodChem,2018,66, 9269-containing 9281; Mingming Wang et al chem. Eng. J,2019,370, 1274-containing 1285). In addition, U.S. Pat. No. 6 to Andrea Filipini et al (US patent No.4565582, lactulosemification process) first proposed the separation and purification of lactulose from a mixture of 50% lactulose and other sugars by using a boric acid-based resin column chromatography, and the purity of the final lactulose could be increased from 32% to 83% by using a multi-column chromatography, but the recovery rate of lactulose was only 62.5%.
It is well known that lactulose is used in the pharmaceutical and health food additive industries and has high purity requirements, especially for most lactose intolerant patients or consumers. The research and production of lactulose in China are late, in early 1984, lactulose is developed by the research institute of commercial science in Liaoning province and is put into production, and the blank in China is filled up, but the production of lactulose in China still depends on a chemical method until now, the product is low-purity lactulose syrup, and pharmaceutical grade high-purity lactulose almost completely depends on import, so that the research and development of a high-purity lactulose preparation technology suitable for the pharmaceutical industry and the food field are realized, a core preparation process is mastered, and the lactulose preparation method has positive significance for the production and application of lactulose in China.
Disclosure of Invention
Aiming at the problems of low catalytic efficiency, a plurality of byproducts, difficult separation and removal of a catalyst, low recovery rate, high cost, long time, potential safety hazard and the like in the separation and purification process of high-purity lactulose prepared by the existing chemical isomerization method, the invention utilizes the principle that the lactulose containing a unique ortho-cis hydroxyl structure and a boric acid group can be complexed under a specific environment (pH is greater than pKa), and the complexation force between different saccharides and boric acid groups has different strength and priority order, utilizes the special property of a phenylboronic acid group loaded by a phenylboronic acid group carrier to apply the phenylboronic acid group to the reaction for preparing lactulose under one lactose flow, realizes the selective adsorption of lactulose, promotes the chemical isomerization reaction to be carried out in the positive direction of generating lactulose, greatly improves the lactulose conversion rate, and desorbs the phenylboronic acid carrier after adsorption, the high-purity lactulose solution can be obtained, so that the separation and purification of the high-purity lactulose are completed while the directional auxiliary lactose isomerization is realized to generate the lactulose.
According to the prior literature, the invention adopts the phenylboronic acid-based carrier to realize the oriented auxiliary catalysis of lactose isomerization for preparing lactulose, and simultaneously completes the separation and purification of high-purity lactulose, which is the first time at home and abroad.
The invention comprises two parts, namely, a phenylboronic acid-based carrier is used for directionally assisting lactose isomerization to generate lactulose, and the method can be realized by the following 2 modes:
A. batch type mode, comprising the steps of:
(1) isomerization of lactose: dissolving lactose and alkaline catalyst (NaOH, phosphate, etc.) to appropriate concentration and pH, and performing isomerization reaction at 30-70 deg.C;
(2) selective adsorption: after reacting for a certain time, adding a certain amount of phenylboronic acid-based polymer carrier, selectively adsorbing the product lactulose, and promoting the reaction to be carried out towards the positive direction of generating lactulose;
(3) separation of the carrier: after adsorbing for a certain time, separating the carrier from a lactose isomerization system by suction filtration, centrifugation and the like;
(4) and (3) desorption of the carrier: the separated phenylboronic acid based carrier loads lactulose generated by the reaction, and the lactulose is eluted by eluent to obtain high-purity lactulose eluent;
(5) and (3) recycling of the carrier: the eluted phenylboronic acid-based carrier is further dried and can be recycled as a new adsorption carrier.
(6) Batch type directional auxiliary lactose isomerization reaction: and (3) adding a certain amount of new phenylboronic acid-based carrier into the lactose isomerization solution from which the phenylboronic acid-based carrier is separated in the step (3), and repeating the steps (2) to (5) until the lactose in the final system is basically reacted completely.
(7) On the basis of (6), adding a certain amount of new phenylboronic acid-based carrier into the lactose isomerization solution after separation of the phenylboronic acid-based carrier in the step (3), supplementing lactose until the concentration of the lactose in the solution reaches an initial concentration level, and repeating the steps (2) to (5) until a desired lactulose production is achieved.
B. A column chromatography continuous mode comprising the steps of:
(1) loading a carrier into a column: loading a certain amount of phenylboronic acid-based carrier into a chromatographic column and connecting the whole chromatographic column system;
(2) selective adsorption: dissolving lactose and an alkaline catalyst (NaOH, phosphate and the like) to proper concentration and pH, introducing a lactose isomerization system solution from the bottom of a chromatographic column at a certain flow rate at 30-70 ℃, introducing the lactose isomerization system solution to be separated into a new phenylboronic acid-based carrier chromatographic column after the adsorption of a carrier in the chromatographic column is saturated, and continuing isomerization and selective adsorption until the lactose in the final system is basically completely reacted;
(3) and (3) elution: after the step (2) is finished, introducing eluent from the bottom of the chromatographic column at a certain flow rate to elute the adsorbed phenylboronic acid based carrier, and collecting effluent desorption liquid;
(4) and (3) recycling the carrier: and (4) stopping introducing the eluent after the elution in the step (3) is finished, and repeating the steps (1) to (4) to perform directional auxiliary lactose isomerization of a new batch to prepare lactulose.
In one example, lactulose is prepared by batch type mode assisted lactose isomerization, wherein in step (1), disodium hydrogen phosphate and sodium hydroxide are used as alkaline catalysts, isomerization is carried out at 60 ℃ and pH 11.0, 1% (w/v) phenylboronic acid based polymer carrier is added in the initial stage of the reaction, after selective adsorption for 50min, the phenylboronic acid based polymer carrier is separated, and new 1% (w/v) phenylboronic acid based polymer carrier is added for continuous selective adsorption until the lactose concentration in the final isomerization system is less than 10 g/L.
In one example, lactulose is prepared by a column chromatography continuous mode assisted lactose isomerization in which disodium hydrogen phosphate and sodium hydroxide are used as basic catalysts in step (1), the isomerized lactose system has a pH of 11.0, the column temperature is maintained at 50 ℃, and lactulose is selectively adsorbed.
In one example, substrate lactose is continuously replenished during the process of preparing lactulose by the batch mode assisted lactose isomerization, wherein disodium hydrogen phosphate and sodium hydroxide are used as alkaline catalysts in step (1), and lactose addition is calculated according to conversion in step (3) to perform substrate replenishment. The isomerized lactose system has a pH of 11.0, the temperature of the chromatographic column is kept at 50 ℃, and lactulose is selectively adsorbed.
The second part of the present invention is to desorb phenylboronic acid based polymer carrier selectively adsorbing lactulose in the first step to separate and purify high purity lactulose, and the separation and purification of high purity lactulose in the second part is different according to the auxiliary isomerization reaction mode selected in the first step, and is described as follows:
(1) for batch type auxiliary lactose isomerization to produce lactulose, the corresponding separation and purification operation steps are as follows: collecting multiple batches of phenylboronic acid-based polymer carriers after selectively adsorbing lactulose, adding desorption liquid with the volume of 1-5 times, desorbing for 10min to 2h at room temperature, collecting the desorption liquid, and performing subsequent treatment. The desorbed phenylboronic acid-based polymer carrier can be recycled as a new batch of adsorption carrier after vacuum drying at 60 ℃.
(2) For column chromatography continuous type auxiliary lactose isomerization to produce lactulose, the corresponding separation and purification operation steps are as follows: and stopping introducing the lactose isomerization system solution after the adsorption of the phenylboronic acid-based polymer carrier in the chromatographic column reaches saturation, introducing desorption liquid with a certain volume from the bottom, and collecting the permeated desorption liquid for subsequent treatment. The desorbed chromatographic column can be used as a new chromatographic packing column for carrying out new cycle selective adsorption.
In one example, a 2-fold volume of desorption solution (aqueous hydrochloric acid, pH 1.0) was added to the collected selectively adsorbed phenylboronic acid-based carrier, and the mixture was desorbed at 25 ℃ and 150rpm for 30min, and the eluate was collected
In one example, a desorption solution (40% ethanol aqueous solution, pH adjusted to 3.0) in a volume 3 times that of the phenylboronic acid-based carrier chromatography column after saturation of adsorption was introduced, and the column was desorbed at room temperature and the eluate was collected.
In one embodiment, the collected high purity lactulose eluate is spray dried to obtain a high purity lactulose powder.
In one embodiment, the collected high-purity lactulose eluate is crystallized by cooling to obtain high-purity fructose crystals.
The invention has the following beneficial effects:
(1) according to the method, the specific affinity adsorption effect between the phenylboronic acid functional group loaded by the phenylboronic acid-based carrier and the lactulose containing an ortho-position cis-hydroxyl structure is utilized, so that the lactulose in a lactose isomerization system is directionally and selectively adsorbed, the isomerization reaction can be promoted to be carried out in the direction of generating the lactulose, and the yield of the final lactulose is greatly improved;
(2) after the phenylboronic acid-based carrier directionally and selectively adsorbs lactulose in a reaction system, a high-purity lactulose desorption solution can be obtained through desorption operation, separation and purification of the high-purity lactulose are completed while directional auxiliary lactose isomerization is realized to generate lactulose, and the preparation, separation and purification processes of the lactulose are greatly simplified;
(3) in the alkaline isomerization reaction process, the phenylboronic acid-based carrier can adsorb lactulose in a directional and selective manner, so that the lactulose can be protected, and the lactulose generated in the isomerization reaction is prevented from being degraded in a strong alkaline environment, so that the lactulose yield is improved, and the generation of byproducts is effectively reduced;
(4) the phenylboronic acid-based carrier has strong stability in the processes of preparing lactulose and separating and purifying high-purity lactulose by directionally assisting lactose isomerization, can be recycled for a long time, effectively reduces the production cost, is simple and efficient, is suitable for industrial large-batch continuous preparation of high-purity lactulose, and can almost completely recover lactulose;
(6) the preparation method provided by the invention meets the production requirements of resource conservation and environmental friendliness, and provides a beneficial reference for promoting clean, efficient and environment-friendly industrial production of lactulose.
Drawings
FIG. 1 is a process flow chart of the process flow of preparing lactulose and separating and purifying high-purity lactulose by using phenylboronic acid based carrier orientation auxiliary lactose isomerization.
FIG. 2 is a schematic diagram of the principle of lactose isomerization assisted by column chromatography continuous mode of the present invention.
FIG. 3 is a graph of HP L C of the solution before and after the isomerization reaction using phenylboronic acid groups to assist lactose.
FIG. 4 shows the HP L C spectrum of the stripping solution.
Detailed description of the preferred embodiments
The purity detection method of target saccharides (lactulose, lactose and the like) comprises the following steps:
the specific procedure for determining the purity of the target saccharide by using HP L C was as follows, sampling and centrifuging (12000rpm, 20min), filtering the supernatant with a 0.22 μm microporous membrane, and subjecting the filtrate to HP L C detection analysis.
The specific detection conditions for determining the purity of the target saccharide using HP L C were as follows:
chromatograph: waters 2695 high performance liquid chromatograph;
chromatographic column Ashipak NH 2P-504E (4.6 × 250 mm);
mobile phase: 75% acetonitrile: 25% aqueous solution (v/v);
the flow rate of the mobile phase is 1m L/min;
temperature: 40 ℃;
a detector: water 2414 as a differential refractive detector;
the sample size is 10 mu L.
The present invention will be described in detail below.
Example 1: batch type mode-assisted lactose isomerization preparation of lactulose
100g of lactose was dissolved in 900M L M disodium hydrogen phosphate buffer (concentration: 0.5M), NaOH was added to adjust the pH of the system to 11.0, and then deionized water was added to the system volume of 1L, which was used as a lactose isomerization system solution.
10g of phenylboronic acid-based carrier is added in the initial stage of the reaction, after the reaction is carried out for 50min at 60 ℃ and 150rpm, the adsorbed phenylboronic acid-based carrier is removed by suction filtration, then 10g of new phenylboronic acid-based carrier is added into the lactose isomerization system solution, and the operation is continued until the lactose content in the final system is lower than 10 g/L.
Compared with the lactulose conversion rate of about 15% under a single NaOH catalytic system, the lactulose conversion rate can reach about 55% finally by adopting the batch type auxiliary catalytic mode of the phenylboronic acid-based carrier.
Example 2: batch type mode-assisted lactose isomerization preparation of lactulose
100g of lactose was dissolved in 900M L M disodium hydrogen phosphate buffer (concentration: 0.5M), NaOH was added to adjust the pH of the system to 11.0, and then deionized water was added to the system volume of 1L, which was used as a lactose isomerization system solution.
Adding 10g of phenylboronic acid-based polymer carrier at the initial stage of reaction, reacting at 60 ℃ and 150rpm for 25min, performing suction filtration to remove the adsorbed phenylboronic acid-based polymer carrier, adding 10g of new phenylboronic acid-based polymer carrier and 10-15 g of lactose (the 25min lactose conversion rate is about 15% -20%) into the lactose isomerization system solution, continuing the above operation, performing 10 rounds of sugar supplement circulation, and stopping sugar supplement, wherein the mode operation in example 1 is adopted until the lactose content in the final system is lower than 10 g/L.
Compared with the lactulose conversion rate weakened by the reduction of the substrate concentration under the batch intermittent auxiliary mode, the adoption of the continuous sugar-supplementing auxiliary catalytic mode of the phenylboronic acid-based polymer carrier can eliminate the influence of the substrate concentration, and the final lactulose conversion rate can reach about 68%.
Example 3: column chromatography continuous mode assisted lactose isomerization preparation of lactulose
250g of lactose is dissolved in 800M L disodium hydrogen phosphate buffer solution (the concentration is 0.5M), NaOH is added to adjust the pH of the system to 11.0, and then deionized water is added until the volume of the whole system is 1L, wherein the solution is lactose isomerization system solution and is used as the next feeding solution.
Respectively filling 60-80g of phenylboronic acid-based carriers into 4 chromatographic columns with the length of 100cm, heating the feed liquid to 50 ℃, slowly introducing the feed liquid into a chromatographic column No. 1 from the bottom of the chromatographic column to enable the feed liquid to flow through the phenylboronic acid-based carriers, stopping introducing the feed liquid into the chromatographic column No. 1 after the uppermost layer of liquid is extracted for 30min, introducing the feed liquid into a chromatographic column No. 2, and performing the same operation until the 4 chromatographic columns are completely adsorbed and saturated, wherein the lactose concentration in the reaction liquid is less than 10 g/L and can be regarded as reaching the reaction end point.
Example 4: batch intermittent type mode separation and purification of lactulose
The phenylboronic acid-based polymer carrier (about 100g) after selective adsorption of lactulose collected in example 1 was placed in a 500m L conical flask, 200m L aqueous hydrochloric acid (eluent) with pH 1.0 was added to desorb at 150rpm and 25 ℃ for 30min, and the desorbed solution was collected and spray-dried to obtain a high-purity lactulose powder sample, and about 35g of the high-purity lactulose powder sample was finally obtained with a purity of 90% or more.
Example 5: column chromatography continuous mode separation and purification of lactulose
300m of L desorption solution (40% ethanol aqueous solution, pH is adjusted to 3.0) is respectively introduced into the chromatographic columns of the phenylboronic acid-based polymer carriers 1-4 after saturated adsorption in the example 2, desorption is carried out at room temperature, the effluent liquid at the upper part is collected, and about 120g of high-purity lactulose crystals are obtained by a cooling crystallization mode, wherein the purity of the lactulose crystals reaches more than 95%.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A method for preparing, separating and purifying lactulose by utilizing oriented auxiliary lactose isomerization of a phenylboronic acid-based carrier is characterized in that the structure of the phenylboronic acid-based polymer carrier is as follows:
wherein: p is the bulk (substrate) structure of the support, including the polymer structure or magnetic nanoparticles, and is covalently bound to the phenylboronic acid group.
2. A method for preparing, separating and purifying lactulose by directionally assisting lactose isomerization by using phenylboronic acid-based carrier is characterized in that the process of preparing lactulose by directionally assisting lactose isomerization is that the phenylboronic acid-based carrier in claim 1 is added into a lactose isomerization system to selectively adsorb lactulose and promote reaction to be carried out forward, so that the aim of preparing lactulose by directionally assisting lactose isomerization is fulfilled.
3. The lactose isomerization system of claim 2, characterized in that: lactose is used as a substrate; the catalyst is an alkaline agent, including but not limited to sodium hydroxide, calcium hydroxide, phosphate, sodium metaaluminate, boric acid/sodium hydroxide, and the like; the pH of the isomerization system is basic (pH > 8.0).
4. The addition of phenylboronic acid based carriers to a lactose isomerization system as set forth in claim 2 can be accomplished by the following 2 modes:
A. batch-batch type mode, characterized by: directly adding a certain amount of phenylboronic acid-based carrier according to claim 1 at the initial or any stage of isomerization reaction, after reacting for a certain time, separating the added phenylboronic acid-based carrier from the system, desorbing the adsorbed lactulose with eluent, drying the desorbed carrier, and recycling. And (3) continuing to add the phenylboronic acid-based carrier into the lactose isomerization system after the carrier is separated, and repeating the operation until the reaction is finished.
B. A column chromatography continuous mode characterized by: loading a certain amount of phenylboronic acid-based carrier according to claim 1 into a chromatographic column, connecting the chromatographic column with the whole column chromatographic system, introducing a lactose isomerization system solution from the bottom of the chromatographic column at a certain flow rate, introducing the lactose isomerization system solution to be separated into a new phenylboronic acid-based carrier chromatographic column after the adsorption of the chromatographic column is saturated, and continuing isomerization and adsorption until the reaction is finished. Desorbing the chromatographic column with saturated adsorption with eluent, and circulating adsorbing lactulose with the desorbed chromatographic column.
5. A method for preparing, separating and purifying lactulose by utilizing a phenylboronic acid-based carrier to directionally assist lactose isomerization is characterized in that the separation and purification of lactulose refers to the desorption of the phenylboronic acid-based carrier after absorbing lactulose in claim 4, and the final desorption solution is high-purity lactulose.
6. Lactulose desorption according to claims 4 and 5, characterized in that: the eluent is aqueous solution, alcoholic solution or acidic solution. Wherein the alcoholic solution is methanol solution or ethanol solution or mixed solution of methanol and ethanol at any ratio, the pH of acidic solution is less than 7.0, and can be adjusted by adding inorganic acid such as hydrochloric acid, acetic acid, sulfuric acid, etc., or organic acid such as lactic acid, citric acid, etc.
7. The method for preparing and separating and purifying lactulose by utilizing the oriented auxiliary lactose isomerization of the phenylboronic acid based carrier according to the claims 1-6, wherein the oriented auxiliary catalysis of the lactose and the separation and purification of the lactulose are realized based on the selective adsorption and desorption of the lactulose on the phenylboronic acid based carrier.
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