CN116425810B - Purification method of 3-fucosyllactose in mixed solution - Google Patents
Purification method of 3-fucosyllactose in mixed solution Download PDFInfo
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- 239000011259 mixed solution Substances 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000000746 purification Methods 0.000 title claims abstract description 33
- AUNPEJDACLEKSC-ZAYDSPBTSA-N 3-fucosyllactose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)O[C@H](CO)[C@@H]1O AUNPEJDACLEKSC-ZAYDSPBTSA-N 0.000 title claims abstract 13
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- 238000000855 fermentation Methods 0.000 claims abstract description 34
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- 125000002091 cationic group Chemical group 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
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- WJPIUUDKRHCAEL-YVEAQFMBSA-N 3-fucosyllactose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](CO)OC(O)[C@@H]1O WJPIUUDKRHCAEL-YVEAQFMBSA-N 0.000 description 70
- 235000019441 ethanol Nutrition 0.000 description 16
- 235000010633 broth Nutrition 0.000 description 13
- 238000004128 high performance liquid chromatography Methods 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 7
- 239000012535 impurity Substances 0.000 description 6
- 229920001542 oligosaccharide Polymers 0.000 description 6
- 150000002482 oligosaccharides Chemical class 0.000 description 6
- 235000020256 human milk Nutrition 0.000 description 5
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- 238000001514 detection method Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
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- 102100026189 Beta-galactosidase Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108010059881 Lactase Proteins 0.000 description 2
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- 238000010362 genome editing Methods 0.000 description 2
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- 238000001694 spray drying Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 229940062827 2'-fucosyllactose Drugs 0.000 description 1
- HWHQUWQCBPAQQH-UHFFFAOYSA-N 2-O-alpha-L-Fucosyl-lactose Natural products OC1C(O)C(O)C(C)OC1OC1C(O)C(O)C(CO)OC1OC(C(O)CO)C(O)C(O)C=O HWHQUWQCBPAQQH-UHFFFAOYSA-N 0.000 description 1
- HWHQUWQCBPAQQH-BWRPKUOHSA-N 2-fucosyllactose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O HWHQUWQCBPAQQH-BWRPKUOHSA-N 0.000 description 1
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 description 1
- 240000000103 Potentilla erecta Species 0.000 description 1
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- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
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- 239000000758 substrate Substances 0.000 description 1
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- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Classifications
-
- 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
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for purifying 3-fucosyllactose in mixed liquor. The purification method comprises the steps of filtering to remove thalli, proteins and the like by adopting a ceramic membrane, desalting by adopting ion resin, decoloring by adopting active carbon to obtain a mixed solution containing 3-fucosyllactose and lactose, and then evaporating, precipitating by adding alcohol, filtering, washing and drying a crude product. The purity of the obtained 3-fucosyl lactose is more than 99%, and the purity of the 3-fucosyl lactose is greatly improved.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for purifying 3-fucosyllactose in mixed liquor.
Background
Human milk oligosaccharides (Human milk oligosaccharides, HMO) are important immunologically active components in breast milk, and have a significant promoting effect on infant health. 3-Fucosyllactose (3-Fucosyl lactose, 3-FL; CAS:41312-47-4, molecular weight 488.4 g/mol) is one of HMO, and has great application value. The studies and applications of 3-FL are still in an early stage, and there are few reports on them. The commercial 3-FL product has a purity of about 90%, and is a white to ivory powder containing impurities including lactose (about 5%) and the like. The U.S. food and drug administration describes a published product declaration document (GRN 000925) from Escherichia coli DSM33491 after fermentation and purification. The purification steps of the 3-FL in the fermentation broth are as follows: filtering-ion exchange-electrodialysis-decolorization-spray drying.
3-FL has many physicochemical properties similar to lactose, thus making purification of 3-FL difficult. Breast milk itself is also a natural mixture containing 3-FL and lactose. Foreign Raj Mehra et al (Raj Mehra, daniela Barile, mariarosaria Marotta,et al. Novel High-Molecular Weight Fucosylated Milk Oligosaccharides Identified in Dairy Streams[J]. Plos one, 2014, 9(5): e96040. ) Separating and enriching oligosaccharide from mother liquor by membrane filtration technology, removing other impurities by microporous membrane filtration, ultrafiltration and other steps until the mixed liquor mainly contains oligosaccharide, lactose and mineral substances, and further purifying by lactose crystallization-membrane filtration-drying step, wherein researchers find that 3-FL in the product is lost. The main reason may be loss in the lactose crystallization link.
Therefore, it is still a technical problem to be solved how to purify 3-FL from lactose-containing mixed solutions such as fermentation solutions to further improve the purity of 3-FL. Because of the lack of adequate literature reports, there are two approaches to solve the problem at present, one is to make lactose as depleted as possible during fermentation, i.e. to reduce lactose content from the fermentation process in order to reduce purification difficulty and increase purity of 3-FL. But this method is more difficult to control. Another idea is to use enzymatic degradation of lactose. For example CN202111179170.7, lactase is added after removing the thalli during purification, lactose is degraded under specific temperature and pH conditions, and then protein denaturation, filtration, ion exchange and spray drying are performed. However, when used as a pharmaceutical product, lactase is added and degraded, resulting in the need for detection and quality control of newly introduced impurities. The purity of the 3-FL in the obtained product is 89% -95.3%, and the purity of the product is still lower.
Disclosure of Invention
In view of the above problems, the invention provides a method for purifying 3-fucosyllactose in a mixed solution by examining the influence of a non-one-step drying method on the purity of 3-fucosyllactose.
Specifically, the technical scheme adopted by the invention is as follows:
a purification method of 3-fucosyllactose in a mixed solution at least comprises 3-fucosyllactose and lactose, wherein the mass percentage of 3-fucosyllactose in the mixed solution is F1, and the mass percentage of lactose in the mixed solution is L1, wherein F1 is L1, and the range C of L1 is 6< C <99; the solvent of the mixed solution is water or an organic solvent aqueous solution with the concentration of less than 1 percent; the purification method comprises the following steps:
s4, evaporating at 50-80 ℃ and vacuum degree of less than or equal to-0.090 mpa to ensure that F1 in the mixed solution is more than or equal to 60% and less than or equal to 90%, and the content of the organic solvent is less than or equal to 1%, thus obtaining an evaporation mixed solution;
s5, maintaining the evaporation mixed solution obtained in the step S4 at 40-80 ℃, and adding ethanol with the concentration of more than 90% in an amount which is 1-2 times of the volume of the evaporation mixed solution under the stirring condition of 80-300 rpm; standing for 3-5 h, and continuously feeding ethanol with the concentration of more than 90% to obtain an ethanol-added mixed solution;
s6, filtering the alcohol-added mixed solution obtained in the step S5 to obtain a 3-fucosyllactose crude product, washing the 3-fucosyllactose crude product by ethanol with the concentration of more than 90%, and drying to obtain a 3-fucosyllactose finished product; the purity of the 3-fucosyl lactose finished product is more than or equal to 99 percent.
Preferably, the temperature before the ethanol is added in the step S5 is maintained at 50-70 ℃.
Preferably, for the step S5, the flow rate of the ethanol added after standing is 0.5-1 times of the volume of the evaporated mixed solution/h, and the volume of the ethanol added after standing is 2-8 times of the volume of the evaporated mixed solution.
Preferably, for the step S5, the temperature of the alcohol-added mixed solution is reduced to 10 ℃ to 40 ℃ after the end of the alcohol-added mixed solution.
Preferably, the ethanol with a concentration of >90% in the step S6 is absolute ethanol.
Preferably, the volume of the ethanol for washing in the step S6 is 0.1-0.2 times of the ethanol adding mixed solution in the step S5; the number of times of ethanol washing in the step S6 is 1 to 3.
Preferably, the drying temperature in the step S6 is 40-80 ℃; the drying time is 4-12 h.
Further, the mixed solution is prepared from an escherichia coli fermentation broth, and the escherichia coli fermentation broth at least contains 3-fucosyllactose, lactose, glycerol, proteins, thalli and mineral salts; the range of C is 6< C <99, and the solvent of the escherichia coli fermentation broth is water; the purification method further comprises the following steps:
s1, heating the escherichia coli fermentation liquor until protein is denatured, passing through a 10 nm-200 nm ceramic membrane, and dialyzing with purified water to obtain a ceramic membrane clear liquid.
S2, sequentially passing the ceramic membrane clear liquid obtained in the step S1 through 001 x 7 cationic resin and 330 anionic resin, washing with purified water, and collecting resin permeation liquid.
S3, adding 1-3% of active carbon into the resin permeation liquid obtained in the step S2, stirring for 1.0-3.0 h at 50-60 ℃, and filtering to obtain the mixed liquid.
Preferably, in the step S1, the temperature is maintained at 10℃to 40℃when the Escherichia coli fermentation broth passes through the ceramic membrane.
Preferably, in the step S2, the feeding flow rate is 0.5 times to 3.0 times of resin filling amount/h, and purified water with 0.5 times to 1.0 times of resin filling amount is used for top washing; the feeding temperature is controlled between 10 ℃ and 40 ℃.
Preferably, the strain for fermentation production used in the fermentation broth of Escherichia coli in the step S1 is selected from one of Escherichia coli K-12 MG1655 and Escherichia coli HCYJ-08.
Wherein the Escherichia coli HCYJ-08 accession number is: CGMCC No. 27189; the preservation date is: 2023, 04, 23; the preservation units are as follows: china general microbiological culture Collection center (China Committee for culture Collection); the preservation address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. The taxonomic name of the escherichia coli HCYJ-08 is escherichia coli @Escherichia coli). The Escherichia coli HCYJ-08 is obtained by using Escherichia coli K12 MG1655 as an original strain, performing preliminary gene editing according to patent document CN112501106A to obtain Escherichia coli W2, and further performing gene editing. It can use glycerol as a carbon source for growth and convert the substrate lactose to 3-fucosyllactose.
It is apparent that when referring to drying of the finished product, a variety of drying means may be used in the art to remove moisture from the finished product. Preferably, the drying can be carried out at 40-80 ℃ for 4-12 h.
Obviously, when referring to an organic solvent solution, the aforementioned "concentrations" are all volume percent concentrations.
The beneficial effects are that:
1. when the 3-fucosyl lactose is prepared by adopting a fermentation method, the escherichia coli fermentation liquor has deep chromaticity and high viscosity, and the traditional plate-frame filtration needs to be added with flocculating agent and filter aid, so that the filter cloth is easy to block, the filtration is difficult, and the labor intensity is high. By using ceramic membrane filtration, parameter automatic control can be realized, the degree of automation is high, the yield is high, and the labor cost is saved;
2. the invention provides a method for desalting escherichia coli fermentation broth by using an ion exchange method, so that the yield of 3-fucosyllactose is improved, and the problems of complex installation process, high maintenance cost, low sugar yield and the like caused by using electrodialysis are avoided;
3. the invention adopts the activated carbon for decolorization, can effectively remove impurities such as chromaticity, protein and the like of the escherichia coli fermentation liquor, and is simple and effective;
4. the purification method only uses an organic reagent which is easy to remove by a solid-liquid separation method, does not adopt a lactose degradation method, is not easy to introduce other novel small molecular impurities, and is environment-friendly;
5. the purification method has simple steps, relatively common purification process equipment, small investment and cost saving;
6. according to the purification method, the purity of the 3-fucosyllactose can reach more than 99%;
7. the purification method of the invention is subjected to pilot scale experiments, and is more suitable for process amplification to industrial production.
Drawings
FIG. 1 is an HPLC chromatogram of 3-fucosyllactose obtained in experiment 7 of example 1 of the present invention.
Detailed Description
The technology of the present invention will be described with reference to the following specific embodiments, which are not to be construed as limiting the scope of the present invention.
In the following examples, the content of 3-fucosyllactose in a liquid such as a mixed solution or an Escherichia coli fermentation liquid was measured by HPLC. The detection conditions for the HPLC method are as follows:
using HPX-87P cation exchange chromatographic column (300 mm ×7.8 mm, particle size 9 μm) or other type chromatographic column with equivalent performance; purified water is taken as a mobile phase; the detector is a differential refraction detector, the temperature of the pool is 50 ℃, the temperature of the column temperature box is 80 ℃, the flow rate is 0.4mL/min, and the sample injection amount is 10 mu L.
Taking a proper amount of 3-fucosyllactose standard substance (about 20mg containing 3-fucosyllactose), precisely weighing, placing into a 20ml measuring flask, adding purified water for dissolving, diluting to scale, and shaking uniformly to obtain a standard substance solution (0.1 g/L). Standard solutions (0.1 g/L) with different volumes are respectively taken, diluted into standard solutions (0.1 g/L, 0.25g/L, 0.5g/L, 0.75g/L and 1.0 g/L) with different concentrations by water, and standard curves are drawn. Weighing about 25mg of the product, precisely weighing, placing into a 50ml measuring flask, adding purified water for dissolution, diluting to scale, and shaking to obtain sample solution; two samples are weighed and used as parallel samples, the parallel samples are respectively injected into a liquid chromatograph, the area of the chromatogram is recorded, and the corresponding concentration is calculated by using a drawn standard curve.
The lactose content detection method is the same as that of 3-fucosyllactose, and the content of 3-fucosyllactose and lactose can be detected by adopting a method reported in literature: christensen AS, skov SH, lendal SE,et al. Quantifying the human milk oligosaccharides 2'-fucosyllactose and 3-fucosyllactose in different food applications by high-performance liquid chromatography with refractive index detection[J]. Journal of Food Science, 2020, 85(2):332-338。
the purity of 3-fucosyllactose in the following examples was measured by obtaining a liquid chromatogram by HPLC, and calculating the content of 3-fucosyllactose by external standard method.
EXAMPLE 1 purification of 3-fucosyllactose in fermentation broths
The 3-fucosyllactose is fermented by using Escherichia coli HCYJ-08 strain.
Fermentation medium: glycerol 20 g/L, KH 2 PO 4 5 g/L、K 2 HPO 4 5g/L、(NH 4 ) 2 SO 4 4.0 g/L, citric acid monohydrate 1.7. 1.7 g/L, mgSO 4 ·7H 2 O1.4 g/L, thiamine 4.5 mg/L and trace element 1% (v/v), and pH was adjusted to 6.8 with sodium hydroxide.
Microelement liquid: feSO 4 ·7H 2 O 10 g/L、ZnSO 4 ·7H 2 O 2.2 g/L、CuSO 4 ·5H 2 O 1.0 g/L、MnSO 4 ·H 2 O 0.38 g/L、Na 2 B 4 O 7 ·10H 2 O 0.02 g/L、(NH 4 ) 6 Mo 7 O 24 0.1g/L and CaCl 2 2.0 g/L, dissolved in 5 mol/L hydrochloric acid.
The mass percent of the 3-fucosyllactose in the escherichia coli fermentation broth is 5 percent and the mass percent of the lactose is 0.5 percent.
The purification steps of the 3-fucosyllactose in the fermentation broth are as follows:
s1, heating escherichia coli fermentation liquor until protein is denatured, passing through a ceramic membrane, and dialyzing with purified water to obtain ceramic membrane clear liquid;
s2, sequentially passing the ceramic membrane clear liquid through 001 x 7 cation resin, 330 anion resin and purified water through a column, and collecting resin permeate;
s3, adding active carbon into the resin permeation solution, stirring, and filtering to obtain a mixed solution;
s4, performing reduced pressure rotary evaporation on the mixed solution under the condition that the vacuum degree is less than or equal to-0.090 mpa until the mass percentage of 3-fucosyl lactose in the mixed solution is 70%, so as to obtain an evaporation mixed solution;
s5, adding 95% ethanol into the evaporation mixed solution under stirring, and continuously adding 95% ethanol after standing to obtain an ethanol-added mixed solution;
and S6, carrying out suction filtration on the alcohol-added mixed solution by using a Buchner funnel to obtain a 3-fucosyl lactose crude product, washing the 3-fucosyl lactose crude product by using absolute ethyl alcohol, and drying a solid substance in an oven after washing to obtain a 3-fucosyl lactose finished product, wherein the purity of the 3-fucose is detected by using an HPLC method.
The purity detection results of the 3-fucosyllactose finished product under different parameter conditions are shown in tables 1 and 2 according to the method.
TABLE 1 purity of 3-fucosyllactose finished products under different purification conditions
TABLE 2 purity of 3-fucosyllactose finished products under different purification conditions (Table 1, supra)
As can be seen from tables 1 and 2, the purity of the 3-fucosyllactose finished product obtained under the conditions of the method and parameters is more than 99.00%. FIG. 1 is an HPLC chromatogram of the final product obtained in experiment 7. As shown in figure 1, the HPLC chromatogram of the product has no lactose chromatogram peak and no other impurity peaks, and the purity of the product is more than or equal to 99.99 percent.
Comparative example 1 purification of 3-fucosyllactose in fermentation broth
Step S3 was removed in the same manner as in experiment 9 of example 1. The color of the obtained 3-fucosyl lactose finished product turns yellow, and the purity of the 3-fucosyl lactose finished product measured by an HPLC method is 90.82%.
Comparative example 2 purification of 3-fucosyllactose in fermentation broth
In the same manner as in experiment 9 of example 1, 4 times of 95% ethanol was added to the evaporation mixture at a time in step S5, and the other conditions were unchanged. The 3-fucosyl lactose finished product is obtained, and the purity of the 3-fucosyl lactose finished product measured by an HPLC method is 88.79%.
Comparative example 3 purification of 3-fucosyllactose in fermentation broth
The same procedure as in experiment 9 of example 1 was followed, wherein the drying temperature in step S6 was 90℃and the drying time period was 10 hours, and the other conditions were unchanged. The obtained 3-fucosyl lactose finished product has yellow color and sticky crystal, and the purity of the 3-fucosyl lactose finished product measured by an HPLC method is 94.27%.
As can be seen from comparative examples 1 to 3, the purity of the finished 3-fucosyllactose product can reach 90% when step S3 is removed; when the drying temperature in the step S6 is too high, the purity of the finished 3-fucosyl lactose product is not more than 95%; when ethanol is added at a time in step S5 (comparative example 2), the purity of the finished 3-fucosyllactose product is the lowest, which is lower than 90%.
EXAMPLE 2 purification of 3-fucosyllactose in Mixed liquor
The mixed solution is prepared by purified water, 3-fucosyllactose, lactose and ethanol, so that the ethanol concentration (v/v) in the mixed solution is 0.5%.
And (3) measuring the content of 3-fucosyllactose and lactose in the mixed solution by an HPLC method, and calculating the mass percent of the 3-fucosyllactose in the mixed solution to be F1 and the mass percent of the lactose in the mixed solution to be L1 and F1 to L1 range C. Purification was performed according to the methods of steps S4 to S6 by using the method of experiment 9 of example 1 and comparative example 3 described above, and the purity of the 3-fucosyllactose product was measured by HPLC after the purification was completed, and the results are shown in table 3.
TABLE 3 purity of finished 3-fucosyllactose product from different mixtures
As shown in Table 3, when the initial value of C is 6-96 and F1 is 60-90 in the step S4, the purity of the product obtained by the experiment 9 method in example 1 is 99.16-99.77% and the purity of the product obtained by the comparison 3 method is 92.72-96.86%. Example 1 experiment 9 resulted in a higher purity product than comparative example 3.
Claims (9)
1. A purification method of 3-fucosyllactose in a mixed solution at least comprises 3-fucosyllactose and lactose, wherein the mass percentage of 3-fucosyllactose in the mixed solution is F1, and the mass percentage of lactose in the mixed solution is L1, wherein the range C of F1 to L1 is 6< C <99; the solvent of the mixed solution is water or an organic solvent aqueous solution with the concentration of less than 1 percent; the mixed solution is prepared from escherichia coli fermentation liquor, and the escherichia coli fermentation liquor at least contains 3-fucosyllactose, lactose, glycerol, proteins, thalli and mineral salts; the range of C is 6< C <99, and the solvent of the escherichia coli fermentation broth is water; the purification method is characterized by comprising the following steps:
s1, heating the escherichia coli fermentation liquor until protein is denatured, passing through a 10 nm-200 nm ceramic membrane, and dialyzing with purified water to obtain a ceramic membrane clear solution;
s2, sequentially passing the ceramic membrane clear liquid obtained in the step S1 through 001 x 7 cationic resin and 330 anionic resin, washing with purified water, and collecting resin permeation liquid;
s3, adding 1% -3% of active carbon into the resin permeation solution obtained in the step S2, stirring for 1.0-3.0 h at 50-60 ℃, and filtering to obtain the mixed solution;
s4, evaporating at 50-80 ℃ and vacuum degree of less than or equal to-0.090 mpa to ensure that F1 in the mixed solution is more than or equal to 60% and less than or equal to 90%, and the content of the organic solvent is less than or equal to 1%, thus obtaining an evaporation mixed solution;
s5, maintaining the evaporation mixed solution obtained in the step S4 at 40-80 ℃, and adding ethanol with the concentration of more than 90% in an amount which is 1-2 times of the volume of the evaporation mixed solution under the stirring condition of 80-300 rpm; standing for 3-5 h, and continuously feeding ethanol with the concentration of more than 90% to obtain an ethanol-added mixed solution;
s6, filtering the alcohol-added mixed solution obtained in the step S5 to obtain a 3-fucosyllactose crude product, washing the 3-fucosyllactose crude product by ethanol with the concentration of more than 90%, and drying to obtain a 3-fucosyllactose finished product; the purity of the 3-fucosyl lactose finished product is more than or equal to 99 percent.
2. The purification method according to claim 1, wherein the temperature before the ethanol is added in step S5 is maintained at 50 ℃ to 70 ℃; in the step S5, the flow rate of the ethanol added after standing is 0.5-1 time of the volume of the evaporation mixed solution per hour, and the volume of the ethanol added after standing is 2-8 times of the volume of the evaporation mixed solution.
3. The purification method according to claim 1, wherein the alcohol-added mixed solution in step S5 is cooled to 10 ℃ to 40 ℃ after the end of the ethanol addition.
4. The method according to claim 1, wherein the ethanol with a concentration of >90% in step S6 is absolute ethanol.
5. The purification method according to claim 1, wherein the volume of the ethanol for washing in the step S6 is 0.1 to 0.2 times/time the ethanol-added mixture in the step S5; the number of times of ethanol washing in the step S6 is 1 to 3.
6. The purification method according to claim 1, wherein the drying temperature in step S6 is 40 ℃ to 80 ℃; the drying time is 4-12 h.
7. The method according to claim 1, wherein in the step S1, the temperature is maintained at 10℃to 40℃when the fermentation broth of Escherichia coli passes through the ceramic membrane.
8. The purification method according to claim 1, wherein in the step S2, the feed flow rate is 0.5 to 3.0 times the resin loading per hour, and the purified water is top-washed with 0.5 to 1.0 times the resin loading; the feeding temperature is controlled between 10 ℃ and 40 ℃.
9. The method according to claim 1, wherein in the step S1, the strain for fermentation production used for the fermentation broth of Escherichia coli is selected from one of Escherichia coli K-12 MG1655 and Escherichia coli HCYJ-08; the Escherichia coli HCYJ-08 has a preservation number of: CGMCC No. 27189; the preservation date is: 2023, 04, 23; the preservation units are as follows: china general microbiological culture Collection center (China Committee for culture Collection); the preservation address is: the taxonomy of the escherichia coli HCYJ-08 is named as escherichia coli @ No. 3 of North Silu 1, the Korean region of Beijing, the city of the sunEscherichia coli)。
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014069625A1 (en) * | 2012-11-01 | 2014-05-08 | 協和発酵バイオ株式会社 | Crystal of oligosaccharide, and method for producing same |
| WO2014086373A1 (en) * | 2012-12-07 | 2014-06-12 | Glycom A/S | Crystallisation of human milk oligosaccharides (hmo) |
| CN106132977A (en) * | 2014-01-20 | 2016-11-16 | 詹内怀恩生物技术股份有限公司 | For effective purification from the method for the neutral human milk oligosaccharides (HMO) of fermentable |
| WO2018164937A1 (en) * | 2017-03-06 | 2018-09-13 | Dupont Nutrition Biosciences Aps | Process for crystallizing 2'-fucosyllactose and related compositions |
| CN111094311A (en) * | 2017-08-29 | 2020-05-01 | 詹尼温生物技术有限责任公司 | Method for purifying sialylated oligosaccharides |
| CN111164090A (en) * | 2017-09-29 | 2020-05-15 | 菲仕兰坎皮纳荷兰公司 | Method for purifying neutral Human Milk Oligosaccharides (HMOs) from microbial fermentation |
| CN113004347A (en) * | 2021-02-19 | 2021-06-22 | 中国科学院合肥物质科学研究院 | Method for separating and purifying 2' -fucosyllactose |
| CN113195509A (en) * | 2018-12-18 | 2021-07-30 | 因比奥斯公司 | Alpha-1, 3-fucosyltransferase for producing 3-fucosyllactose and converting lactose |
| CN114828652A (en) * | 2019-11-15 | 2022-07-29 | 科汉森母乳低聚糖股份有限公司 | Method for drying human milk oligosaccharides |
| CN116171329A (en) * | 2020-08-10 | 2023-05-26 | 因比奥斯公司 | Method for purifying oligosaccharide solutions produced by cell culture or microbial fermentation |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3098403C (en) * | 2011-02-16 | 2022-05-10 | Glycosyn LLC | Biosynthesis of human milk oligosaccharides in engineered bacteria |
| EP3227309A4 (en) * | 2014-12-05 | 2019-01-16 | Glycom A/S | DIFUCOSYLLACTOSE CRYSTALLINE |
-
2023
- 2023-06-14 CN CN202310700483.5A patent/CN116425810B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014069625A1 (en) * | 2012-11-01 | 2014-05-08 | 協和発酵バイオ株式会社 | Crystal of oligosaccharide, and method for producing same |
| WO2014086373A1 (en) * | 2012-12-07 | 2014-06-12 | Glycom A/S | Crystallisation of human milk oligosaccharides (hmo) |
| CN106132977A (en) * | 2014-01-20 | 2016-11-16 | 詹内怀恩生物技术股份有限公司 | For effective purification from the method for the neutral human milk oligosaccharides (HMO) of fermentable |
| WO2018164937A1 (en) * | 2017-03-06 | 2018-09-13 | Dupont Nutrition Biosciences Aps | Process for crystallizing 2'-fucosyllactose and related compositions |
| CN111094311A (en) * | 2017-08-29 | 2020-05-01 | 詹尼温生物技术有限责任公司 | Method for purifying sialylated oligosaccharides |
| CN111164090A (en) * | 2017-09-29 | 2020-05-15 | 菲仕兰坎皮纳荷兰公司 | Method for purifying neutral Human Milk Oligosaccharides (HMOs) from microbial fermentation |
| CN113195509A (en) * | 2018-12-18 | 2021-07-30 | 因比奥斯公司 | Alpha-1, 3-fucosyltransferase for producing 3-fucosyllactose and converting lactose |
| CN114828652A (en) * | 2019-11-15 | 2022-07-29 | 科汉森母乳低聚糖股份有限公司 | Method for drying human milk oligosaccharides |
| CN116171329A (en) * | 2020-08-10 | 2023-05-26 | 因比奥斯公司 | Method for purifying oligosaccharide solutions produced by cell culture or microbial fermentation |
| CN113004347A (en) * | 2021-02-19 | 2021-06-22 | 中国科学院合肥物质科学研究院 | Method for separating and purifying 2' -fucosyllactose |
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