CN111004826A - Preparation method of galactooligosaccharide powder - Google Patents
Preparation method of galactooligosaccharide powder Download PDFInfo
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- CN111004826A CN111004826A CN201911115468.4A CN201911115468A CN111004826A CN 111004826 A CN111004826 A CN 111004826A CN 201911115468 A CN201911115468 A CN 201911115468A CN 111004826 A CN111004826 A CN 111004826A
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- galactooligosaccharide
- galactosidase
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- 235000021255 galacto-oligosaccharides Nutrition 0.000 title claims abstract description 81
- 150000003271 galactooligosaccharides Chemical class 0.000 title claims abstract description 81
- 239000000843 powder Substances 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 66
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 66
- 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 claims abstract description 31
- 239000008101 lactose Substances 0.000 claims abstract description 31
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical group [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims abstract description 26
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 26
- 239000006188 syrup Substances 0.000 claims abstract description 18
- 235000020357 syrup Nutrition 0.000 claims abstract description 18
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 16
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 16
- 239000002245 particle Substances 0.000 claims abstract description 11
- 238000001914 filtration Methods 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 44
- 238000003756 stirring Methods 0.000 claims description 31
- 239000012528 membrane Substances 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 10
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 229920001661 Chitosan Polymers 0.000 claims description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 238000001728 nano-filtration Methods 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 229920002774 Maltodextrin Polymers 0.000 claims description 5
- 239000005913 Maltodextrin Substances 0.000 claims description 5
- 229920002472 Starch Polymers 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 229940035034 maltodextrin Drugs 0.000 claims description 5
- 239000008107 starch Substances 0.000 claims description 5
- 235000019698 starch Nutrition 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 3
- 238000005185 salting out Methods 0.000 claims description 3
- 229930182830 galactose Natural products 0.000 abstract description 8
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 3
- 150000001720 carbohydrates Chemical class 0.000 abstract description 2
- 229920002521 macromolecule Polymers 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 230000003301 hydrolyzing effect Effects 0.000 abstract 1
- 230000003100 immobilizing effect Effects 0.000 abstract 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 17
- 239000008103 glucose Substances 0.000 description 17
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 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 4
- 238000001514 detection method Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000010298 pulverizing process Methods 0.000 description 4
- 241000228245 Aspergillus niger Species 0.000 description 3
- 240000006439 Aspergillus oryzae Species 0.000 description 3
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 3
- 244000285963 Kluyveromyces fragilis Species 0.000 description 3
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 3
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 229920001542 oligosaccharide Polymers 0.000 description 3
- 150000002482 oligosaccharides Chemical class 0.000 description 3
- 241001138401 Kluyveromyces lactis Species 0.000 description 2
- 125000002519 galactosyl group Chemical group C1([C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 240000005384 Rhizopus oryzae Species 0.000 description 1
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000020244 animal milk Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- 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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- 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
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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Abstract
The invention discloses a preparation method of galactooligosaccharide powder in the related technical field of saccharide preparation, which comprises the steps of preparing activated macroporous alkalescent acrylic acid anion exchange resin, preparing immobilized β -galactosidase, preparing immobilized β -galactosidase particles, preparing galactooligosaccharide clear solution, preparing galactooligosaccharide syrup and the like to finally obtain the galactooligosaccharide powder, wherein the method comprises the steps of hydrolyzing lactose by using extracellular β -galactosidase to improve the production efficiency and save energy, immobilizing β -galactosidase and then producing to facilitate subsequent separation and purification, adding saccharomyces cerevisiae in the latter half of lactose hydrolysis reaction to improve the content of galactose and improve the quality of products, and filtering and nano-filtering to remove macromolecules in galactooligosaccharide before concentrating the galactooligosaccharide solution to further improve the purity of the products.
Description
Technical Field
The invention relates to the technical field related to saccharide preparation, in particular to a preparation method of galactooligosaccharide powder.
Background
Galacto-oligosaccharide is one of the functional oligosaccharides which have been developed internationally at present, and is the only oligosaccharide in various functional oligosaccharide varieties which is derived from animal milk at present. The molecular structure is generally that 1-7 galactosyl groups are connected on galactose or glucose molecules.
The preparation method of the galactooligosaccharide mainly comprises five methods, namely extraction from natural raw materials, acid hydrolysis of natural polysaccharide, chemical synthesis, fermentation synthesis and enzymatic synthesis, wherein the enzymatic synthesis is a mainstream production method, lactose is used as a raw material in the enzymatic synthesis, the lactose is hydrolyzed by β -galactosidase to generate glucose and galactose, and then 1-4 galactose molecules are connected on a galactosyl group by β -galactosidase catalysis to obtain the galactooligosaccharide.
The strains used for producing β -galactosidase at present are microorganisms such as Kluyveromyces fragilis (Kluyveromyces fragilis), Aspergillus niger (Aspergillus niger) Aspergillus oryzae (Aspergillus oryzae), and Kluyveromyces lactis (Kluyveromyces lactis), wherein β -galactosidase produced by Kluyveromyces fragilis is intracellular enzyme, and β -galactosidase produced by lactic acid yeast, Aspergillus niger, Aspergillus oryzae, Rhizopus oryzae, and the like is extracellular enzyme.
However, lactose is a disaccharide formed by combining glucose and galactose, the formation of galactose is accompanied with the formation of glucose along with the hydrolysis of lactose, and the excessive glucose causes the proportion of synthesized galacto-oligosaccharide to fail to meet the standard. In addition, the purity of the enzymatically synthesized galactooligosaccharides is limited by the current separation and purification techniques.
Disclosure of Invention
Aiming at the technical problems of overhigh glucose content and limited purity in galactose in the prior art, the invention provides a preparation method of galactooligosaccharide powder.
In order to achieve the purpose, the technical scheme of the invention is as follows:
1. a preparation method of galactooligosaccharide powder comprises the following steps:
s1) stirring glutaraldehyde and macroporous weakly basic acrylic acid anion exchange resin for 6-12 h at 16-32 ℃ to prepare activated macroporous weakly basic acrylic acid anion exchange resin;
s2) stirring β -galactosidase solution and the activated macroporous weak base acrylic acid anion exchange resin prepared in S1 to react for 12-24 h at 16-32 ℃ to prepare immobilized β -galactosidase;
s3) crushing the immobilized β -galactosidase prepared in the step S2, adding the crushed immobilized β -galactosidase into a mixed solution with the pH of 5-7 and containing 1-5% by mass of calcium chloride and 0.15-0.30% by mass of chitosan, and stirring the mixed solution at 48-52 ℃ for 24-36 hours to prepare immobilized β -galactosidase particles;
s4) mixing the immobilized β -galactosidase particles prepared in the S3 with a lactose solution with the concentration of 200-500 g/L, stirring and reacting for 6-8 h at the temperature of 35-40 ℃, adding Saccharomyces cerevisiae (Saccharomyces cerevisiae) to continue reacting for 4-6 h, and consuming glucose in the Saccharomyces cerevisiae to prepare galactooligosaccharide clear liquid with the purity of more than 57%;
s5) filtering to remove macromolecular proteins in the galactooligosaccharide clear solution prepared in the S4, and performing nanofiltration by using an organic membrane with the membrane aperture of 200-1000 Da to prepare a galactooligosaccharide solution;
s6) concentrating the galactooligosaccharide solution prepared in S5 to prepare galactooligosaccharide syrup;
s7) slowly adding maltodextrin and/or starch to the galactooligosaccharide syrup prepared in S6 under stirring until the galactooligosaccharide syrup becomes powder, namely, the galactooligosaccharide powder.
Further, the β -galactosidase in S2 was subjected to salting out purification treatment before use.
Furthermore, the ratio of the β -galactosidase to the macroporous weakly basic acrylic acid anion exchange resin in S2 is 100-200U/g, namely the enzyme activity of the β -galactosidase contained in each gram of the macroporous weakly basic acrylic acid anion exchange resin is 100-200U.
Further, the concentration of the β -galactosidase solution added in the S2 is 0.4-2.0U/mL.
Further, the stirring reaction in S4 needs 4000-20000U of the immobilized β -galactosidase to transport lactose solution to the reactor for reaction per kilogram of lactose on a dry basis.
Further, in the concentration in S6, the galactooligosaccharide solution prepared in S5 is concentrated by a plate evaporator until the solid content is 70-75%, and the concentration is stopped.
The invention has the following advantages:
1. the β -galactosidase outside the cells is utilized to hydrolyze the lactose, so that the production efficiency is improved, and the energy is saved;
2.β -galactosidase is immobilized and then produced, so that subsequent β -galactosidase can be conveniently separated from the product;
3. adding Saccharomyces cerevisiae (Saccharomyces cerevisiae) in the latter half of lactose hydrolysis reaction, and selectively consuming glucose generated by lactose hydrolysis by using the characteristic that the Saccharomyces cerevisiae preferentially utilizes glucose as a carbon source, so as to improve the content of galactose and the quality of the product;
4. and (3) filtering and nano-filtering the galactooligosaccharide solution before concentration to remove macromolecules in the galactooligosaccharide, thereby further improving the purity of the product.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example one
S1) stirring glutaraldehyde and macroporous weakly basic acrylic acid anion exchange resin for 6-12 h at 16-32 ℃ to prepare activated macroporous weakly basic acrylic acid anion exchange resin;
s2) mixing β -galactosidase solution with the concentration of 0.4U/mL with the activated macroporous weak base acrylic acid anion exchange resin prepared by S1 according to the proportion that the enzyme activity of β -galactosidase contained in each gram of macroporous weak base acrylic acid anion exchange resin is 100U, and then stirring and reacting for 12h at 16 ℃ to prepare immobilized β -galactosidase;
s3) pulverizing the immobilized β -galactosidase prepared in S2, adding the pulverized immobilized β -galactosidase into a mixed solution with the pH of 5 and containing 1% by mass of calcium chloride and 0.15% by mass of chitosan, and stirring the mixed solution at 48 ℃ for 24 hours to prepare immobilized β -galactosidase granules;
s4) mixing the immobilized β -galactosidase particles prepared in the S3 with a lactose solution with the concentration of 200g/L, conveying the lactose solution into a reactor according to the consumption of 4000U of immobilized β -galactosidase per kilogram of lactose on a dry basis, stirring and reacting for 6 hours at 35 ℃, adding saccharomyces cerevisiae, continuing to react for 4 hours according to the original conditions, and consuming glucose in the saccharomyces cerevisiae to prepare a galactooligosaccharide clear solution with the purity of 57%;
s5) filtering to remove macromolecular proteins in the galactooligosaccharide clear solution prepared by the S4, and performing nanofiltration by using an organic membrane with the membrane aperture of 200Da to prepare a galactooligosaccharide solution;
s6) concentrating the galacto-oligosaccharide solution prepared in S5 by a plate evaporator until the solid content is 70%, and stopping concentrating to prepare galacto-oligosaccharide syrup;
s7) slowly adding maltodextrin to the galactooligosaccharide syrup prepared in the S6 under the stirring state until the galactooligosaccharide syrup becomes powder, thus obtaining galactooligosaccharide powder, wherein the mass content of the galactooligosaccharide in the powder is 90% in total sugar (galactooligosaccharide, glucose and lactose) through HPLC detection.
Example two
S1) stirring glutaraldehyde and macroporous weakly basic acrylic acid anion exchange resin for 9h at 24 ℃ to prepare activated macroporous weakly basic acrylic acid anion exchange resin;
s2) mixing β -galactosidase solution with the concentration of 1.2U/mL with the activated macroporous weak base acrylic acid anion exchange resin prepared by S1 according to the proportion that the enzyme activity of β -galactosidase contained in each gram of macroporous weak base acrylic acid anion exchange resin is 150U, and then stirring and reacting for 18h at 20 ℃ to prepare immobilized β -galactosidase;
s3) pulverizing the immobilized β -galactosidase prepared in S2, adding the pulverized immobilized β -galactosidase into a mixed solution with the pH of 6 and containing 3% by mass of calcium chloride and 0.20% by mass of chitosan, and stirring the mixed solution at 50 ℃ for 30 hours to prepare immobilized β -galactosidase particles;
s4) mixing the immobilized β -galactosidase particles prepared in the S3 with a lactose solution with the concentration of 300g/L, conveying the lactose solution into a reactor according to the consumption of 10000U of immobilized β -galactosidase per kilogram of lactose on a dry basis, stirring and reacting for 7 hours at 37 ℃, adding saccharomyces cerevisiae, continuing to react for 5 hours according to the original conditions, and consuming glucose in the saccharomyces cerevisiae to prepare a galactooligosaccharide clear solution with the purity of 60%;
s5) filtering to remove macromolecular proteins in the galactooligosaccharide clear solution prepared by the S4, and performing nanofiltration by using an organic membrane with the membrane aperture of 600Da to prepare a galactooligosaccharide solution;
s6) concentrating the galactooligosaccharide solution prepared in S5 by a plate evaporator until the solid content is 73%, and stopping concentrating to prepare galactooligosaccharide syrup;
s7) slowly adding starch into the galactooligosaccharide syrup prepared in S6 under stirring until the galactooligosaccharide syrup becomes powder, thus obtaining galactooligosaccharide powder, wherein the mass content of the galactooligosaccharide in the powder is 92% in total sugar (galactooligosaccharide, glucose and lactose) through HPLC detection.
EXAMPLE III
S1) stirring glutaraldehyde and macroporous weakly basic acrylic acid anion exchange resin for 10h at 28 ℃ to prepare activated macroporous weakly basic acrylic acid anion exchange resin;
s2) mixing β -galactosidase solution with the concentration of 1.8U/mL with the activated macroporous weak base acrylic acid anion exchange resin prepared by S1 according to the proportion that the enzyme activity of β -galactosidase contained in each gram of macroporous weak base acrylic acid anion exchange resin is 180U, and then stirring and reacting for 20h at 28 ℃ to prepare immobilized β -galactosidase;
s3) pulverizing the immobilized β -galactosidase prepared in S2, adding the pulverized immobilized β -galactosidase into a mixed solution with pH of 6 and containing 4% by mass of calcium chloride and 0.25% by mass of chitosan, and stirring the mixed solution at 50 ℃ for 32 hours to prepare immobilized β -galactosidase particles;
s4) mixing the immobilized β -galactosidase particles prepared in the S3 with a lactose solution with the concentration of 400g/L, conveying the lactose solution into a reactor according to the dosage of immobilized β -galactosidase with the requirement of 16000U per kilogram of lactose on a dry basis, stirring and reacting for 7 hours at 39 ℃, adding saccharomyces cerevisiae, continuing to react for 5 hours according to the original conditions, and consuming glucose in the saccharomyces cerevisiae to prepare a galactooligosaccharide clear solution with the purity of 65%;
s5) filtering to remove macromolecular proteins in the galactooligosaccharide clear solution prepared from S4, and performing nanofiltration by using an organic membrane with the membrane aperture of 800Da to prepare a galactooligosaccharide solution;
s6) concentrating the galacto-oligosaccharide solution prepared in S5 by a plate evaporator until the solid content is 72%, and stopping concentrating to prepare galacto-oligosaccharide syrup;
s7) slowly adding maltodextrin and starch into the galactooligosaccharide syrup prepared in S6 under stirring until the galactooligosaccharide syrup becomes powder, thus obtaining galactooligosaccharide powder, wherein the mass content of the galactooligosaccharide in the powder is 91% in total sugar (galactooligosaccharide, glucose and lactose) through HPLC detection.
Example four
S1) stirring glutaraldehyde and macroporous weakly basic acrylic acid anion exchange resin for 12h at 32 ℃ to prepare activated macroporous weakly basic acrylic acid anion exchange resin;
s2) mixing β -galactosidase solution with the concentration of 2.0U/mL with the activated macroporous weak base acrylic acid anion exchange resin prepared by S1 according to the proportion that the enzyme activity of β -galactosidase contained in each gram of macroporous weak base acrylic acid anion exchange resin is 200U, and then stirring and reacting for 24h at 32 ℃ to prepare immobilized β -galactosidase;
s3) pulverizing the immobilized β -galactosidase prepared in S2, adding the pulverized immobilized β -galactosidase into a mixed solution with the pH of 7 and containing 5% by mass of calcium chloride and 0.30% by mass of chitosan, and stirring the mixed solution at 52 ℃ for 36 hours to prepare immobilized β -galactosidase granules;
s4) mixing the immobilized β -galactosidase particles prepared in the S3 with a lactose solution with the concentration of 500g/L, conveying the lactose solution into a reactor according to the dosage of 20000U immobilized β -galactosidase required by each kilogram of lactose on a dry basis, stirring and reacting for 8 hours at 40 ℃, adding saccharomyces cerevisiae, continuing to react for 6 hours according to the original conditions, and consuming glucose in the reaction to prepare galacto-oligosaccharide clear liquid with the purity of more than 67%;
s5) filtering to remove macromolecular proteins in the galactooligosaccharide clear solution prepared by the S4, and performing nanofiltration by using an organic membrane with the membrane aperture of 1000Da to prepare a galactooligosaccharide solution;
s6) concentrating the galacto-oligosaccharide solution prepared in S5 by a plate evaporator until the solid content is 75%, and stopping concentrating to prepare galacto-oligosaccharide syrup;
s7) slowly adding a mixture of maltodextrin and starch into the galactooligosaccharide syrup prepared in S6 under stirring until the galactooligosaccharide syrup becomes powder, thus obtaining galactooligosaccharide powder, wherein the mass content of the galactooligosaccharide in the powder is 93% in total sugar (galactooligosaccharide, glucose and lactose) through HPLC detection.
EXAMPLE five
On the basis of any of the first to fourth embodiments, β -galactosidase in S2 was purified by salting out before use, and other conditions were the same as those in the previous embodiment.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in the embodiments without departing from the principles and spirit of the invention, and these embodiments are within the scope of the invention.
Claims (6)
1. A preparation method of galactooligosaccharide powder is characterized in that: the method comprises the following steps:
s1) stirring glutaraldehyde and macroporous weakly basic acrylic acid anion exchange resin for 6-12 h at 16-32 ℃ to prepare activated macroporous weakly basic acrylic acid anion exchange resin;
s2) stirring β -galactosidase solution and the activated macroporous weak base acrylic acid anion exchange resin prepared in S1 to react for 12-24 h at 16-32 ℃ to prepare immobilized β -galactosidase;
s3) crushing the immobilized β -galactosidase prepared in the step S2, adding the crushed immobilized β -galactosidase into a mixed solution with the pH of 5-7 and containing 1-5% by mass of calcium chloride and 0.15-0.30% by mass of chitosan, and stirring the mixed solution at 48-52 ℃ for 24-36 hours to prepare immobilized β -galactosidase particles;
s4) mixing the immobilized β -galactosidase particles prepared in the S3 with a lactose solution with the concentration of 200-500 g/L, stirring and reacting for 6-8 h at the temperature of 35-40 ℃, adding saccharomyces cerevisiae, and continuing to react for 4-6 h to prepare galactooligosaccharide clear liquid with the purity of more than 57%;
s5) filtering to remove macromolecular proteins in the galactooligosaccharide clear solution prepared in the S4, and performing nanofiltration by using an organic membrane with the membrane aperture of 200-1000 Da to prepare a galactooligosaccharide solution;
s6) concentrating the galactooligosaccharide solution prepared in S5 to prepare galactooligosaccharide syrup;
s7) adding maltodextrin and/or starch to the galactooligosaccharide syrup prepared in S6 to obtain a galactooligosaccharide powder.
2. The method for preparing galactooligosaccharide powder of claim 1, wherein the β -galactosidase in S2 is purified by salting out before use.
3. The method for preparing galactooligosaccharide powder of claim 1, wherein the ratio of the β -galactosidase to the macroporous weakly basic acrylic acid anion exchange resin in S2 is 100-200U/g.
4. The method for preparing galactooligosaccharide powder according to any one of claims 1 to 3, wherein the concentration of the β -galactosidase solution added to S2 is 0.4 to 2.0U/mL.
5. The method for preparing galactooligosaccharide powder of claim 1, wherein the stirring reaction in S4 requires 4000-20000U of the immobilized β -galactosidase to transport lactose solution to a reactor for reaction per kg of lactose on a dry basis.
6. The method for preparing galactooligosaccharide powder according to claim 1, wherein: and in the S6, concentrating the galactooligosaccharide solution prepared in the S5 by using a plate evaporator until the solid content is 70-75%, and stopping concentrating.
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| CN119242737B (en) * | 2024-12-04 | 2025-09-19 | 浙江晟格生物科技有限公司 | Preparation method of galacto-oligosaccharide |
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