CN102676614A - Method for preparing galacto-oligosaccharide - Google Patents
Method for preparing galacto-oligosaccharide Download PDFInfo
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- CN102676614A CN102676614A CN2012101729772A CN201210172977A CN102676614A CN 102676614 A CN102676614 A CN 102676614A CN 2012101729772 A CN2012101729772 A CN 2012101729772A CN 201210172977 A CN201210172977 A CN 201210172977A CN 102676614 A CN102676614 A CN 102676614A
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- Prior art keywords
- beta
- galactosidase enzymes
- preparation
- galactooligosaccharide
- lactose
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- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 53
- 235000021255 galacto-oligosaccharides Nutrition 0.000 title claims abstract description 21
- 150000003271 galactooligosaccharides Chemical class 0.000 title claims abstract description 21
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 84
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 84
- 239000007788 liquid Substances 0.000 claims abstract description 44
- 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 35
- 239000008101 lactose Substances 0.000 claims abstract description 35
- 239000011347 resin Substances 0.000 claims abstract description 22
- 229920005989 resin Polymers 0.000 claims abstract description 22
- 238000003756 stirring Methods 0.000 claims abstract description 20
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims description 45
- 108090000790 Enzymes Proteins 0.000 claims description 45
- 241000193752 Bacillus circulans Species 0.000 claims description 34
- 238000002360 preparation method Methods 0.000 claims description 33
- 238000000855 fermentation Methods 0.000 claims description 24
- 230000004151 fermentation Effects 0.000 claims description 24
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 239000012531 culture fluid Substances 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 12
- 239000006052 feed supplement Substances 0.000 claims description 10
- 230000004913 activation Effects 0.000 claims description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 9
- 229910052760 oxygen Inorganic materials 0.000 claims description 9
- 239000001301 oxygen Substances 0.000 claims description 9
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 7
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 235000005822 corn Nutrition 0.000 claims description 7
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 7
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 7
- 235000017550 sodium carbonate Nutrition 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 5
- 238000013019 agitation Methods 0.000 claims description 3
- 238000001471 micro-filtration Methods 0.000 claims description 3
- 238000010899 nucleation Methods 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 241000170793 Phalaris canariensis Species 0.000 claims description 2
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 15
- 239000006188 syrup Substances 0.000 abstract description 14
- 235000020357 syrup Nutrition 0.000 abstract description 14
- 238000001914 filtration Methods 0.000 abstract description 13
- 230000008569 process Effects 0.000 abstract description 8
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 abstract description 3
- 238000005265 energy consumption Methods 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 229930182830 galactose Natural products 0.000 description 35
- 108010093096 Immobilized Enzymes Proteins 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- 210000001822 immobilized cell Anatomy 0.000 description 12
- 230000000694 effects Effects 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 239000008367 deionised water Substances 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 235000008504 concentrate Nutrition 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 238000000151 deposition Methods 0.000 description 4
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 238000011085 pressure filtration Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 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
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 241001138401 Kluyveromyces lactis Species 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004061 bleaching Methods 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 235000010410 calcium alginate Nutrition 0.000 description 2
- 239000000648 calcium alginate Substances 0.000 description 2
- 229960002681 calcium alginate Drugs 0.000 description 2
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
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- 238000007738 vacuum evaporation Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 240000005384 Rhizopus oryzae Species 0.000 description 1
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
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- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
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- 230000000968 intestinal effect Effects 0.000 description 1
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- 230000003834 intracellular effect Effects 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
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- 239000012528 membrane Substances 0.000 description 1
- 238000011169 microbiological contamination Methods 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
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- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- -1 o-nitrophenol galactoside Chemical class 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a method for preparing galacto-oligosaccharide. The method comprises the following steps: (1) reacting glutaraldehyde with a macroporous weak basic styrene resin for 16 to 24 hours with stirring at the temperature of between 15 and 35DEG C to activate the resin; (2) reacting liquid beta-galactosidase with the activated macroporous weak basic styrene resin obtained in the step (1) for 16 to 24 hours with stirring at the temperature of between 15 and 35DEG C to obtain cured beta-galactosidase; and (3) adding the cured beta-galactosidase into a lactose solution, and reacting for 24 to 36 hours with stirring at the temperature of between 48 and 52DEG C. By the method for producing the galacto-oligosaccharide, the filter pressure can be kept stable in a syrup filtering process, the yield of the galacto-oligosaccharide is improved, the energy consumption is reduced and the industrial production requirement can be met.
Description
Technical field
The present invention relates to a kind of Preparation of Galactooligosaccharide method, particularly a kind of method of utilizing immobilized enzyme to produce oligomeric galactose.
Background technology
(galacto-oligosaccharide is one type of natural functional oligose that is present in the animal breast milk GOS) to oligomeric galactose, has outstanding moisture holding capacity and extremely strong acid heat stability; Can not be digested and assimilated by people's small intestine, but can be fermented, can promote bifidus bacillus propagation by colonic microflora; Suppress harmful pathogenic bacteria and spoilage organism growth; Promote the absorption of calcium, magnesium, potassium, help the generation of vitamin B group, nicotinic acid and folic acid, ability effective stimulus intestines peristalsis; Reduce and prevent the generation of constipation, reduce the adjusting intestinal microecologies such as content of toxic substance in the colon, the effect of promotion intestinal health.
Oligomeric galactose is to be that raw material makes through beta-galactosidase enzymes (EC3.2.1.23) catalysis with the lactose, is the oligosaccharides class mixture that connects 1～4 galactose molecule on the oligomeric galactose base in lactose molecule with β (1 → 2) key or β (1 → 3), β (1 → 4) key, β (1 → 6) key.
Usually use free cell or free beta-galactosidase enzymes enzyme to produce oligomeric galactose at present.In process of production, product can be introduced impurity such as free cell or protein, and needing increases complicated purifying technique, is unfavorable for cleaner production.For head it off, worked out cell has been carried out immobilization, and utilized the method for immobilized cells produce oligomeric galactose.This method can be simplified production technique, and the immobilized cell that contains beta-galactosidase enzymes can reuse, and has saved production cost.The calcium alginate embedded method of the normal employing of immobilized cell; Utilizing immobilized cell to transform in the process of lactose; Because the higher and alginate calcium bad mechanical strength of temperature causes immobilized cell broken easily, thereby introduces in the syrup; Cause syrup easy blocking filtration unit in follow-up filtration operation, it is very fast that filter pressure is raise.
In addition, the bacterial classification that is used to produce beta-galactosidase enzymes at present has Kluyveromyces fragilis (Kluyveromyces fragilis), black mold (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), Kluyveromyces lactis mikrobes such as (Kluyveromyces lactis).Wherein, the beta-galactosidase enzymes that Kluyveromyces fragilis produced is an intracellular enzyme, and the beta-galactosidase enzymes that saccharomyces lactis, black mold, aspergillus oryzae, Rhizopus oryzae etc. are produced is an extracellular enzyme.Obtaining of beta-galactosidase enzymes need be carried out broken thalline, separated step such as purifications in the born of the same parents, and this has increased production link and production unit, also increase of power consumption simultaneously.If bacterial strain directly is secreted into beta-galactosidase enzymes outside the born of the same parents, then greatly facilitates the separation and the utilization of enzyme, and save production cost.The beta-galactosidase enzymes that known Bacillus circulans produced is an extracellular enzyme, and has resistant to elevated temperatures character, but utilizes report that Bacillus circulans produces beta-galactosidase enzymes seldom.
Summary of the invention
One object of the present invention is to provide a kind of Preparation of Galactooligosaccharide method, utilizes this method can obtain higher oligomeric galactose yield.
Another object of the present invention is to provide a kind of preparation method of immobilization beta-galactosidase enzymes; Utilize the immobilized enzyme of this method preparation to have advantages such as physical strength height, physical and chemical performance be stable, can effectively solve in the filtration operation that the immobilized cell that adopts calcium alginate embedded method preparation or immobilized enzyme produce oligomeric galactose the filter pressure problem faster that rises.
Another purpose of the present invention is to provide a kind of method of utilizing Bacillus circulans (Bacillus circulans) to produce beta-galactosidase enzymes, and this method can significantly improve the output of beta-galactosidase enzymes.
The technical scheme that the present invention adopted is:
A kind of Preparation of Galactooligosaccharide method may further comprise the steps:
(1) use LUTARALDEHYDE 15-35 ℃ down and macropore weak base styron under agitation react 16-24h, make the resin activation;
(2) the activatory macropore weak base styron of getting liquid beta-galactosidase enzymes and step (1) gained obtains immobilized beta-galactosidase enzymes at 15-35 ℃ of following stirring reaction 16-24h;
(3) immobilized beta-galactosidase enzymes is added in the lactose solution, in 48-52 ℃ of stirring reaction 24-36h.
Preferably, the described beta-galactosidase enzymes of step (2) is purified through saltouing before use.
Preferably, the proportioning of beta-galactosidase enzymes and macropore weak base styron is the 100-200U/g resin in the step (2); The lactose concn that is added in the step (3) is 45%-52% (w/w), and adds the immobilization beta-galactosidase enzymes of 6000-10000U according to every kilogram of lactose (by butt).
Preferably, beta-galactosidase enzymes is produced by Bacillus circulans described in the step (2).
Preferably, beta-galactosidase enzymes prepares through following steps described in the step (2):
A. the Bacillus circulans that preserves is seeded to the LB solid medium, cultivates 16-24h down, make actication of culture in 36-38 ℃;
B. get the activatory bacterial classification inoculation to the LB liquid nutrient medium of 20-50ml, be seeded to behind the shaking culture 46h in the seeding tank that the LB substratum is housed, under 36-38 ℃, cultivate 4-6h, obtain seed culture fluid;
C. seed culture fluid is inserted in the fermentor tank that fermention medium is housed and cultivate 36-48h, obtain the Bacillus circulans fermented liquid;
D. with fermented liquid through micro-filtration or the centrifugal thalline of removing, again gained enzyme liquid is concentrated, obtain the liquid beta-galactosidase enzymes.
Preferably, above-mentioned Bacillus circulans is Bacillus circulans ATCC No.31382.
Preferably, the said best cultivation of step C is: with seed culture fluid insert in the fermentor tank cultivate 16-24h after, with feed supplement amount 4-6gL
-1And feed supplement speed is 0.4-0.6gL
-1H
-1Mode add lactose, continue fermentation 20-28h.
Preferably, the said fermention medium of step C is: lactose 5-15g/L, semi-lactosi 5-10g/L, soy peptone 10-30g/L, corn extract 4-6g/L, yeast extract 2-4g/L, Secondary ammonium phosphate 2-4g/L, yellow soda ash 1-2g/L, VT 18 8-15g/L, skimmer 0.5-2g/L.
Preferably, culture condition is described in the step C: temperature 36-38 ℃, pH6.8-7.5, dissolved oxygen>=30%, air flow 0.5-2VV
-1Min
-1, stirring velocity 120-220rpm.
A kind of oligomeric galactose that is obtained by method for preparing comprises pulpous state product and powder-like product.
The invention has the beneficial effects as follows: the present invention adopts the immobilization beta-galactosidase enzymes to produce oligomeric galactose; The resin physical strength that immobilization adopted is high, physical and chemical performance is stable; In the conversion process of lactose, can not change, be not easy fragmentation yet, thereby can not stop up filtration unit; Can make pressure keep stable, overcome pressure that immobilized cell the brings problem faster that rises.Immobilized enzyme used in the production process can reclaim repeated use, and the beta-galactosidase enzymes of its service efficiency specific ionization is much higher, has the characteristics of suitable commercial scale prodn.It is simple that the present invention prepares the production technique of oligomeric galactose, and operation is convenient, can satisfy the requirement of industrialized production, the yield of oligomeric galactose >=57%.The present invention adopts Bacillus circulans to produce beta-galactosidase enzymes, because the beta-galactosidase enzymes that Bacillus circulans produced is an extracellular enzyme, therefore the separation and purification process of follow-up enzyme is greatly simplified, thereby has saved the equipment and the cost of separation and purification.Simultaneously, the beta-galactosidase enzymes that this bacterial classification produced has resistant to elevated temperatures character, therefore, produces the process of oligomeric galactose and can under comparatively high temps, carry out, and brings following at least benefit thus: the first, and reaction at high temperature can reduce microbiological contamination; The second, the solubleness of lactose is higher under the high temperature, and the yield of high more lactose starting point concentration oligomeric galactose is just high more; The 3rd, beta-galactosidase enzymes generally shows as the activity of hydrolyzes lactose under low temperature, lower lactose concn, and the synthetic oligomeric galactose is less.In addition, the present invention improves fermentation mode, substratum and the fermentation condition that Bacillus circulans produces beta-galactosidase enzymes, makes the enzymatic productivity of beta-galactosidase enzymes obtain certain raising.
Embodiment
The bacterial strain Bacillus circulans of the product beta-galactosidase enzymes that uses in the following embodiment (Bacillus circulans) is numbered 31382 available from American Type Culture Collecti (ATCC).But the used bacterial strain of the present invention is not limited to Bacillus circulans ATCC No.31382, and other Bacillus circulans also is applicable to the present invention.
Below describe describing several notion of the present invention.
Free beta-galactosidase enzymes enzyme activity determination: contain (pH 6.0) adding 1mL beta-galactosidase enzymes enzyme liquid in the ONPG 0.1M phosphoric acid buffer that concentration is 4mg/mL at 1mL; At 50 ℃ of reaction 10min; Add 2mL10% sodium carbonate solution termination reaction, through measuring the amount of hydrolysis of o-nitrophenol cubage o-nitrophenol galactoside (ONPG) in the product.The required enzyme amount of PM hydrolysis 1 μ M ONPG is defined as enzyme unit (U) alive under this condition.
The assay of oligomeric galactose: with reference to AOAC Official Method 2001.02 " Determination of trans-Galacto-oligosaccharides (TGOS) in Selected Food Products ".
The invention provides a kind of Preparation of Galactooligosaccharide method, may further comprise the steps:
(1) use LUTARALDEHYDE 15-35 ℃ down and macropore weak base styron under agitation react 16-24h, make the resin activation;
(2) the activatory macropore weak base styron of getting liquid beta-galactosidase enzymes and step (1) gained obtains immobilized beta-galactosidase enzymes at 15-35 ℃ of following stirring reaction 16-24h;
(3) immobilized beta-galactosidase enzymes is added in the lactose solution, in 48-52 ℃ of stirring reaction 24-36h.
The present invention also provides a kind of Preparation of Galactooligosaccharide method, and its step comprises the preparation of liquid beta-galactosidase enzymes, the preparation and immobilization beta-galactosidase enzymes production oligomeric galactose three parts of immobilization beta-galactosidase enzymes.Below describe to each part.
1. the preparation of liquid beta-galactosidase enzymes
(Bacillus circulans) is the bacterial classification that sets out with Bacillus circulans, activated, seed tank culture, fermentation, removes thalline, concentrates and obtain the liquid beta-galactosidase enzymes, and concrete steps are following:
1) actication of culture: the Bacillus circulans that glycerine is preserved is inoculated in the flat board that the LB solid medium is housed, and cultivates 16 ~ 24 hours, and obtains the activation Bacillus circulans for 36 ~ 38 ℃.
2) seed tank culture: bacterial classification inoculation is in the triangular flask that 20 ~ 50mLLB liquid nutrient medium is housed after picking one to the two ring activation; After the shaking culture 4 ~ 6 hours; Insert by the inoculum size of 1 ~ 3% (v/v) again and be equipped with in the seeding tank of LB substratum, in 36 ~ 38 ℃, dissolved oxygen>=30%, air flow 0.5 ~ 2V.V
-1.min
-1, stirring velocity 120 ~ 400rpm cultivated 4 ~ 6 hours, obtained seed tank culture liquid.
3) fermentation culture: the fermentor tank that seed tank culture liquid is equipped with fermention medium by the inoculum size access of 1 ~ 3% (v/v) carried out fermentation culture after 16 ~ 20 hours, was 5g.L by the feed supplement amount
-1, feed supplement speed 0.5g.L
-1.h
-1Mode add lactose to fermentor tank, continue fermentation after 20 ~ 28 hours, obtain the Bacillus circulans fermented liquid.Wherein, Fermentation comprises following several kinds of materials with substratum: lactose 5 ~ 15g/L, semi-lactosi 5 ~ 10g/L, soy peptone 10 ~ 30g/L, corn extract 4 ~ 6g/L, yeast extract 2 ~ 4g/L, Secondary ammonium phosphate 2 ~ 4g/L, yellow soda ash 1 ~ 2g/L, VT 18 8 ~ 15g/L, skimmer 0.5 ~ 2g/L, its culture condition are at 36 ℃ ~ 38 ℃, pH7.0 ~ 7.5, dissolved oxygen>=30%, air flow 0.5 ~ 2V.V
-1.min
-1, stirring velocity 120 ~ 220rpm.
4) remove thalline, concentrated enzyme liquid: fermented liquid through micro-filtration or centrifugal removal thalline, is concentrated with cryogenic vacuum or the concentrated enzyme liquid of ultrafiltration and concentration method again, and obtaining enzyme work is the liquid beta-galactosidase enzymes of 80 ~ 120U/mL.
2. the preparation of immobilization beta-galactosidase enzymes
The free beta-galactosidase enzymes is processed the immobilized enzyme of definite shape with immobilization technology.
The preparation of immobilization beta-galactosidase enzymes is through the ammonium sulfate precipitation preliminary purification with the liquid beta-galactosidase enzymes; Use chemical coupling method and resin crosslinks then; Ammonium sulfate use final concentration is 25 ~ 30% in the said ammonium sulfate precipitation process, and solid-liquid separation can adopt filter paper plate pressure filtration centrifugal or 0.2 ~ 1 μ m aperture; Used resin is a macropore weak base styron; Used linking agent is a LUTARALDEHYDE.Immobilization way is first activated resin, adsorbs crosslinked with enzyme again.The resin activation condition is: it is 0.5 ~ 1.5% that LUTARALDEHYDE uses final concentration, and temperature is 15 ~ 35 ℃, and rotating speed is 50 ~ 100rpm, and the reaction times is 16 ~ 24 hours.The enzyme immobilization condition is: the proportioning of enzyme and resin is that the required enzyme amount of every gram macropore weak base styron is 100 ~ 200U, and temperature is 15 ~ 35 ℃, and rotating speed is 50 ~ 100rpm, and the reaction times is 16 ~ 24 hours.
3. the immobilization beta-galactosidase enzymes is produced oligomeric galactose
With immobilization beta-galactosidase enzymes production batch reactions method that oligomeric galactose adopted is the lactose solution that in retort, adds 45 ~ 52% (w/w); The immobilization beta-galactosidase enzymes that adds 6000 ~ 10000U enzyme amount by every kilogram of lactose (butt calculation); Controlled temperature is at 48 ~ 52 ℃, and mixing speed is the conditioned response of 50 ~ 100rpm 24 ~ 36 hours, after reaction is accomplished; Reclaim immobilized enzyme; Be used for the production of next batch raw material, it is the oligomeric galactose syrup more than 57% that reaction solution obtains oligomeric galactose content through filtration, vacuum concentration, and perhaps further drying, interpolation auxiliary material, drying obtain the powdery oligomeric galactose with spissated syrup.
Below in conjunction with embodiment the present invention is further explained.
The preparation of embodiment 1 beta-galactosidase enzymes
1, actication of culture: glycerine is guaranteed that the Bacillus circulans of depositing is seeded to the LB solid medium, cultivate 18h in 37 ℃.
2, seed liquor is cultivated: with Bacillus circulans after the dull and stereotyped activation of LB; Picking one ring bacterial classification inoculation is in the triangular flask that 25mL LB substratum is housed; In 37 ℃, 180rpm shaking culture 4 hours, be transferred to then in the fermentor tank that 2.5L LB substratum is housed, in 37 ℃, stirring velocity 120 ~ 220rpm, air flow 1.25L/min; Cultivated 4 hours through adjustment rotating speed control dissolved oxygen >=30%, obtain seed culture fluid.
3, fermentation culture:
1) fermentation in advance: seed culture fluid is inoculated in the 500L fermentor tank that lactose 1250g, semi-lactosi 2000g, soy peptone 4000g, corn extract 1300g, yeast extract 650g, Secondary ammonium phosphate 650g, yellow soda ash 325g, VT 18 2275g, skimmer 125g, water 250kg are housed; Under 37 ℃; Stirring velocity 120 ~ 220rpm, air flow 125L/min; Control dissolved oxygen >=30% usefulness salt acid for adjusting pH value 7.4 through the adjustment rotating speed, fermentation culture 18 hours.Wherein the various compositions of substratum comprise that soy peptone, corn extract and yeast extract etc. all can buy from the market.And skimmer can use polyethers, polyether-modified silicon defoaming agent etc.
2) the fed-batch fermentation stage: in the fermentation system of step 1), begin to add 12.5% lactose solution 10L by 20mL/min stream.After the beginning feed supplement, every separated 2h sampling fermented liquid is measured the beta-galactosidase enzymes enzyme and is lived, and treats to stop to ferment after enzyme work stops to rise.
4, crude enzyme liquid preparation:
Get above-mentioned fermented liquid, under 4 ℃, the centrifugal 10min of 10000rpm collects middle part enzyme liquid, with enzyme liquid-0.01 ~-the 0.009MPa condition under evaporation concentration, obtaining enzyme activity is the beta-galactosidase enzymes crude enzyme liquid of 115U/mL.
The immobilization of embodiment 2 beta-galactosidase enzymess
Prepare the immobilization beta-galactosidase enzymes according to following steps:
1, thick enzyme is refining
Get embodiment 1 and concentrate the enzyme liquid 100mL that obtains; After wherein slowly add ammonium sulfate 43.6g, treating that ammonium sulfate dissolves fully, in 4 ℃ leave standstill 2 hours after; Under 4 ℃; The centrifugal 10min collecting precipitation of 10000rpm is used 100mL pH6.0 phosphoric acid buffer dissolution precipitation then, and obtaining enzyme activity is 100U/mL beta-galactosidase enzymes refined liquid.
2, the immobilization of enzyme
(1) takes by weighing macropore weak base styron A103s100g, after washed with de-ionized water 2 ~ 3 times, add deionized water 2000mL; The glutaraldehyde solution that adds 40mL 50% again, 30 ℃ are stirred 16h, filter; Use the washed with de-ionized water resin then, filtration is dried moisture and is promptly got activated resin.
(2) in activated resin, add the beta-galactosidase enzymes refined liquid that contains 100mL, in 30 ℃ of shaking table vibrations 16 hours, filter, use the washed with de-ionized water resin then, filtration is dried moisture and is obtained the immobilization beta-galactosidase enzymes 100g that enzyme activity is 85U/g.
Embodiment 3 immobilized enzyme are produced oligomeric galactose
The adding mass concentration is 50% lactose solution 2000g in the 5L retort; The immobilized enzyme 80g that adds embodiment 2; After transforming 24 hours under 50 ℃, 80rpm condition; Adopt filter bag to collect immobilized enzyme, the syrup that is separated to through filter, vacuum concentrator concentrates that to obtain oligomeric galactose content be that 57.6% Brix is 76 syrup.
The preparation of embodiment 4 beta-galactosidase enzymess
1, actication of culture: glycerine is guaranteed that the Bacillus circulans of depositing is seeded to the LB solid medium, cultivate 18h in 37 ℃.
2, seed liquor is cultivated: with Bacillus circulans after the dull and stereotyped activation of LB; Picking two ring bacterial classifications are inoculated into respectively in the triangular flask of the 2 bottled 50mL of having LB substratum; In 37 ℃, 180rpm shaking culture 4 hours, be transferred to then in the fermentor tank that 10L LB substratum is housed, in 37 ℃, stirring velocity 120 ~ 220rpm, air flow 5L/min; Cultivated 4 hours through adjustment rotating speed control dissolved oxygen >=30%, obtain seed culture fluid.
3, fermentation culture:
1) fermentation in advance: seed culture fluid is inoculated in the 2000L fermentor tank that lactose 5kg, semi-lactosi 8kg, soy peptone 16kg, corn extract 5.2kg, yeast extract 2.6kg, Secondary ammonium phosphate 2.6kg, yellow soda ash 1.3kg, VT 18 9.1kg, skimmer 0.5kg, water 1000kg are housed; Under 37 ℃; Stirring velocity 120 ~ 220rpm, air flow 500L/min; With salt acid for adjusting pH value to 7.4, fermentation culture 18 hours.
2) the fed-batch fermentation stage: 1) begin in the fermentation system to add 20% lactose solution 25L by 25mL/min stream.After the beginning feed supplement, every separated 2h sampling fermented liquid is measured the beta-galactosidase enzymes enzyme and is lived, and treats to stop to ferment after enzyme work stops to rise.
3, crude enzyme liquid preparation
With above-mentioned fermented liquid below 30 ℃, to advance film pressure be under 0.3 ~ 0.4MPa condition, removes thalline with the ceramic membrane in the 0.5 μ m aperture cross flow filter that circulates, an amount of water that adds washes out enzyme in this process, finally obtains clear clear enzyme solution.Then with clear clear enzyme solution below 30 ℃, to advance film pressure be under 0.25 ~ 0.3MPa condition, uses the rolled film ultrafiltration and concentration of molecular weight cut-off as 10KD, obtain enzyme and live and be the crude enzyme liquid of 98.5U/mL.
The immobilization of embodiment 5 beta-galactosidase enzymess
Prepare the immobilization beta-galactosidase enzymes according to following steps:
1, beta-galactosidase enzymes is refining
The crude enzyme liquid 200L that gets embodiment 4 preparations adds 87.2kg ammonium sulfate; After stirring at room to ammonium sulfate dissolves fully; After leaving standstill 2 hours; Deposition is held back in clarification cardboard pressure filtration with 0.2 μ m aperture, will precipitate wash cycles with the phosphoric acid buffer of 200LpH6.0 then and get off, and promptly getting enzyme activity is 85U/mL beta-galactosidase enzymes refined liquid.
2, the preparation of immobilization beta-galactosidase enzymes
Take by weighing macropore weak base styron A103s 150kg; After washed with de-ionized water 2 ~ 3 times, add deionized water to 3000L, add 60L concentration again and be 50% glutaraldehyde solution; Stirred 24 hours in 15 ℃, 80rpm; Filter, remove remaining LUTARALDEHYDE with the washed with de-ionized water resin then, centrifuge dripping promptly gets activated resin.Activated resin is joined in the beta-galactosidase enzymes refined liquid of 200L step 1 preparation; Stirred 24 hours in 15 ℃, 80rpm, filter, use the washed with de-ionized water resin then; Centrifuge dripping moisture, promptly getting enzyme activity is the immobilization beta-galactosidase enzymes 150kg of 100U/g.
Embodiment 6 immobilized enzyme are produced oligomeric galactose
At 4m
3The adding mass concentration is 50% lactose solution 3500kg in jar; The immobilized enzyme 150kg that adds embodiment 5 preparations; Under 48 ℃, 80rpm condition, transform 36 hours; Adopt the enzyme strainer to reclaim immobilized enzyme, the syrup that is separated to further filters, vacuum concentrator concentrates that to obtain oligomeric galactose content be that 57.4% Brix is 75 syrup.
Embodiment 7 immobilized cells and immobilized enzyme are produced the comparison of oligomeric galactose
1, immobilized cell is produced oligomeric galactose
At 8m
3The adding mass concentration is 50% lactose solution 6000kg in jar; The immobilized cell that adds the preparation of patent CN201110049829.7 method; Under 60 ℃, 80rpm condition, transform 20 hours; Adopt the enzyme strainer to reclaim immobilized cell, the syrup that is separated to is further long-pending through filtering surface to be 5 ㎡ flame filter press pressure filtrations, obtains clarifying syrup and carries out desalination bleaching, the concentrated oligomeric galactose that obtains of rotation vacuum-evaporation again.
2, immobilized enzyme is produced oligomeric galactose
According to the inventive method, at 8m
3The adding mass concentration is 50% lactose solution 6000kg in jar; Add immobilized enzyme according to the invention; Under 50 ℃, 80rpm condition, transform 24 hours; Adopt the enzyme strainer to reclaim immobilized enzyme, the syrup that is separated to is further long-pending through filtering surface to be 5 ㎡ flame filter press pressure filtrations, obtains clarifying syrup and carries out desalination bleaching, the concentrated oligomeric galactose that obtains of rotation vacuum-evaporation again.
The Plate Filtration effect is following in the above-mentioned dual mode production oligomeric galactose process:
Can know by last table; Compare with immobilized cell; The syrup that adopts immobilized enzyme method to produce oligomeric galactose filters in the operation; Be not more than under the 0.4MPa condition at the control feed pressure, filtration area is that the syrupy ability of the disposable processing of the plate-and-frame filter press of 5 ㎡ is brought up to 60000kg by 6000kg, and filtration velocity is brought up to 6000kg/h by 3000kg/h.Therefore adopt immobilized enzyme method of the present invention to produce oligomeric galactose, the plate-and-frame filter press of unit surface filters syrupy ability and speed all is significantly improved.
Embodiment 8 fermentation process produce the influence of beta-galactosidase enzymes ability to Bacillus circulans
1, adopts the inventive method fermentative prepn beta-galactosidase enzymes
The method fermentative prepn beta-galactosidase enzymes of describing according to embodiment 1 was whenever measured the beta-galactosidase enzymes enzyme at a distance from 2h sampling fermented liquid and is lived in the fed-batch fermentation stage, and to reach that the highest enzyme lives in 42 hours be 47U/mL through measuring, ferment.
2, adopt ordinary method fermentative prepn beta-galactosidase enzymes
1) actication of culture: glycerine is guaranteed that the Bacillus circulans of depositing is seeded to the LB solid medium, cultivate 18h in 37 ℃.
2) seed liquor is cultivated: with Bacillus circulans after the dull and stereotyped activation of LB; Picking one ring bacterial classification inoculation is in the triangular flask that 25mL LB substratum is housed; In 37 ℃, 180rpm shaking culture 4 hours, be transferred to then in the fermentor tank that 2.5L LB substratum is housed, in 37 ℃, stirring velocity 120 ~ 220rpm, air flow 1.25L/min; Cultivated 4 hours through adjustment rotating speed control dissolved oxygen >=30%, obtain seed culture fluid.
3) fermentation culture: seed culture fluid is inoculated in the 500L fermentor tank that lactose 2500g, semi-lactosi 2000g, soy peptone 4000g, corn extract 1300g, yeast extract 650g, primary ammonium phosphate 650g, yellow soda ash 325g, VT 18 2275g, skimmer 125g, water 250kg are housed; Under 37 ℃; Stirring velocity 120 ~ 220rpm, air flow 125L/min; Control dissolved oxygen >=30% usefulness salt acid for adjusting pH value 7.4 through the adjustment rotating speed; Through measuring, fermentation culture reached the highest enzyme 33U/mL of being alive in 40 hours.
Therefore; The present invention has adopted lactose feed supplement mode to ferment and ordinary method is not carried out feed supplement during the fermentation; The result shows; Adopt the inventive method can make the enzyme work of gained beta-galactosidase enzymes bring up to 47U/mL from 33U/mL, the enzymatic productivity of Bacillus circulans is significantly improved.
Claims (10)
1. Preparation of Galactooligosaccharide method may further comprise the steps:
(1) use LUTARALDEHYDE 15-35 ℃ down and macropore weak base styron under agitation react 16-24h, make the resin activation;
(2) the activatory macropore weak base styron of getting liquid beta-galactosidase enzymes and step (1) gained obtains immobilized beta-galactosidase enzymes at 15-35 ℃ of following stirring reaction 16-24h;
(3) immobilized beta-galactosidase enzymes is added in the lactose solution, in 48-52 ℃ of stirring reaction 24-36h.
2. a kind of Preparation of Galactooligosaccharide method according to claim 1, wherein the described beta-galactosidase enzymes of step (2) is purified through saltouing before use.
3. a kind of Preparation of Galactooligosaccharide method according to claim 1, wherein the proportioning of beta-galactosidase enzymes and macropore weak base styron is the 100-200U/g resin in the step (2); The lactose concn that is added in the step (3) is 45%-52% (w/w), and adds 6000-10000U immobilization beta-galactosidase enzymes according to every kilogram of lactose (by butt).
4. a kind of Preparation of Galactooligosaccharide method according to claim 1, wherein beta-galactosidase enzymes is produced by Bacillus circulans described in the step (2).
5. a kind of Preparation of Galactooligosaccharide method according to claim 1, wherein beta-galactosidase enzymes prepares through following steps described in the step (2):
A. the Bacillus circulans that preserves is seeded to the LB solid medium, cultivates 16-24h down, make actication of culture in 36-38 ℃;
B. get the activatory bacterial classification inoculation to the LB liquid nutrient medium of 20-50ml, be seeded to behind the shaking culture 4-6h in the seeding tank that the LB substratum is housed, under 36-38 ℃, cultivate 4-6h, obtain seed culture fluid;
C. seed culture fluid is inserted in the fermentor tank that fermention medium is housed and cultivate 36-48h, obtain the Bacillus circulans fermented liquid;
D. with fermented liquid through micro-filtration or the centrifugal thalline of removing, again gained enzyme liquid is concentrated, obtain the liquid beta-galactosidase enzymes.
6. a kind of Preparation of Galactooligosaccharide method according to claim 5, wherein the said best cultivation of step C is: with seed culture fluid insert in the fermentor tank cultivate 16-20h after, add lactose, continue fermentation 20-28h.
7. a kind of Preparation of Galactooligosaccharide method according to claim 5, wherein said Bacillus circulans are Bacillus circulans ATCC No.31382.
8. a kind of Preparation of Galactooligosaccharide method according to claim 5, the wherein said mode of adding lactose is: feed supplement amount 4 ~ 6gL
-1, feed supplement speed is 0.4 ~ 0.6gL
-1H
-1
9. a kind of Preparation of Galactooligosaccharide method according to claim 5, wherein the said fermention medium of step C is: lactose 5-15g/L, semi-lactosi 5-10g/L, soy peptone 10-30g/L, corn extract 4-6g/L, yeast extract 2-4g/L, primary ammonium phosphate 2-4g/L, yellow soda ash 1-2g/L, VT 18 8-15g/L, skimmer 0.5-2g/L.
10. a kind of Preparation of Galactooligosaccharide method according to claim 5, wherein culture condition is described in the step C: temperature 36-38 ℃, pH6.8-7.5, dissolved oxygen>=30%, air flow 0.5-2VV
-1Min
-1, stirring velocity 120-220rpm.
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| CN102994591A (en) * | 2012-12-19 | 2013-03-27 | 山东龙力生物科技股份有限公司 | Method for preparing galactooligosaccharide co-produced ethanol |
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| CN105274164A (en) * | 2015-11-20 | 2016-01-27 | 保龄宝生物股份有限公司 | Preparation method of galactooligosaccharides |
| CN105441365B (en) * | 2015-12-31 | 2019-05-31 | 南通励成生物工程有限公司 | A kind of bacterial strain producing beta galactosidase |
| CN105441365A (en) * | 2015-12-31 | 2016-03-30 | 南通励成生物工程有限公司 | Beta-galactosidase producing strain |
| CN108384821A (en) * | 2017-12-18 | 2018-08-10 | 江苏省农业科学院 | A kind of preparation method of the oligosaccharide of promotion proliferation of intestinal probiotics |
| CN108384821B (en) * | 2017-12-18 | 2021-09-14 | 江苏省农业科学院 | Preparation method of oligosaccharide for promoting proliferation of intestinal probiotics |
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