CN102676614B - Method for preparing galacto-oligosaccharide - Google Patents
Method for preparing galacto-oligosaccharide Download PDFInfo
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- CN102676614B CN102676614B CN201210172977.2A CN201210172977A CN102676614B CN 102676614 B CN102676614 B CN 102676614B CN 201210172977 A CN201210172977 A CN 201210172977A CN 102676614 B CN102676614 B CN 102676614B
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- galactosidase enzymes
- lactose
- enzyme
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- 238000000034 method Methods 0.000 title abstract description 37
- 235000021255 galacto-oligosaccharides Nutrition 0.000 title abstract description 5
- 150000003271 galactooligosaccharides Chemical class 0.000 title abstract description 5
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 83
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 83
- 239000007788 liquid Substances 0.000 claims abstract description 42
- 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 34
- 229920005989 resin Polymers 0.000 claims abstract description 34
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000003756 stirring Methods 0.000 claims abstract description 22
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 102000004190 Enzymes Human genes 0.000 claims description 44
- 108090000790 Enzymes Proteins 0.000 claims description 44
- 229930182830 galactose Natural products 0.000 claims description 40
- 241000193752 Bacillus circulans Species 0.000 claims description 32
- 238000000855 fermentation Methods 0.000 claims description 32
- 230000004151 fermentation Effects 0.000 claims description 32
- 238000002360 preparation method Methods 0.000 claims description 19
- 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 12
- 239000012531 culture fluid Substances 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 12
- 230000004913 activation Effects 0.000 claims description 9
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- 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
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- 235000005822 corn Nutrition 0.000 claims description 7
- 239000013530 defoamer Substances 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
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- 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
- 238000004321 preservation Methods 0.000 claims description 2
- 239000006188 syrup Substances 0.000 abstract description 16
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- 238000005265 energy consumption Methods 0.000 abstract 1
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- 210000001822 immobilized cell Anatomy 0.000 description 11
- 230000000694 effects Effects 0.000 description 9
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- 230000001580 bacterial effect Effects 0.000 description 7
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- 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
- 210000004027 cell Anatomy 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
- 238000001556 precipitation Methods 0.000 description 4
- 238000011085 pressure filtration Methods 0.000 description 4
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- 241000228245 Aspergillus niger Species 0.000 description 3
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- 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
- 238000005352 clarification Methods 0.000 description 3
- 235000008504 concentrate Nutrition 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 150000003839 salts Chemical class 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
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- 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
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- 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
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- 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
- 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
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- 238000013467 fragmentation Methods 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (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 method of oligomeric galactose, particularly a kind of method utilizing immobilized enzyme to produce oligomeric galactose.
Background technology
Oligomeric galactose (galacto-oligosaccharide, GOS) be the natural functional oligose be present in animal breast milk of a class, there is 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 by colonic microflora, can promote that bifidus bacillus breeds, suppress harmful pathogenic bacteria and spoilage organism growth, promote calcium, magnesium, the absorption of potassium, be conducive to vitamin B group, the generation of nicotinic acid and folic acid, energy effective stimulus intestines peristalsis, reduce and prevent the generation of constipation, reduce the regulating intestinal canal Tiny ecosystem such as the content of toxic substance in colon, promote the effect of intestinal health.
Oligomeric galactose is that raw material obtains through beta-galactosidase enzymes (EC3.2.1.23) catalysis with lactose, is the oligosaccharide kind mixture oligomeric galactose base in lactose molecule connecting 1 ~ 4 galactose molecule with β (1 → 2) key or β (1 → 3), β (1 → 4) key, β (1 → 6) key.
Usually free cell or free beta-galactosidase enzymes enzyme is used to produce oligomeric galactose at present.In process of production, product can introduce the impurity such as free cell or protein, and needing increases complicated purifying technique, is unfavorable for cleaner production.In order to head it off, work out being fixed of cell, and utilized the method for immobilized cells produce oligomeric galactose.The method can simplify production technique, and the immobilized cell containing beta-galactosidase enzymes can reuse, and saves production cost.Immobilized cell often adopts calcium alginate embedded method, in the process utilizing Cell of Anmrobe lactose, due to the higher and alginate calcium bad mechanical strength of temperature, cause immobilized cell easily broken, thus in introducing syrup, cause syrup easy blocking filtering device in subsequent filter operation, filter pressure is raised very fast.
In addition, the microorganisms such as Kluyveromyces fragilis (Kluyveromycesfragilis), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), Kluyveromyces lactis (Kluyveromyces lactis) are had for the production of the bacterial classification of beta-galactosidase enzymes at present.Wherein, the beta-galactosidase enzymes that Kluyveromyces fragilis produces is intracellular enzyme, and the beta-galactosidase enzymes that saccharomyces lactis, aspergillus niger, aspergillus oryzae, Rhizopus oryzae etc. produce is extracellular enzyme.In born of the same parents, the acquisition of beta-galactosidase enzymes needs to carry out the steps such as broken thalline, separating-purifying, which increases production link and production unit, consumes energy simultaneously and also increase.If beta-galactosidase enzymes is directly secreted into outside born of the same parents by bacterial strain, then greatly facilitates separation and the utilization of enzyme, and save production cost.The beta-galactosidase enzymes that known Bacillus circulans produces is extracellular enzyme, and has resistant to elevated temperatures character, but utilizes the report of Bacillus circulans production beta-galactosidase enzymes little.
Summary of the invention
One object of the present invention is the preparation method providing a kind of oligomeric galactose, utilizes the method can obtain higher oligomeric galactose yield.
Another object of the present invention is the preparation method providing a kind of immobilization beta-galactosidase enzymes, the advantages such as the immobilized enzyme utilizing the method to prepare has that physical strength is high, physical and chemical performance is stable, effectively can solve and adopt the standby immobilized cell of calcium alginate embedded legal system or immobilized enzyme to produce filter pressure in the filter progress of oligomeric galactose to rise problem faster.
Another object of the present invention is to provide one to utilize Bacillus circulans (Bacillus circulans) to produce the method for beta-galactosidase enzymes, and the method can significantly improve the output of beta-galactosidase enzymes.
The technical solution adopted in the present invention is:
A preparation method for oligomeric galactose, comprises the following steps:
(1) use glutaraldehyde under agitation to react 16-24h with macropore weak base styrene resin at 15-35 DEG C, make resin activated;
(2) macropore weak base styrene resin stirring reaction 16-24h at 15-35 DEG C of the activation of liquid beta-galactosidase enzymes and step (1) gained is got, the beta-galactosidase enzymes of being fixed;
(3) immobilized beta-galactosidase enzymes is added in lactose solution, in 48-52 DEG C of stirring reaction 24-36h.
Preferably, the beta-galactosidase enzymes described in step (2) is purified through saltouing before use.
Preferably, in step (2), the proportioning of beta-galactosidase enzymes and macropore weak base styrene resin is 100-200U/g resin; The lactose concn added in step (3) is 45%-52%(w/w), and the immobilization beta-galactosidase enzymes of 6000-10000U is added according to every kg lactose (by butt).
Preferably, described in step (2), beta-galactosidase enzymes is produced by Bacillus circulans.
Preferably, described in step (2), beta-galactosidase enzymes is prepared through following steps:
A. the Bacillus circulans of preservation is seeded to LB solid medium, at 36-38 DEG C, cultivates 16-24h, make actication of culture;
The strain inoculation of B. getting activation, in the LB liquid nutrient medium of 20-50ml, is seeded in the seeding tank that LB substratum is housed, cultivates 4-6h, obtain seed culture fluid at 36-38 DEG C after shaking culture 46h;
C. seed culture fluid access is equipped with in the fermentor tank of fermention medium and cultivates 36-48h, obtain Bacillus circulans fermented liquid;
D. by fermentation liquor micro-filtration or centrifugal removing thalline, then gained enzyme liquid is concentrated, obtain liquid beta-galactosidase enzymes.
Preferably, above-mentioned Bacillus circulans is Bacillus circulans ATCC No.31382.
Preferably, the mode of cultivating described in step C is: accessed by seed culture fluid after cultivating 16-24h in fermentor tank, with feed supplement amount 4-6gL
-1and feed rate is 0.4-0.6gL
-1h
-1mode add lactose, continue fermentation 20-28h.
Preferably, fermention medium described in 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, sodium carbonate 1-2g/L, soybean oil 8-15g/L, defoamer 0.5-2g/L.
Preferably, the condition of cultivating described in step C is: temperature 36-38 DEG C, pH6.8-7.5, dissolved oxygen>=30%, air flow 0.5-2VV
-1min
-1, stirring velocity 120-220rpm.
The oligomeric galactose prepared by aforesaid method, comprises pasty products and powder-like product.
The invention has the beneficial effects as follows: the present invention adopts immobilization beta-galactosidase enzymes to produce oligomeric galactose, the resin physical strength that immobilization adopts is high, physical and chemical performance is stablized, can not change in the conversion process of lactose, also fragmentation is not easy, thus can not blocking filtering device, can pressure remained steady be made, overcome pressure increase that immobilized cell brings problem faster.Immobilized enzyme used in production process can reclaim and reuse, and the beta-galactosidase enzymes of its service efficiency specific ionization is much higher, has the feature of applicable commercial scale production.The production technique that the present invention prepares oligomeric galactose is simple, and operation is convenient, can meet the requirement of industrialized production, yield >=57% of oligomeric galactose.The present invention adopts Bacillus circulans to produce beta-galactosidase enzymes, and the beta-galactosidase enzymes produced due to Bacillus circulans is extracellular enzyme, and the separation and purification process of therefore follow-up enzyme greatly simplifies, thus saves equipment and the cost of separation and purification.Meanwhile, the beta-galactosidase enzymes that this bacterial classification produces has resistant to elevated temperatures character, and therefore, the process of producing oligomeric galactose can be carried out at relatively high temperatures, brings at least following benefit thus: the first, and at high temperature reaction can reduce microbiological contamination; The second, under high temperature, the solubleness of lactose is higher, and the yield of higher lactose starting point concentration oligomeric galactose is higher; 3rd, beta-galactosidase enzymes generally shows as the activity of hydrolyzes lactose under low temperature, lower lactose concn, and the oligomeric galactose of synthesis is less.In addition, the present invention produces the fermentation mode of beta-galactosidase enzymes, substratum and fermentation condition to Bacillus circulans and improves, and makes the enzymatic productivity of beta-galactosidase enzymes obtain certain raising.
Embodiment
Bacterial strain Bacillus circulans (Bacilluscirculans) purchased from American DSMZ (ATCC) of the product beta-galactosidase enzymes used in following embodiment, is numbered 31382.But the present invention's bacterial strain used is not limited to Bacillus circulans ATCC No.31382, and other Bacillus circulans is also applicable to the present invention.
Below description several concept of the present invention is described.
Free beta-galactosidase enzymes enzyme activity determination: containing concentration at 1mL is that in the ONPG 0.1M phosphoric acid buffer of 4mg/mL, (pH 6.0) adds 1mL beta-galactosidase enzymes enzyme liquid, at 50 DEG C of reaction 10min, add 2mL10% sodium carbonate solution termination reaction, by measuring the amount of hydrolysis of o-nitrophenol cubage o-nitrophenol galactoside (ONPG) in product.The per minute enzyme amount be hydrolyzed needed for 1 μM of ONPG is defined as a Ge Meihuo unit (U) under this condition.
The assay of oligomeric galactose: with reference to AOAC Official Method 2001.02 " Determination of trans-Galacto-oligosaccharides (TGOS) in Selected FoodProducts ".
The invention provides a kind of preparation method of oligomeric galactose, comprise the following steps:
(1) use glutaraldehyde under agitation to react 16-24h with macropore weak base styrene resin at 15-35 DEG C, make resin activated;
(2) macropore weak base styrene resin stirring reaction 16-24h at 15-35 DEG C of the activation of liquid beta-galactosidase enzymes and step (1) gained is got, the beta-galactosidase enzymes of being fixed;
(3) immobilized beta-galactosidase enzymes is added in lactose solution, in 48-52 DEG C of stirring reaction 24-36h.
Present invention also offers a kind of preparation method of oligomeric galactose, its step comprises the preparation of liquid beta-galactosidase enzymes, the preparation of immobilization beta-galactosidase enzymes and immobilization beta-galactosidase enzymes and produces oligomeric galactose three part.Be described for each part below.
1. the preparation of liquid beta-galactosidase enzymes
With Bacillus circulans (Bacillus circulans) for the bacterial classification that sets out, activated, seed tank culture, fermentation, except thalline, concentratedly obtain liquid beta-galactosidase enzymes, concrete steps are as follows:
1) actication of culture: be inoculated in the flat board that LB solid medium is housed by the Bacillus circulans that glycerine is preserved, cultivates 16 ~ 24 hours for 36 ~ 38 DEG C, obtains activating Bacillus circulans.
2) seed tank culture: after the activation of picking one to two ring, strain inoculation is in the triangular flask that 20 ~ 50mLLB liquid nutrient medium is housed, shaking culture is after 4 ~ 6 hours, again by 1 ~ 3%(v/v) inoculum size access be equipped with in the seeding tank of LB substratum, in 36 ~ 38 DEG C, dissolved oxygen>=30%, air flow 0.5 ~ 2V.V
-1.min
-1, stirring velocity 120 ~ 400rpm cultivate 4 ~ 6 hours, obtain seed tank culture liquid.
3) fermentation culture: by seed tank culture liquid by 1 ~ 3%(v/v) inoculum size access fermentor tank that fermention medium is housed carry out fermentation culture after 16 ~ 20 hours, be 5g.L by feed supplement amount
-1, feed rate 0.5g.L
-1.h
-1mode add lactose to fermentor tank, continue fermentation after 20 ~ 28 hours, obtain Bacillus circulans fermented liquid.Wherein, fermentation substratum comprises following several material: 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, sodium carbonate 1 ~ 2g/L, soybean oil 8 ~ 15g/L, defoamer 0.5 ~ 2g/L, its culture condition be 36 DEG C ~ 38 DEG C, pH7.0 ~ 7.5, dissolved oxygen>=30%, air flow 0.5 ~ 2V.V
-1.min
-1, stirring velocity 120 ~ 220rpm.
4) except thalline, concentrated enzyme liquid: by fermentation liquor micro-filtration or centrifugal segregation thalline, then with cryogenic vacuum, concentrated or ultrafiltration and concentration method concentrates enzyme liquid, obtains the liquid beta-galactosidase enzymes that enzyme work is 80 ~ 120U/mL.
2. the preparation of immobilization beta-galactosidase enzymes
Free beta-galactosidase enzymes immobilization technology is made the immobilized enzyme of definite shape.
The preparation of immobilization beta-galactosidase enzymes is through ammonium sulfate precipitation preliminary purification by liquid beta-galactosidase enzymes, then chemical coupling method and resin crosslinks is used, in described ammonium sulfate precipitation process, ammonium sulfate uses final concentration to be 25 ~ 30%, and solid-liquid separation can adopt filter paper plate pressure filtration that is centrifugal or 0.2 ~ 1 μm of aperture; Resin used is macropore weak base styrene resin; Linking agent used is glutaraldehyde.Immobilization way is first activated resin, then carries out cross-linking with enzyme.Resin activated condition is: glutaraldehyde uses final concentration to be 0.5 ~ 1.5%, and temperature is 15 ~ 35 DEG C, and rotating speed is 50 ~ 100rpm, and the reaction times is 16 ~ 24 hours.Enzyme immobilization condition is: the proportioning of enzyme and resin is that needed for every gram of macropore weak base styrene resin, enzyme amount is 100 ~ 200U, and temperature is 15 ~ 35 DEG C, and rotating speed is 50 ~ 100rpm, and the reaction times is 16 ~ 24 hours.
3. immobilization beta-galactosidase enzymes produces oligomeric galactose
Producing the batch reactions method that adopts of oligomeric galactose with immobilization beta-galactosidase enzymes is add 45 ~ 52%(w/w in retort) lactose solution, the immobilization beta-galactosidase enzymes of 6000 ~ 10000U enzyme amount is added by every kg lactose (butt calculation), control temperature is at 48 ~ 52 DEG C, mixing speed is in the conditioned response 24 ~ 36 hours of 50 ~ 100rpm, after having reacted, reclaim immobilized enzyme, for the production of next batch raw material, reaction solution after filtration, vacuum concentration obtains the oligomeric galactose syrup that Determination of galactooligosacchariin is more than 57%, or by dry further for concentrated syrup, add auxiliary material, drying obtains powdery oligomeric galactose.
Below in conjunction with embodiment, the present invention is further detailed.
The preparation of embodiment 1 beta-galactosidase enzymes
1, actication of culture: the Bacillus circulans that glycerine pipe is preserved is seeded to LB solid medium, cultivates 18h in 37 DEG C.
2, seed liquor is cultivated: by Bacillus circulans after the activation of LB flat board, picking one ring strain inoculation is in the triangular flask that 25mL LB substratum is housed, in 37 DEG C, 180rpm shaking culture 4 hours, then be transferred in the fermentor tank that 2.5L LB substratum is housed, in 37 DEG C, stirring velocity 120 ~ 220rpm, air flow 1.25L/min, control dissolved oxygen >=30% by adjustment rotating speed to cultivate 4 hours, obtain seed culture fluid.
3, fermentation culture:
1) pre fermentation: 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, sodium carbonate 325g, soybean oil 2275g, defoamer 125g, water 250kg are housed, at 37 DEG C, stirring velocity 120 ~ 220rpm, air flow 125L/min, dissolved oxygen >=30% salt acid for adjusting pH value is controlled 7.4, fermentation culture 18 hours by adjustment rotating speed.Wherein the various compositions of substratum comprise soy peptone, corn extract and yeast extract etc. and all can buy from market.Further, defoamer can use polyethers, polyether-modified silicon defoaming agent etc.
2) the fed-batch fermentation stage: start the lactose solution 10L adding 12.5% by 20mL/min stream in the fermentation system of step 1).After beginning feed supplement, sample fermented liquid mensuration beta-galactosidase enzymes enzyme every 2h and live, stopping fermentation after enzyme stopping alive being risen.
4, crude enzyme liquid preparation:
Get above-mentioned fermented liquid, at 4 DEG C, the centrifugal 10min of 10000rpm, collects middle part enzyme liquid, by enzyme liquid evaporation concentration under-0.01 ~-0.009MPa condition, obtains the beta-galactosidase enzymes crude enzyme liquid that enzyme activity is 115U/mL.
The immobilization of embodiment 2 beta-galactosidase enzymes
Immobilization beta-galactosidase enzymes is prepared according to following steps:
1, thick enzyme is refining
The concentrated enzyme liquid 100mL obtained of Example 1, slowly add ammonium sulfate 43.6g wherein, after ammonium sulfate dissolves completely, after leaving standstill 2 hours in 4 DEG C, at 4 DEG C, the centrifugal 10min collecting precipitation of 10000rpm, then uses 100mL pH6.0 phosphoric acid buffer dissolution precipitation, and obtaining enzyme activity is 100U/mL beta-galactosidase enzymes refined liquid.
2, enzyme immobilizatio
(1) take macropore weak base styrene resin A103s100g, after washed with de-ionized water 2 ~ 3 times, add deionized water 2000mL, add the glutaraldehyde solution of 40mL 50% again, 30 DEG C are stirred 16h, filter, then use washed with de-ionized water resin, filtration is dried moisture and is namely obtained activated resin.
(2) in activated resin, add the beta-galactosidase enzymes refined liquid containing 100mL, vibrate 16 hours in 30 DEG C of shaking tables, filter, then use washed with de-ionized water resin, filtration is dried moisture and is obtained the immobilization beta-galactosidase enzymes 100g that enzyme activity is 85U/g.
Embodiment 3 immobilized enzyme produces oligomeric galactose
The lactose solution 2000g that mass concentration is 50% is added in 5L retort, add the immobilized enzyme 80g of embodiment 2, in 50 DEG C, transform 24 hours under 80rpm condition after, adopt filter bag collect immobilized enzyme, the syrup be separated to after filtration, vacuum concentrator concentrate obtain Determination of galactooligosacchariin be 57.6% Brix be the syrup of 76.
The preparation of embodiment 4 beta-galactosidase enzymes
1, actication of culture: the Bacillus circulans that glycerine pipe is preserved is seeded to LB solid medium, cultivates 18h in 37 DEG C.
2, seed liquor is cultivated: by Bacillus circulans after the activation of LB flat board, picking two ring bacterial classification is inoculated into 2 respectively bottledly to be had in the triangular flask of 50mL LB substratum, in 37 DEG C, 180rpm shaking culture 4 hours, then be transferred in the fermentor tank that 10L LB substratum is housed, in 37 DEG C, stirring velocity 120 ~ 220rpm, air flow 5L/min, control dissolved oxygen >=30% by adjustment rotating speed to cultivate 4 hours, obtain seed culture fluid.
3, fermentation culture:
1) pre fermentation: 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, sodium carbonate 1.3kg, soybean oil 9.1kg, defoamer 0.5kg, water 1000kg are housed, at 37 DEG C, 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) start the lactose solution 25L adding 20% by 25mL/min stream in fermentation system.After beginning feed supplement, sample fermented liquid mensuration beta-galactosidase enzymes enzyme every 2h and live, stopping fermentation after enzyme stopping alive being risen.
3, crude enzyme liquid preparation
By above-mentioned fermented liquid below 30 DEG C, to enter film pressure be under 0.3 ~ 0.4MPa condition, carries out circulation cross flow filter removing thalline with the ceramic membrane in 0.5 μm of aperture, add water in the process in right amount and enzyme washed out, finally obtain clear clear enzyme solution.Then by clear clear enzyme solution below 30 DEG C, to enter film pressure be under 0.25 ~ 0.3MPa condition, is the rolled film ultrafiltration and concentration of 10KD with molecular weight cut-off, obtain enzyme and live as the crude enzyme liquid of 98.5U/mL.
The immobilization of embodiment 5 beta-galactosidase enzymes
Immobilization beta-galactosidase enzymes is prepared according to following steps:
1, beta-galactosidase enzymes is refining
Crude enzyme liquid 200L prepared by Example 4 adds 87.2kg ammonium sulfate, after stirring at room temperature to ammonium sulfate dissolves completely, leave standstill after 2 hours, precipitation is retained with the clarification cardboard pressure filtration in 0.2 μm of aperture, then precipitation wash cycles got off with the phosphoric acid buffer of 200LpH6.0, obtaining enzyme activity is 85U/mL beta-galactosidase enzymes refined liquid.
2, the preparation of immobilization beta-galactosidase enzymes
Take macropore weak base styrene resin A103s 150kg, after washed with de-ionized water 2 ~ 3 times, add deionized water to 3000L, add the glutaraldehyde solution that 60L concentration is 50% again, in 15 DEG C, 80rpm stirs 24 hours, filter, then remove remaining glutaraldehyde with washed with de-ionized water resin, namely centrifuge dripping obtains activated resin.Activated resin is joined in beta-galactosidase enzymes refined liquid prepared by 200L step 1, in 15 DEG C, 80rpm stirs 24 hours, filter, then use washed with de-ionized water resin, centrifuge dripping moisture, obtains the immobilization beta-galactosidase enzymes 150kg that enzyme activity is 100U/g.
Embodiment 6 immobilized enzyme produces oligomeric galactose
At 4m
3the lactose solution 3500kg that mass concentration is 50% is added in tank, add immobilized enzyme 150kg prepared by embodiment 5, in 48 DEG C, transform 36 hours under 80rpm condition, adopt enzyme strainer to reclaim immobilized enzyme, the syrup be separated to filter further, vacuum concentrator concentrate obtain Determination of galactooligosacchariin be 57.4% Brix be the syrup of 75.
Embodiment 7 immobilized cell produces comparing of oligomeric galactose with immobilized enzyme
1, immobilized cell produces oligomeric galactose
At 8m
3the lactose solution 6000kg that mass concentration is 50% is added in tank, add immobilized cell prepared by patent CN201110049829.7 method, in 60 DEG C, transform 20 hours under 80rpm condition, enzyme strainer is adopted to reclaim immobilized cell, it is 5 ㎡ flame filter press pressure filtrations that the syrup be separated to amasss further across filtering surface, and syrup carries out desalination bleaching again, rotatory vacuum evaporation concentration obtains oligomeric galactose to obtain clarification.
2, immobilized enzyme produces oligomeric galactose
According to the inventive method, at 8m
3the lactose solution 6000kg that mass concentration is 50% is added in tank, add immobilized enzyme of the present invention, in 50 DEG C, transform 24 hours under 80rpm condition, enzyme strainer is adopted to reclaim immobilized enzyme, it is 5 ㎡ flame filter press pressure filtrations that the syrup be separated to amasss further across filtering surface, and syrup carries out desalination bleaching again, rotatory vacuum evaporation concentration obtains oligomeric galactose to obtain clarification.
It is as follows that above-mentioned two kinds of modes produce Plate Filtration effect in oligomeric galactose process:
As seen from the above table, compared with immobilized cell, immobilized enzyme method is adopted to produce in the syrup filter progress of oligomeric galactose, under control feed pressure is not more than 0.4MPa condition, filtration area is that the ability of the disposable process syrup of plate-and-frame filter press of 5 ㎡ brings up to 60000kg by 6000kg, and filtration velocity brings up to 6000kg/h by 3000kg/h.Therefore adopt immobilized enzyme method of the present invention to produce oligomeric galactose, ability and the speed of the plate-and-frame filter press filtration syrup of unit surface are all significantly improved.
Embodiment 8 fermentation process produces the impact of beta-galactosidase enzymes ability to Bacillus circulans
1, adopt the inventive method fermentation for beta-galactosidase enzymes
The method fermentation described according to embodiment 1, for beta-galactosidase enzymes, samples fermented liquid in the fed-batch fermentation stage every 2h and measures beta-galactosidase enzymes enzyme and live, and after measured, ferment and within 42 hours, reaching that the highest enzyme lives is 47U/mL.
2, adopt ordinary method fermentation for beta-galactosidase enzymes
1) actication of culture: the Bacillus circulans that glycerine pipe is preserved is seeded to LB solid medium, cultivates 18h in 37 DEG C.
2) seed liquor is cultivated: by Bacillus circulans after the activation of LB flat board, picking one ring strain inoculation is in the triangular flask that 25mL LB substratum is housed, in 37 DEG C, 180rpm shaking culture 4 hours, then be transferred in the fermentor tank that 2.5L LB substratum is housed, in 37 DEG C, stirring velocity 120 ~ 220rpm, air flow 1.25L/min, control dissolved oxygen >=30% by adjustment rotating speed to cultivate 4 hours, 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, sodium carbonate 325g, soybean oil 2275g, defoamer 125g, water 250kg are housed, at 37 DEG C, stirring velocity 120 ~ 220rpm, air flow 125L/min, dissolved oxygen >=30% salt acid for adjusting pH value is controlled 7.4 by adjustment rotating speed, after measured, fermentation culture reaches the highest enzyme for 40 hours alive is 33U/mL.
Therefore, present invention employs that lactose feed profile carries out fermenting and ordinary method does not carry out feed supplement during the fermentation, result shows, adopt the inventive method that the enzyme work of gained beta-galactosidase enzymes can be made to bring up to 47U/mL from 33U/mL, the enzymatic productivity of Bacillus circulans is significantly improved.
Claims (2)
1. a preparation method for oligomeric galactose, comprises the following steps:
(1) use glutaraldehyde under agitation to react 16-24h with macropore weak base styrene resin at 15-35 DEG C, make resin activated;
(2) macropore weak base styrene resin stirring reaction 16-24h at 15-35 DEG C of the activation of liquid beta-galactosidase enzymes and step (1) gained is got, the beta-galactosidase enzymes of being fixed;
(3) immobilized beta-galactosidase enzymes is added in lactose solution, in 48-52 DEG C of stirring reaction 24-36h,
Wherein, the described lactose solution concentration of step (3) is 45%-52%(w/w), and by butt, add 6000-10000U immobilization beta-galactosidase enzymes according to every kg lactose; Beta-galactosidase enzymes described in step (2) is purified through saltouing before immobilization;
Wherein, described in step (2), beta-galactosidase enzymes is prepared through following steps:
A. the Bacillus circulans of preservation is seeded to LB solid medium, at 36-38 DEG C, cultivates 16-24h, make actication of culture;
The strain inoculation of B. getting activation, in the LB liquid nutrient medium of 20-50ml, is seeded in the seeding tank that LB substratum is housed, cultivates 4-6h, obtain seed culture fluid at 36-38 DEG C after shaking culture 4-6h;
C. seed culture fluid access is equipped with in the fermentor tank of fermention medium and cultivates 36-48h, obtain Bacillus circulans fermented liquid;
D. by fermentation liquor micro-filtration or centrifugal removing thalline, then gained enzyme liquid is concentrated, obtain liquid beta-galactosidase enzymes;
Wherein, the preserving number of described Bacillus circulans is ATCC No.31382;
The mode of cultivating described in step C is: accessed by seed culture fluid after cultivating 16-20h in fermentor tank, add lactose, and continue fermentation 20-28h, the wherein said mode adding lactose is: feed supplement amount 4 ~ 6gL
-1, feed rate is 0.4 ~ 0.6gL
-1h
-1;
The solute component of fermention medium described in 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, sodium carbonate 1-2g/L, soybean oil 8-15g/L, defoamer 0.5-2g/L;
The condition of cultivating described in step C is: temperature 36-38 DEG C, pH6.8-7.5, dissolved oxygen>=30%, air flow 0.5-2VV
-1min
-1, stirring velocity 120-220rpm.
2. the preparation method of a kind of oligomeric galactose according to claim 1, wherein in step (2), the proportioning of beta-galactosidase enzymes and macropore weak base styrene resin is 100-200U/g resin.
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| CN102994591B (en) * | 2012-12-19 | 2015-01-28 | 山东龙力生物科技股份有限公司 | Method for preparing galactooligosaccharide co-produced ethanol |
| CN104560706B (en) * | 2013-10-17 | 2018-03-16 | 丰益(上海)生物技术研发中心有限公司 | Enzyme reactor, enzymatic reaction system and fat processing method |
| CN105274086A (en) * | 2015-10-19 | 2016-01-27 | 量子高科(中国)生物股份有限公司 | High-throughput screening method for bacillus circulans |
| 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 |
| 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 |
| CN108949856B (en) * | 2018-07-05 | 2021-12-28 | 量子高科(广东)生物有限公司 | Preparation method of galacto-oligosaccharide |
| EP4190898A1 (en) * | 2020-07-28 | 2023-06-07 | Quantum Hi-Tech (Guangdong) Biological Co., Ltd. | Method for preparing beta-galactosidase and application thereof |
| CN111849940B (en) * | 2020-07-28 | 2022-03-25 | 量子高科(广东)生物有限公司 | Preparation method and application of beta-galactosidase |
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