CN106191157A - A kind of preparation method of high-purity oligomeric lactulose - Google Patents
A kind of preparation method of high-purity oligomeric lactulose Download PDFInfo
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- CN106191157A CN106191157A CN201610752459.6A CN201610752459A CN106191157A CN 106191157 A CN106191157 A CN 106191157A CN 201610752459 A CN201610752459 A CN 201610752459A CN 106191157 A CN106191157 A CN 106191157A
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- arthrobacterium
- sucrose
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- oligomeric lactulose
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- JCQLYHFGKNRPGE-FCVZTGTOSA-N lactulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-FCVZTGTOSA-N 0.000 title claims abstract description 58
- 229960000511 lactulose Drugs 0.000 title claims abstract description 58
- PFCRQPBOOFTZGQ-UHFFFAOYSA-N lactulose keto form Natural products OCC(=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O PFCRQPBOOFTZGQ-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 239000007788 liquid Substances 0.000 claims abstract description 64
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 46
- 229930006000 Sucrose Natural products 0.000 claims abstract description 46
- 239000005720 sucrose Substances 0.000 claims abstract description 46
- 238000000855 fermentation Methods 0.000 claims abstract description 42
- 230000004151 fermentation Effects 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 24
- 239000008101 lactose Substances 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 17
- 241000186073 Arthrobacter sp. Species 0.000 claims abstract description 13
- 239000011259 mixed solution Substances 0.000 claims abstract description 11
- 239000012141 concentrate Substances 0.000 claims abstract description 9
- 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 claims abstract description 8
- 238000007445 Chromatographic isolation Methods 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000005696 Diammonium phosphate Substances 0.000 claims description 13
- 229940041514 candida albicans extract Drugs 0.000 claims description 13
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 13
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 13
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 13
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 13
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 13
- 239000012138 yeast extract Substances 0.000 claims description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 10
- 239000000376 reactant Substances 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 9
- 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 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 7
- 239000000306 component Substances 0.000 claims description 6
- 239000012533 medium component Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000006199 nebulizer Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000002834 transmittance Methods 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000004042 decolorization Methods 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 12
- 102000004190 Enzymes Human genes 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 9
- FVVCFHXLWDDRHG-UPLOTWCNSA-N (2s,3r,4s,5r,6r)-2-[(2r,3s,4r,5r,6r)-6-[(2s,3s,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[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](CO)O2)O)[C@@H](CO)O1 FVVCFHXLWDDRHG-UPLOTWCNSA-N 0.000 abstract description 3
- 229930091371 Fructose Natural products 0.000 abstract description 2
- 239000005715 Fructose Substances 0.000 abstract description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 abstract description 2
- 108010090785 inulinase Proteins 0.000 abstract description 2
- 235000008504 concentrate Nutrition 0.000 description 6
- 108010036940 Levansucrase Proteins 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000002689 soil Substances 0.000 description 5
- ISPYQTSUDJAMAB-UHFFFAOYSA-N 2-chlorophenol Chemical compound OC1=CC=CC=C1Cl ISPYQTSUDJAMAB-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 3
- 235000013361 beverage Nutrition 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 238000005352 clarification Methods 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 235000001497 healthy food Nutrition 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241001656232 Pseudarthrobacter chlorophenolicus Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 235000011073 invertase Nutrition 0.000 description 2
- 230000002906 microbiologic effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 235000019605 sweet taste sensations Nutrition 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 235000021185 dessert Nutrition 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000008944 intestinal immunity Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000013615 non-nutritive sweetener Nutrition 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical group 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The present invention relates to the preparation method of a kind of high-purity oligomeric lactulose.Step is as follows: arthrobacterium fermentation liquid is inoculated in the mixed solution of sucrose and lactose by (1), after reaction, prepares the thick liquid of oligomeric lactulose；Described arthrobacterium is arthrobacterium (Arthrobacter sp.) BLCY 004, preserving number CGMCC No.12855；(2) by thick for oligomeric lactulose liquid through decolouring, filter, from friendships, chromatographic isolation, concentrate, be dried prepared oligomeric lactulose.The present invention utilizes arthrobacterium (Arthrobacter sp.) BLCY 004 fermentation liquid to produce oligomeric lactulose, produced by this strain, activity more traditional arthrobacterium institute inulinase-producing activity of fructose transfer base enzyme improves more than 50%, substantially increase sucrose inversion and become the ability of oligomeric lactulose, use stream with the method for sucrose simultaneously, make lactosucrose content in finished product reach 98%, be significantly better than the existing product prepared.
Description
Technical field
The present invention relates to the preparation method of a kind of high-purity oligomeric lactulose, belong to technical field of biotechnology.
Background technology
Oligomeric lactulose is a kind of functional oligose, has promotion intestinal bifidobacteria and breeds, regulating intestinal canal Tiny ecosystem,
Improving the special physiological functions such as intestinal immunity, sugariness is the 30% of sucrose, and sweet taste characteristic is similar to sucrose, and sweet taste quality is each
Plant in oligosaccharide optimal, therefore can apply in food industry without worrying that it produces impact to product special flavour.Business
The oligomeric lactulose that metaplasia is produced, due to containing other compositions such as sucrose, lactose, thus sugariness is slightly higher.Oligomeric with other
Sugar is compared, and oligomeric lactulose has higher stability to acid, heat, and its stability is similar to sucrose, stable in neutral conditions,
The most more stable, pH 3.0 times 80 DEG C of heating 2h of 7.0 times 80 DEG C of heating 2h and pH, occur the most hardly to divide
Solving, under conditions of pH 4.5, its heating-up temperature is even up to 120 DEG C.Oligomeric lactulose also has high moisture retention, can make
Food keeps moistening, and the starch food products such as bread, dessert can be prevented aging, extends Food Shelf-life.
Investigation in 2005 shows, oligomeric lactulose has become Japanese the third-largest functional oligose consumer goods.Permitted in recent years
Many researcheres find that oligomeric lactulose has many specific physiological functions, are expected to become after oligofructose, oligomeric galactose
Another functional food additives approved by the whole world.
Enzymatic clarification is the main path of industrialized production oligomeric lactulose, and the productivity of synthesizing lactosucrose by enzyme method is low by one
Being directly the important problem of restriction enzymatic clarification development, the purity the most how improving enzymatic clarification productivity and oligomeric lactulose is urgently
Problem to be solved.
As Chinese patent literature CN104073456A (application number 201410327028.6) discloses a strain chlorophenol arthrobacterium
(Arthrobacter chlorophenolicus) SK33.001, has been preserved in China typical culture collection center, and preservation is compiled
Number it is CCTCC NO:M2013387.With this arthrobacterium as starting strain, with sucrose, lactose as carbon source, with nitrogen source and inorganic salt group
Become fermentation medium, fermenting and producing levansucrase.Fermentation medium, with sucrose, lactose as primary carbon source, with peptone is
Main nitrogen, through detection after fermentation, in fermentation liquid, the work of levansucrase enzyme reaches 2～200U/mL.Levansucrase is added
Producing oligomeric lactulose in sucrose to 20%～60%, lactose solution, convert 3～36h, conversion ratio reaches more than 35%.
Chinese patent literature CN104480164A (application number 201410726466.X) discloses a kind of Production by Enzymes breast fruit
Oligosaccharide, comprises the following steps: accessed by chlorophenol arthrobacterium SK33.001 in seed culture medium, cultivates 12h at 30 DEG C；Seed
Liquid inoculum concentration is 1%, and ferment in fermentation medium 0.8～2h production levansucrase；It is respectively 27% to mass concentration
Sucrose, lactose solution add levansucrase and 50mg/L Zn2+Catalyzed conversion obtains the enzyme reaction containing oligomeric lactulose
Liquid；Decolouring；Concentrate, obtain oligomeric lactulose syrup.
All use due to technique scheme chlorophenol arthrobacterium SK33.001 as the enzyme source of enzyme process, due to the office of its enzyme
The sex-limited purity causing productivity and oligomeric lactulose is relatively low, it is impossible to meet actual demand.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that the preparation method of a kind of high-purity oligomeric lactulose.
Technical solution of the present invention is as follows:
A kind of preparation method of oligomeric lactulose, step is as follows:
(1) arthrobacterium fermentation liquid is inoculated in the mixed solution of sucrose and lactose, under the conditions of 50～60 DEG C, reaction 10～
14 hours, then in reactant liquor, stream added sucrose solution, and maintaining sucrose mass concentration in reactant liquor is 30%～40%, continues anti-
Answer 12～24h, stopped reaction, prepare the thick liquid of oligomeric lactulose；
Described arthrobacterium is arthrobacterium (Arthrobacter sp.) BLCY-004, and this bacterial strain is on August 16th, 2016
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: BeiChen West Road, Chaoyang District, BeiJing City 1
No. 3 Institute of Microorganism, Academia Sinica of institute, preserving number CGMCC No.12855；
(2) by step (1) prepare the thick liquid of oligomeric lactulose through decolouring, filter, from friendships, chromatographic isolation, concentrate, be dried make
Obtain oligomeric lactulose.
According to currently preferred, in described step (1), sucrose with the Solute mass concentration of the mixed solution of lactose is
20%～60%, sucrose is (1.2～1.5) with the mass ratio of lactose: 1, pH 4～6.
According to currently preferred, in described step (1), the mass concentration of sucrose solution is 35～45%；The most excellent
Choosing, the mass concentration of sucrose solution is 40%.
According to currently preferred, in described step (1), arthrobacterium fermentation liquid preparation method is as follows:
I, arthrobacterium (Arthrobacter sp.) BLCY-004 is inoculated in seed culture medium, at the bar of 28～35 DEG C
Under part, enrichment culture 24～48h, prepare seed liquor；
Described seed culture medium component is as follows, is all weight percentage:
Glucose 1～2%, yeast extract 0.5～1.5%, potassium dihydrogen phosphate 0.1～0.2%, diammonium phosphate 0.3～
0.6%, Magnesium sulfate heptahydrate 0.01～0.05%, excess water, pH6.0～7.2；
II, the seed liquor that step I is prepared by volume 1～10% ratio be inoculated in fermentation medium, 28～
35 DEG C of fermentation culture 36～60h, prepare arthrobacterium fermentation liquid；
Described fermentation medium component is as follows, is all weight percentage:
Sucrose 2～4%, lactose 0.5～1%, yeast extract 0.5～1.2%, peptone 0.4～0.8%, Magnesium sulfate heptahydrate
0.05～0.1%, diammonium phosphate 0.1～0.4%, excess water, pH 6.0～7.2.
According to currently preferred, in described step (1), the inoculum concentration of arthrobacterium fermentation liquid is mixed liquor volume percentage
The 5% of ratio.
According to currently preferred, in described step (2), decolorization process is as follows: oligomeric lactulose step (1) prepared
Thick liquid, by mass percentage 0.25～1% ratio add activated carbon, 80～85 DEG C of stirrings 30～40min.
According to currently preferred, in described step (2), filter and use plate-and-frame filtration, filter pressure 0.2～0.4Mpa,
Discharge 5.0～6.0t/h.
According to currently preferred, in described step (2), from handing over as using continuous ionic exchange system to process feed liquid, from
Feed liquid light transmittance >=98% after friendship.Feed liquid after process is as clear as crystal, free from extraneous odour.
According to currently preferred, in described step (2), chromatrographic separation step is:
Chromatographic run pressure 0.20～0.30MPa, temperature 60～70 DEG C, water loss-rate 1:1.3～1:1.6, feed per hour
1.5～2.0m3, collect oligomeric lactulose.
According to currently preferred, in described step (2), concentrate for be concentrated into original volume 60～65%.
According to currently preferred, in described step (2), being dried as being spray-dried, step is as follows:
Liquid after concentration enters in drying tower, inlet temperature 130～150 DEG C, starts nebulizer, and liquid is through spray dried
Dry for pulverulent solids.
Beneficial effect
1, the present invention obtains the arthrobacterium with high saccharase activity with screening from soil
(Arthrobacter sp.) BLCY-004, and utilize its fermentation liquid to produce oligomeric lactulose, fructose produced by this strain turns
The activity more traditional arthrobacterium institute inulinase-producing activity moving base enzyme improves more than 50%, substantially increases sucrose inversion and becomes oligomeric lactulose
Ability, simultaneously use stream with the method for sucrose, make lactosucrose content in finished product reach 98%, be significantly better than existing system
The standby product obtained；
2, the present invention uses arthrobacterium fermentation liquid to be directly produced, and eliminates the process extracting enzyme, makes production cost significantly drop
Low, relatively conventional production methods reduces cost and reaches about 20%, makes the competitiveness of product be greatly enhanced；
3, the present invention uses chromatographic isolation and spray drying process to produce solid high-purity oligomeric lactulose product, more traditional
Method for crystallising is time saving and energy saving, and cost-saved about 20%；
4, high product purity lactulose of the present invention has higher stability as low calorie sweetener to acid, heat, can be wide
General food and drink, cosmetics, the medicine and other fields of being applied to, improves added value of product.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is further elaborated, but institute of the present invention protection domain is not limited to
This.
Embodiment 1
One strain arthrobacterium (Arthrobacter sp.) BLCY-004, is stored in China Microbiological bacterium on August 16th, 2016
Plant preservation administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is micro-
Biological study institute, preserving number preserving number CGMCC No.12855；
The original strain of arthrobacterium of the present invention (Arthrobacter sp.) BLCY-004 is located away from Dezhou, Shandong hundred
Soil near the workshop of Long Chuan garden, and obtain after mutation, concrete separation process is as follows:
(1) enrichment culture
Choose the soil near the workshop of Long Chuan garden, Dezhou, Shandong hundred, remove table soil with little scoop, take overhead 5-
Soil at 15cm about 10g, dilutes 10 times with sterilized water, adds culture medium and carries out enrichment culture, medium component: glucose
2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium phosphate 0.6%, Magnesium sulfate heptahydrate 0.01%, pH is 6.5；Cultivate
Temperature is 30 DEG C, cultivates 36h.
(2) Pure strain separation
Use line partition method, take a Boiling tube filling 5ml sterilized water, take the bacterium after enrichment culture in step (1)
Liquid 2ml puts into and wherein dilutes, abundant vibrating dispersion, with inoculating loop with sterile working's picking diluent one ring first at plating medium
While doing parallel scribing 3-4 bar for the first time, being rotated further by about 60 degree of angles of culture dish, residue on inoculating loop being burnt, after cooling
Do second time line with a scribble method, take turns doing third time and the 4th line with method.Rule complete, cover ware lid, will
Culture dish is inverted, and cultivates after 36h for 30 DEG C, the single colony inoculation of picking on 10 slant mediums, numbering 01-10 respectively.
01-10 inclined-plane seed is inoculated in Shake flask medium cultivation 32 DEG C cultivate 36h, to 01-10 shake flask fermentation liquid β-
Fructofuranosidase enzyme is lived and is measured, and No. 04 shaking flask enzyme is lived the highest, reaches 505U/ml.
Plating medium composition: glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium phosphate 0.6%,
Magnesium sulfate heptahydrate 0.01%, agar 2%, pH is pH6.5.
Shake-flask culture based component: sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, Magnesium sulfate heptahydrate
0.1%, diammonium phosphate 0.4%, pH6.5.
(3) mutagenesis screening
No. 04 strain carries out ultraviolet mutagenesis, and ultraviolet mutagenesis uses 15W Burdick lamp 20cm to irradiate, and irradiation time is
150s, finally gives the named BLCY-004 of superior strain of high yield saccharase, and its enzyme is lived and reached 1100U/ml.
Enzyme is lived and is defined as follows: at pH 6.0,40 DEG C using sucrose as substrate react time, per minute make reducing power increase
The enzyme amount of the amount adding the D-Glucose being equivalent to 2 μm ol schedules be 1 unit of activity (U).
Ecological form
Bacterial strain is creamy white, translucent, size about 2～4 μ m 1.8～3.5 μm.Gram’s staining is positive, quarter butt
Shape, single or arrange in pairs, along with shaft-like, the change of spherical-like morphology in cultivation cycle.
Embodiment 2
A kind of preparation method of oligomeric lactulose, step is as follows:
(1) preparation mass concentration is sucrose and the mixed solution of lactose of 20%, and sucrose is 1.2 with the interpolation ratio of lactose:
1；The pH to 4 of regulation mixed solution, inoculates arthrobacterium fermentation liquid in the ratio of mixeding liquid volume 5%, under the conditions of 50 DEG C, and reaction
12 hours, then in reactant liquor, stream added the sucrose solution that mass concentration is 40%, and in maintenance reactant liquor, sucrose mass concentration is
30%, continue stopped reaction after reaction 12h, prepare the thick liquid of oligomeric lactulose；
(2) by step (1) prepare the thick liquid of oligomeric lactulose by mass percentage 0.5% ratio add activated carbon, 80
DEG C stirring 40min；Then through plate-and-frame filtration, filter pressure 0.4Mpa, discharge 6.0t/h；Use at continuous ionic exchange system
Reason feed liquid, feed liquid light transmittance >=98% after handing over；Through chromatographic isolation, chromatographic run pressure 0.30MPa, temperature 60 C, water loss-rate
1:1.6, feeds 2.0m per hour3, collect oligomeric lactulose；Then concentrate through sextuple effect, be concentrated into the 60% of original volume；
Liquid after concentration enters in drying tower, inlet temperature 140 DEG C, starts nebulizer, and liquid is spray-dried solid for powder
Body prepares oligomeric lactulose.
Arthrobacterium fermentation liquid in described step (2) is prepared as follows:
I, arthrobacterium (Arthrobacter sp.) BLCY-004 is inoculated in seed culture medium, the condition of 28 DEG C
Under, enrichment culture 24h, prepare seed liquor；
Described seed culture medium component is as follows, is all weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium phosphate 0.6%, Magnesium sulfate heptahydrate
0.01%, excess water, pH6.0；
II, by step I prepare seed liquor by volume 1% ratio be inoculated in fermentation medium, 28 DEG C of fermentations
Cultivate 36h, prepare arthrobacterium fermentation liquid；
Described fermentation medium component is as follows, is all weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, Magnesium sulfate heptahydrate 0.1%, diammonium phosphate
0.4%, excess water, pH 6.0；
After testing, the oligomeric lactulose purity prepared reaches 98.2%, and conversion ratio is 55.2%, can be applicable to healthy food
Product, beverage, milk, health product, medicine and other fields.
Embodiment 3
A kind of preparation method of oligomeric lactulose, step is as follows:
(1) preparation mass concentration is sucrose and the mixed solution of lactose of 40%, and sucrose is 1.3 with the interpolation ratio of lactose:
1；The pH to 5 of regulation mixed solution, inoculates arthrobacterium fermentation liquid in the ratio of mixeding liquid volume 5%, under the conditions of 55 DEG C, and reaction
12 hours, then in reactant liquor, stream added the sucrose solution that mass concentration is 40%, and in maintenance reactant liquor, sucrose mass concentration is
35%, continue stopped reaction after reaction 18h, prepare the thick liquid of oligomeric lactulose；
(2) by step (1) prepare the thick liquid of oligomeric lactulose by mass percentage 0.25% ratio add activated carbon, 80
DEG C stirring 40min；Then through plate-and-frame filtration, filter pressure 0.2Mpa, discharge 5.0t/h；Use at continuous ionic exchange system
Reason feed liquid, feed liquid light transmittance >=98% after handing over；Then concentrate through sextuple effect, be concentrated into the 65% of original volume；Through chromatograph
Separate, chromatographic run pressure 0.20MPa, temperature 70 C, water loss-rate 1:1.3, feed 1.5m per hour3, collect oligomeric lactulose；
Liquid after concentration enters in drying tower, inlet temperature 140 DEG C, starts nebulizer, and liquid is spray-dried solid for powder
Body prepares oligomeric lactulose.
Arthrobacterium fermentation liquid in described step (2) is prepared as follows:
I, arthrobacterium (Arthrobacter sp.) BLCY-004 is inoculated in seed culture medium, the condition of 28 DEG C
Under, enrichment culture 24h, prepare seed liquor；
Described seed culture medium component is as follows, is all weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium phosphate 0.6%, Magnesium sulfate heptahydrate
0.01%, excess water, pH6.0；
II, by step I prepare seed liquor by volume 1% ratio be inoculated in fermentation medium, 28 DEG C of fermentations
Cultivate 36h, prepare arthrobacterium fermentation liquid；
Described fermentation medium component is as follows, is all weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, Magnesium sulfate heptahydrate 0.1%, diammonium phosphate
0.4%, excess water, pH 6.0；
After testing, the oligomeric lactulose purity prepared reaches 98.5%, and conversion ratio is 56.3%, can be applicable to healthy food
Product, beverage, milk, health product, medicine and other fields.
Embodiment 4
A kind of preparation method of oligomeric lactulose, step is as follows:
(1) preparation mass concentration is sucrose and the mixed solution of lactose of 60%, and sucrose is 1.5 with the interpolation ratio of lactose:
1；The pH to 6 of regulation mixed solution, inoculates arthrobacterium fermentation liquid in the ratio of mixeding liquid volume 5%, under the conditions of 60 DEG C, and reaction
12 hours, then in reactant liquor, stream added the sucrose solution that mass concentration is 40%, and in maintenance reactant liquor, sucrose mass concentration is
40%, continue stopped reaction after reaction 24h, prepare the thick liquid of oligomeric lactulose；
(2) by step (1) prepare the thick liquid of oligomeric lactulose by mass percentage 1% ratio add activated carbon, 85 DEG C
Stirring 30min；Then through plate-and-frame filtration, filter pressure 0.3Mpa, discharge 5.5t/h；Continuous ionic exchange system is used to process
Feed liquid, feed liquid light transmittance >=98% after handing over；Then concentrate through sextuple effect, be concentrated into the 63% of original volume；Divide through chromatograph
From, chromatographic run pressure 0.25MPa, temperature 65 DEG C, water loss-rate 1:1.4, feed 1.6m per hour3, collect oligomeric lactulose；Dense
Liquid after contracting enters in drying tower, inlet temperature 140 DEG C, starts nebulizer, and liquid is spray-dried for pulverulent solids
Prepare oligomeric lactulose.
Arthrobacterium fermentation liquid in described step (2) is prepared as follows:
I, arthrobacterium (Arthrobacter sp.) BLCY-004 is inoculated in seed culture medium, the condition of 35 DEG C
Under, enrichment culture 48h, prepare seed liquor；
Described seed culture medium component is as follows, is all weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium phosphate 0.6%, Magnesium sulfate heptahydrate
0.01%, excess water, pH6.0；
II, by step I prepare seed liquor by volume 10% ratio be inoculated in fermentation medium, 35 DEG C of fermentations
Cultivate 60h, prepare arthrobacterium fermentation liquid；
Described fermentation medium component is as follows, is all weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, Magnesium sulfate heptahydrate 0.1%, diammonium phosphate
0.4%, excess water, pH 6.0；
After testing, the oligomeric lactulose purity prepared reaches 98.3%, and conversion ratio is 56.5%, can be applicable to healthy food
Product, beverage, milk, health product, medicine and other fields.
Comparative example
The preparation method of oligomeric lactulose as described in Example 2, difference is, utilizes chlorophenol arthrobacterium
(Arthrobacter chlorophenolicus) SK33.001 replaces arthrobacterium (Arthrobacter sp.) BLCY-004.
After testing, the oligomeric lactulose purity prepared reaches 81.2%, and conversion ratio is 36.7%.
Claims (10)
1. the preparation method of an oligomeric lactulose, it is characterised in that step is as follows:
(1) arthrobacterium fermentation liquid is inoculated in the mixed solution of sucrose and lactose, and under the conditions of 50～60 DEG C, reaction 10～14 is little
Time, then in reactant liquor, stream adds sucrose solution, and maintaining sucrose mass concentration in reactant liquor is 30%～40%, continues reaction 12
～24h, stopped reaction, prepare the thick liquid of oligomeric lactulose；
Described arthrobacterium is arthrobacterium (Arthrobacter sp.) BLCY-004, and this bacterial strain is in preservation on August 16 in 2016
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number Institute of Microorganism, Academia Sinica, preserving number CGMCC No.12855；
(2) the thick liquid of oligomeric lactulose that step (1) is prepared through decolouring, filter, from friendships, chromatographic isolation, concentrate, be dried prepared low
Poly-lactulose.
2. preparation method as claimed in claim 1, it is characterised in that in described step (1), sucrose and the mixed solution of lactose
Solute mass concentration be 20%～60%, the mass ratio of sucrose and lactose is (1.2～1.5): 1, pH 4～6.
3. preparation method as claimed in claim 1, it is characterised in that in described step (1), the mass concentration of sucrose solution is
35～45%；
It is further preferred that the mass concentration of sucrose solution is 40%.
4. preparation method as claimed in claim 1, it is characterised in that in described step (1), arthrobacterium fermentation liquid preparation method
As follows:
I, arthrobacterium (Arthrobacter sp.) BLCY-004 is inoculated in seed culture medium, the condition of 28～35 DEG C
Under, enrichment culture 24～48h, prepare seed liquor；
Described seed culture medium component is as follows, is all weight percentage:
Glucose 1～2%, yeast extract 0.5～1.5%, potassium dihydrogen phosphate 0.1～0.2%, diammonium phosphate 0.3～0.6%,
Magnesium sulfate heptahydrate 0.01～0.05%, excess water, pH6.0～7.2；
II, by step I prepare seed liquor by volume 1～10% ratio be inoculated in fermentation medium, at 28～35 DEG C
Fermentation culture 36～60h, prepares arthrobacterium fermentation liquid；
Described fermentation medium component is as follows, is all weight percentage:
Sucrose 2～4%, lactose 0.5～1%, yeast extract 0.5～1.2%, peptone 0.4～0.8%, Magnesium sulfate heptahydrate 0.05
～0.1%, diammonium phosphate 0.1～0.4%, excess water, pH 6.0～7.2.
5. preparation method as claimed in claim 1, it is characterised in that the inoculum concentration of arthrobacterium fermentation liquid in described step (1)
For mixed liquor volume percentage ratio 5%.
6. preparation method as claimed in claim 1, it is characterised in that in described step (2), decolorization process is as follows: by step
(1) prepare the thick liquid of oligomeric lactulose, by mass percentage 0.25～1% ratio add activated carbon, 80～85 DEG C stirring 30
～40min.
7. preparation method as claimed in claim 1, it is characterised in that in described step (2), filter and use plate-and-frame filtration, mistake
Filter pressure 0.2～0.4Mpa, discharge 5.0～6.0t/h.
8. preparation method as claimed in claim 1, it is characterised in that in described step (2), from handing over as using continuous ionic to hand over
System of changing processes feed liquid, feed liquid light transmittance >=98% after handing over.
9. preparation method as claimed in claim 1, it is characterised in that in described step (2), chromatrographic separation step is:
Chromatographic run pressure 0.20～0.30MPa, temperature 60～70 DEG C, water loss-rate 1:1.3～1:1.6, per hour charging 1.5～
2.0m3, collect oligomeric lactulose.
10. preparation method as claimed in claim 1, it is characterised in that in described step (2), concentrate as being concentrated into original volume
60～65%；
Preferably, in described step (2), being dried as being spray-dried, step is as follows:
Liquid after concentration enters in drying tower, inlet temperature 130～150 DEG C, starts nebulizer, and liquid is spray-dried is
Pulverulent solids.
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|---|---|---|---|---|
| CN102168028A (en) * | 2010-02-26 | 2011-08-31 | 江南大学 | Arthrobacter mutant strain, method for producing lactase from mutant strain and method for preparing lactulose by using lactase |
| CN103695501A (en) * | 2013-12-10 | 2014-04-02 | 江南大学 | Method for producing lactosucrose employing levansucrase |
| CN104073456A (en) * | 2014-07-10 | 2014-10-01 | 江南大学 | Bacterial strain for producing levansucrase and method for producing lactosucrose by utilizing lavansucrase |
| CN104480164A (en) * | 2014-12-04 | 2015-04-01 | 山东百龙创园生物科技有限公司 | Enzymic method for preparing lactosucrose |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168028A (en) * | 2010-02-26 | 2011-08-31 | 江南大学 | Arthrobacter mutant strain, method for producing lactase from mutant strain and method for preparing lactulose by using lactase |
| CN103695501A (en) * | 2013-12-10 | 2014-04-02 | 江南大学 | Method for producing lactosucrose employing levansucrase |
| CN104073456A (en) * | 2014-07-10 | 2014-10-01 | 江南大学 | Bacterial strain for producing levansucrase and method for producing lactosucrose by utilizing lavansucrase |
| CN104480164A (en) * | 2014-12-04 | 2015-04-01 | 山东百龙创园生物科技有限公司 | Enzymic method for preparing lactosucrose |
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| TWI624547B (en) * | 2017-01-26 | 2018-05-21 | 國立中興大學 | Preparation method of lactosucrose |
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