CN106191157B - A kind of preparation method of lactosucrose - Google Patents
A kind of preparation method of lactosucrose Download PDFInfo
- Publication number
- CN106191157B CN106191157B CN201610752459.6A CN201610752459A CN106191157B CN 106191157 B CN106191157 B CN 106191157B CN 201610752459 A CN201610752459 A CN 201610752459A CN 106191157 B CN106191157 B CN 106191157B
- Authority
- CN
- China
- Prior art keywords
- lactosucrose
- sucrose
- preparation
- liquid
- arthrobacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 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 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 62
- 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
- 238000006243 chemical reaction Methods 0.000 claims abstract description 31
- 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
- 238000000855 fermentation Methods 0.000 claims abstract description 19
- 230000004151 fermentation Effects 0.000 claims abstract description 19
- 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 17
- 241000186073 Arthrobacter sp. Species 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000011259 mixed solution Substances 0.000 claims abstract description 11
- 238000001914 filtration Methods 0.000 claims abstract description 10
- 238000005342 ion exchange Methods 0.000 claims abstract description 9
- 238000007445 Chromatographic isolation Methods 0.000 claims abstract description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 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
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 13
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 13
- 239000012138 yeast extract Substances 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 11
- 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
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 229910052799 carbon Inorganic materials 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
- 238000001035 drying Methods 0.000 claims description 6
- 239000012533 medium component Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000000306 component Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 238000002834 transmittance Methods 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000004042 decolorization Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 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 claims description 2
- 229960000511 lactulose Drugs 0.000 claims description 2
- 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 claims description 2
- 238000001694 spray drying Methods 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 13
- 102000004190 Enzymes Human genes 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 13
- 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
- -1 after reaction Chemical compound 0.000 abstract 1
- 108010036940 Levansucrase Proteins 0.000 description 5
- 235000013305 food Nutrition 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
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 3
- 235000013361 beverage Nutrition 0.000 description 3
- 238000005352 clarification Methods 0.000 description 3
- 235000013365 dairy product Nutrition 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 235000001497 healthy food Nutrition 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 241001656232 Pseudarthrobacter chlorophenolicus Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000004615 ingredient Substances 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
- 238000007747 plating Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000008223 sterile water Substances 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
- 244000166550 Strophanthus gratus Species 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
- 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
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000006185 dispersion Substances 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
- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 1
- 150000003271 galactooligosaccharides Chemical class 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
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 239000000203 mixture Substances 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
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 150000003839 salts Chemical group 0.000 description 1
- 238000005507 spraying 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
-
- 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
Landscapes
- 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 a kind of preparation methods of lactosucrose.Steps are as follows: pole fermented liquid (1) being inoculated in the mixed solution of sucrose and lactose, after reaction, lactosucrose crude liquid is made；The arthrobacterium is arthrobacterium (Arthrobacter sp.) BLCY-004, deposit number CGMCC No.12855；(2) by lactosucrose crude liquid through decoloration, filtering, ion-exchange, chromatographic isolation, concentration, dry obtained lactosucrose.The present invention produces lactosucrose using arthrobacterium (Arthrobacter sp.) BLCY-004 fermentation liquid, the more traditional arthrobacterium institute's inulinase-producing activity of activity that fructose caused by the strain shifts base enzyme improves 50% or more, sucrose inversion is substantially increased into the ability of lactosucrose, simultaneously using the method for stream plus sucrose, so that lactosucrose content in finished product is reached 98%, is significantly better than the existing product prepared.
Description
Technical field
The present invention relates to a kind of preparation methods of lactosucrose, belong to technical field of biotechnology.
Background technique
Lactosucrose is a kind of functional oligose, has and promotes intestinal bifidobacteria proliferation, adjusts intestinal microecology,
Improve the special physiologicals 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
It is optimal in kind oligosaccharide, therefore can be applied in food industry without worrying that it has an impact product special flavour.Commercially
The lactosucrose that metaplasia produces, due to containing the other compositions such as sucrose, lactose, thus sugariness wants slightly higher.With it is other oligomeric
Sugar is compared, and for lactosucrose to acid, heat stability with higher, stability is similar to sucrose, stable in neutral conditions,
Relatively more stable in acid condition, pH 3.0 lower 80 DEG C of heating 2h of 7.0 lower 80 DEG C of heating 2h and pH are hardly happened point
Solution, under conditions of 4.5 pH, heating temperature is even up to 120 DEG C.Lactosucrose also has high moisture retention, can make
Food keeps wet, can prevent the starch food products aging such as bread, dessert, extends Food Shelf-life.
Investigation in 2005 shows that lactosucrose has become the third-largest functional oligose consumer goods of Japan.Permitted in recent years
More researchers have found that lactosucrose has many specific physiological functions, are expected to become after oligofructose, galactooligosaccharide
Another functional food additives approved by the whole world.
Enzymatic clarification is the main path of industrialized production lactosucrose, the low yield one of synthesizing lactosucrose by enzyme method
It is directly to restrict the important problem of enzymatic clarification development, therefore the purity for how improving enzymatic clarification yield and lactosucrose is urgently
Problem to be solved.
As Chinese patent literature CN104073456A (application number 201410327028.6) discloses one plant of chlorophenol arthrobacterium
(Arthrobacter chlorophenolicus) SK33.001, has been preserved in China typical culture collection center, and preservation is compiled
Number be CCTCC NO:M2013387.Using this arthrobacterium as starting strain, using sucrose, lactose as carbon source, with nitrogen source and inorganic salts group
At fermentation medium, fermenting and producing levansucrase.Fermentation medium is with peptone using sucrose, lactose as primary carbon source
Main nitrogen, through detecting after fermentation, levansucrase enzyme activity is up to 2~200U/mL in fermentation liquid.By levansucrase plus
To 20%~60% sucrose, lactosucrose is produced in lactose solution, convert 3~36h, conversion ratio reaches 35% or more.
Chinese patent literature CN104480164A (application number 201410726466.X) discloses a kind of Production by Enzymes cream fruit
Oligosaccharide, comprising the following steps: chlorophenol arthrobacterium SK33.001 is accessed in seed culture medium, cultivates 12h at 30 DEG C；Seed
Liquid inoculum concentration is 1%, and 0.8~2h of fermentation produces levansucrase in fermentation medium；It is respectively 27% to mass concentration
Levansucrase and 50mg/L Zn are added in sucrose, lactose solution2It is anti-that+catalyzed conversion obtains the enzyme containing lactosucrose
Answer liquid；Decoloration；Concentration is to get lactosucrose syrup.
Since above-mentioned technical proposal is all made of enzyme source of the chlorophenol arthrobacterium SK33.001 as enzyme process, due to the office of its enzyme
The sex-limited purity for leading to yield and lactosucrose is lower, is unable to satisfy actual demand.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of preparation methods of high-purity lactosucrose.
Technical solution of the present invention is as follows:
A kind of preparation method of lactosucrose, steps are as follows:
(1) pole fermented liquid is inoculated in the mixed solution of sucrose and lactose, under the conditions of 50~60 DEG C, reaction 10~
14 hours, then into reaction solution, stream added sucrose solution, and maintaining sucrose mass concentration in reaction solution is 30%~40%, continued anti-
Answer 12~for 24 hours, stop reaction, lactosucrose crude liquid is made；
The arthrobacterium is arthrobacterium (Arthrobacter sp.) BLCY-004, and the 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, institute, deposit number CGMCC No.12855；
(2) by lactosucrose crude liquid made from step (1) through decoloration, filtering, ion-exchange, chromatographic isolation, concentration, dry system
Obtain lactosucrose.
Preferred according to the present invention, in the step (1), the Solute mass concentration of the mixed solution of sucrose and lactose is
20%~60%, the mass ratio of sucrose and lactose is (1.2~1.5): 1, pH 4~6.
Preferred according to the present invention, in the step (1), the mass concentration of sucrose solution is 35~45%；It is further excellent
Choosing, the mass concentration of sucrose solution is 40%.
Preferred according to the present invention, in the step (1), arthrobacterium zymotic fluid preparation method is as follows:
I, arthrobacterium (Arthrobacter sp.) BLCY-004 is inoculated in seed culture medium, in 28~35 DEG C of item
Under part, seed liquor is made in 24~48h of Multiplying culture；
The seed culture medium component is as follows, is weight percentage:
Glucose 1~2%, yeast extract 0.5~1.5%, potassium dihydrogen phosphate 0.1~0.2%, diammonium hydrogen phosphate 0.3~
0.6%, epsom salt 0.01~0.05%, excess water, pH6.0~7.2；
II, by seed liquor made from step I by volume 1~10% ratio be inoculated in fermentation medium, 28~
Pole fermented liquid is made in 35 DEG C of 36~60h of fermented and cultured；
The fermentation medium component is as follows, is weight percentage:
Sucrose 2~4%, lactose 0.5~1%, yeast extract 0.5~1.2%, peptone 0.4~0.8%, epsom salt
0.05~0.1%, diammonium hydrogen phosphate 0.1~0.4%, excess water, pH 6.0~7.2.
Preferred according to the present invention, the inoculum concentration of pole fermented liquid is mixed liquor volume percentage in the step (1)
The 5% of ratio.
Preferred according to the present invention, in the step (2), decolorization process is as follows: by lactosucrose made from step (1)
Crude liquid, active carbon, 80~85 DEG C of 30~40min of stirring are added in 0.25~1% ratio by mass percentage.
It is preferred according to the present invention, in the step (2), filters and use plate-frame filtering, 0.2~0.4Mpa of filter pressure,
5.0~6.0t/h of water flow.
Preferred according to the present invention, in the step (2), ion-exchange is to handle feed liquid using continuous ionic exchange system, from
Feed liquid light transmittance >=98% after friendship.Treated, and feed liquid is as clear as crystal, free from extraneous odour.
It is preferred according to the present invention, in the step (2), chromatrographic separation step are as follows:
Chromatographic run 0.20~0.30MPa of pressure, 60~70 DEG C of temperature, water consume ratio 1:1.3~1:1.6 is fed per hour
1.5~2.0m3, collect lactosucrose.
It is preferred according to the present invention, in the step (2), it is concentrated to be concentrated into the 60~65% of original volume.
Preferred according to the present invention, in the step (2), dry to be spray-dried, steps are as follows:
Liquid after concentration enters in drying tower, 130~150 DEG C of inlet air temperature, starts atomizer, liquid is through spraying dry
Dry is pulverulent solids.
Beneficial effect
1, the present invention is obtained with screening from soil with the active arthrobacterium of high saccharase
(Arthrobacter sp.) BLCY-004, and lactosucrose is produced using its fermentation liquid, fructose caused by the strain turns
The more traditional arthrobacterium institute's inulinase-producing activity of activity for moving base enzyme improves 50% or more, substantially increases sucrose inversion into lactosucrose
Ability, while using stream plus sucrose method, so that lactosucrose content in finished product is reached 98%, be significantly better than existing system
The standby product obtained；
2, the present invention is directly produced using pole fermented liquid, is eliminated the process for extracting enzyme, is dropped production cost significantly
It is low, cost is reduced up to 20% or so compared with conventional production methods, greatly enhances the competitiveness of product；
3, the present invention produces solid lactosucrose product using chromatographic isolation and spray drying process, compared with conventional junction chip
Method is time saving and energy saving, and cost-saved 20% or so；
4, product of the present invention lactulose can be widely applied as low calorie sweetener to acid, heat stability with higher
In food and drink, cosmetics, medicine and other fields, added value of product is improved.
Specific embodiment
Technical solution of the present invention is further elaborated below with reference to embodiment, but institute's protection scope of the present invention is not limited to
This.
Embodiment 1
One plant of arthrobacterium (Arthrobacter sp.) BLCY-004, is stored in China Microbiological bacterium on August 16th, 2016
Kind preservation administration committee common micro-organisms center, address: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is micro-
Biological study institute, deposit number deposit number CGMCC No.12855；
The original strain of arthrobacterium (Arthrobacter sp.) BLCY-004 of the present invention is located away from Shandong Dezhou hundred
Soil near the workshop of the garden Long Chuan, and obtained after mutagenesis, specific separation process is as follows:
(1) enrichment culture
The soil near hundred garden Long Chuan workshop of Shandong Dezhou is chosen, surface soil is removed with small scoop, takes 5- from the ground
Soil about 10g at 15cm dilutes 10 times with sterile water, and culture medium is added and carries out enrichment culture, medium component: glucose
2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%, epsom salt 0.01%, pH 6.5；Culture
Temperature is 30 DEG C, cultivates 36h.
(2) Pure strain separation
Using scribing line partition method, the Boiling tube for filling 5ml sterile water is taken, the bacterium in step (1) after enrichment culture is taken
Liquid 2ml, which is put into, wherein to be diluted, abundant vibrating dispersion, with oese with one ring of sterile working picking dilution first in plating medium
First time parallel scribing 3-4 item is done on one side, is rotated further by about 60 degree of angles of culture dish, residue on oese is burnt up, after cooling
Second of scribing line is done with a scribble method, takes turns doing third time and the 4th scribing line with method.Scribing line finishes, and covers ware lid, will
Culture dish is inverted, and after 30 DEG C of culture 36h, picking single bacterium colony is inoculated on 10 slant mediums, respectively number 01-10.
The inclined-plane 01-10 seed is inoculated in 32 DEG C of culture 36h of culture in Shake flask medium, to 01-10 shake flask fermentation liquid β-
Fructofuranosidase enzyme activity is measured, and No. 04 shaking flask enzyme activity highest reaches 505U/ml.
Plating medium ingredient: glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%,
Epsom salt 0.01%, agar 2%, pH pH6.5.
Shake flask medium ingredient: sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, epsom salt
0.1%, diammonium hydrogen phosphate 0.4%, pH6.5.
(3) mutagenesis screening
Ultraviolet mutagenesis is carried out to No. 04 strain, ultraviolet mutagenesis is irradiated using 15W ultraviolet radiator 20cm, and irradiation time is
150s, the superior strain for finally obtaining high yield saccharase are named as BLCY-004, and enzyme activity reaches 1100U/ml.
Enzyme activity is defined as follows: when being reacted using sucrose as substrate for 6.0,40 DEG C of pH, increasing reducing power per minute
The enzyme amount for the amount of D-Glucose for being equivalent to 2 μm of ol is added to schedule as 1 unit of activity (U).
Ecological form
Bacterial strain is creamy white, translucent, 1.8~3.5 μm of about 2~4 μ m of size.Gram's staining is positive, quarter butt
Shape individually or in pairs arranges, along with rod-shaped, spherical-like morphology variation in cultivation cycle.
Embodiment 2
A kind of preparation method of lactosucrose, steps are as follows:
(1) mixed solution of sucrose and lactose that mass concentration is 20% is prepared, it is 1.2 that the addition of sucrose and lactose, which is compared:
1；The pH to 4 for adjusting mixed solution is inoculated with pole fermented liquid in the ratio of mixeding liquid volume 5%, under the conditions of 50 DEG C, reaction
12 hours, then into reaction solution, stream added mass concentration to be 40% sucrose solution, and sucrose mass concentration is in maintenance reaction solution
Stop reaction after 30%, the reaction was continued 12h, lactosucrose crude liquid is made；
(2) by lactosucrose crude liquid made from step (1) by mass percentage 0.5% ratio be added active carbon, 80
DEG C stirring 40min；Then through plate-frame filtering, filter pressure 0.4Mpa, water flow 6.0t/h；At continuous ionic exchange system
Manage feed liquid, after ion-exchange feed liquid light transmittance >=98%；Through chromatographic isolation, chromatographic run pressure 0.30MPa, temperature 60 C, water consume ratio
1:1.6 feeds 2.0m per hour3, collect lactosucrose；Then it is concentrated through sextuple effect, is concentrated into the 60% of original volume；
Liquid after concentration enters in drying tower, 140 DEG C of inlet air temperature, starts atomizer, spray-dried liquid is powdered solid
Lactosucrose is made in body.
Pole fermented liquid in the step (2) is prepared as follows:
I, arthrobacterium (Arthrobacter sp.) BLCY-004 is inoculated in seed culture medium, in 28 DEG C of condition
Under, for 24 hours, seed liquor is made in Multiplying culture；
The seed culture medium component is as follows, is weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%, epsom salt
0.01%, excess water, pH6.0；
II, by seed liquor made from step I by volume 1% ratio be inoculated in fermentation medium, 28 DEG C ferment
36h is cultivated, pole fermented liquid is made；
The fermentation medium component is as follows, is weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, epsom salt 0.1%, diammonium hydrogen phosphate
0.4%, excess water, pH 6.0；
Through detecting, lactosucrose purity obtained reaches 98.2%, and conversion ratio 55.2% can be applied to healthy food
Product, beverage, dairy products, health care product, medicine and other fields.
Embodiment 3
A kind of preparation method of lactosucrose, steps are as follows:
(1) mixed solution of sucrose and lactose that mass concentration is 40% is prepared, it is 1.3 that the addition of sucrose and lactose, which is compared:
1；The pH to 5 for adjusting mixed solution is inoculated with pole fermented liquid in the ratio of mixeding liquid volume 5%, under the conditions of 55 DEG C, reaction
12 hours, then into reaction solution, stream added mass concentration to be 40% sucrose solution, and sucrose mass concentration is in maintenance reaction solution
Stop reaction after 35%, the reaction was continued 18h, lactosucrose crude liquid is made；
(2) by lactosucrose crude liquid made from step (1) by mass percentage 0.25% ratio be added active carbon, 80
DEG C stirring 40min；Then through plate-frame filtering, filter pressure 0.2Mpa, water flow 5.0t/h；At continuous ionic exchange system
Manage feed liquid, after ion-exchange feed liquid light transmittance >=98%；Then it is concentrated through sextuple effect, is concentrated into the 65% of original volume；Through chromatography
Separation, chromatographic run pressure 0.20MPa, temperature 70 C, water consume ratio 1:1.3 feed 1.5m per hour3, collect lactosucrose；
Liquid after concentration enters in drying tower, 140 DEG C of inlet air temperature, starts atomizer, spray-dried liquid is powdered solid
Lactosucrose is made in body.
Pole fermented liquid in the step (2) is prepared as follows:
I, arthrobacterium (Arthrobacter sp.) BLCY-004 is inoculated in seed culture medium, in 28 DEG C of condition
Under, for 24 hours, seed liquor is made in Multiplying culture；
The seed culture medium component is as follows, is weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%, epsom salt
0.01%, excess water, pH6.0；
II, by seed liquor made from step I by volume 1% ratio be inoculated in fermentation medium, 28 DEG C ferment
36h is cultivated, pole fermented liquid is made；
The fermentation medium component is as follows, is weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, epsom salt 0.1%, diammonium hydrogen phosphate
0.4%, excess water, pH 6.0；
Through detecting, lactosucrose purity obtained reaches 98.5%, and conversion ratio 56.3% can be applied to healthy food
Product, beverage, dairy products, health care product, medicine and other fields.
Embodiment 4
A kind of preparation method of lactosucrose, steps are as follows:
(1) mixed solution of sucrose and lactose that mass concentration is 60% is prepared, it is 1.5 that the addition of sucrose and lactose, which is compared:
1；The pH to 6 for adjusting mixed solution is inoculated with pole fermented liquid in the ratio of mixeding liquid volume 5%, under the conditions of 60 DEG C, reaction
12 hours, then into reaction solution, stream added mass concentration to be 40% sucrose solution, and sucrose mass concentration is in maintenance reaction solution
40%, the reaction was continued stops reaction afterwards for 24 hours, and lactosucrose crude liquid is made；
(2) by lactosucrose crude liquid made from step (1) by mass percentage 1% ratio be added active carbon, 85 DEG C
Stir 30min；Then through plate-frame filtering, filter pressure 0.3Mpa, water flow 5.5t/h；It is handled using continuous ionic exchange system
Feed liquid, after ion-exchange feed liquid light transmittance >=98%；Then it is concentrated through sextuple effect, is concentrated into the 63% of original volume；Through chromatography point
From chromatographic run pressure 0.25MPa, 65 DEG C of temperature, water consume ratio 1:1.4 feeds 1.6m per hour3, collect lactosucrose；It is dense
Liquid after contracting enters in drying tower, 140 DEG C of inlet air temperature, starts atomizer, spray-dried liquid is pulverulent solids
Lactosucrose is made.
Pole fermented liquid in the step (2) is prepared as follows:
I, arthrobacterium (Arthrobacter sp.) BLCY-004 is inoculated in seed culture medium, in 35 DEG C of condition
Under, seed liquor is made in Multiplying culture 48h；
The seed culture medium component is as follows, is weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%, epsom salt
0.01%, excess water, pH6.0；
II, by seed liquor made from step I by volume 10% ratio be inoculated in fermentation medium, 35 DEG C ferment
60h is cultivated, pole fermented liquid is made；
The fermentation medium component is as follows, is weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, epsom salt 0.1%, diammonium hydrogen phosphate
0.4%, excess water, pH 6.0；
Through detecting, lactosucrose purity obtained reaches 98.3%, and conversion ratio 56.5% can be applied to healthy food
Product, beverage, dairy products, health care product, medicine and other fields.
Comparative example
The preparation method of lactosucrose as described in Example 2, the difference is that, utilize chlorophenol arthrobacterium
(Arthrobacter chlorophenolicus) SK33.001 replaces arthrobacterium (Arthrobacter sp.) BLCY-004.
Through detecting, lactosucrose purity obtained reaches 81.2%, conversion ratio 36.7%.
Claims (11)
1. a kind of preparation method of lactosucrose, which is characterized in that steps are as follows:
(1) pole fermented liquid is inoculated in the mixed solution of sucrose and lactose, under the conditions of 50~60 DEG C, reaction 10~14 is small
When, then stream plus sucrose solution into reaction solution, maintaining sucrose mass concentration in reaction solution is 30%~40%, the reaction was continued 12~
For 24 hours, stop reaction, lactosucrose crude liquid is made；
The Solute mass concentration of the mixed solution of the sucrose and lactose is 20%~60%, and the mass ratio of sucrose and lactose is (1.2
~1.5): 1, pH 4~6；
The arthrobacterium is arthrobacterium (Arthrobacter sp.) BLCY-004, which protected on August 16th, 2016
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3 Institute of Microorganism, Academia Sinica, deposit number CGMCC No.12855；
(2) lactosucrose crude liquid made from step (1) is made low through decoloration, filtering, ion-exchange, chromatographic isolation, concentration, drying
Poly- lactulose.
2. preparation method as described in claim 1, which is characterized in that in the step (1), the quality of stream plus sucrose solution is dense
Degree is 35~45%.
3. preparation method as described in claim 1, which is characterized in that in the step (1), the quality of stream plus sucrose solution is dense
Degree is 40%.
4. preparation method as described in claim 1, which is characterized in that in the step (1), arthrobacterium zymotic fluid preparation method
It is as follows:
I, arthrobacterium (Arthrobacter sp.) BLCY-004 is inoculated in seed culture medium, in 28~35 DEG C of condition
Under, seed liquor is made in 24~48h of Multiplying culture；
The seed culture medium component is as follows, is weight percentage:
Glucose 1~2%, yeast extract 0.5~1.5%, potassium dihydrogen phosphate 0.1~0.2%, diammonium hydrogen phosphate 0.3~0.6%, seven
Water magnesium sulfate 0.01~0.05%, excess water, pH6.0~7.2；
II, by seed liquor made from step I by volume 1~10% ratio be inoculated in fermentation medium, 28~35 DEG C send out
Pole fermented liquid is made in ferment 36~60h of culture；
The fermentation medium component is as follows, is weight percentage:
Sucrose 2~4%, lactose 0.5~1%, yeast extract 0.5~1.2%, peptone 0.4~0.8%, epsom salt 0.05~
0.1%, diammonium hydrogen phosphate 0.1~0.4%, excess water, pH 6.0~7.2.
5. preparation method as described in claim 1, which is characterized in that the inoculum concentration of pole fermented liquid in the step (1)
It is the 5% of mixed liquor volume percentage.
6. preparation method as described in claim 1, which is characterized in that in the step (2), decolorization process is as follows: by step
(1) lactosucrose crude liquid made from, by mass percentage 0.25~1% ratio be added active carbon, 80~85 DEG C stirring 30~
40min。
7. preparation method as described in claim 1, which is characterized in that in the step (2), filtering uses plate-frame filtering, mistake
Filtering pressure 0.2~0.4Mpa of power, 5.0~6.0t/h of water flow.
8. preparation method as described in claim 1, which is characterized in that in the step (2), ion-exchange is to be handed over using continuous ionic
Change system processing feed liquid, after ion-exchange feed liquid light transmittance >=98%.
9. preparation method as described in claim 1, which is characterized in that in the step (2), chromatrographic separation step are as follows:
Chromatographic run 0.20~0.30MPa of pressure, 60~70 DEG C of temperature, water consume ratio 1:1.3~1:1.6, charging 1.5 per hour~
2.0m3, collect lactosucrose.
10. preparation method as described in claim 1, which is characterized in that in the step (2), be concentrated to be concentrated into original volume
60~65%.
11. preparation method as described in claim 1, which is characterized in that dry for spray drying, step in the step (2)
It is as follows:
Liquid after concentration enters in drying tower, 130~150 DEG C of inlet air temperature, starts atomizer, liquid is spray-dried to be
Pulverulent solids.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610752459.6A CN106191157B (en) | 2016-08-29 | 2016-08-29 | A kind of preparation method of lactosucrose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610752459.6A CN106191157B (en) | 2016-08-29 | 2016-08-29 | A kind of preparation method of lactosucrose |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106191157A CN106191157A (en) | 2016-12-07 |
| CN106191157B true CN106191157B (en) | 2019-07-09 |
Family
ID=57526663
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610752459.6A Active CN106191157B (en) | 2016-08-29 | 2016-08-29 | A kind of preparation method of lactosucrose |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106191157B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI624547B (en) * | 2017-01-26 | 2018-05-21 | 國立中興大學 | Preparation method of lactosucrose |
Citations (4)
| 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 |
-
2016
- 2016-08-29 CN CN201610752459.6A patent/CN106191157B/en active Active
Patent Citations (4)
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106191157A (en) | 2016-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106520746B (en) | A kind of preparation method of high-purity D psicoses | |
| CN106434494B (en) | One bacillus subtilis and its cultural method and application | |
| US11279961B2 (en) | Aspergillus oryzae BLCY-006 strain and application thereof in preparation of galactooligosaccharide | |
| CN105112306B (en) | One Aspergillus oryzae and its cultural method and application | |
| CN103911322B (en) | Bacillus circulans and the application in symbiotic fermentation technology oligomeric galactose thereof | |
| CN101691538A (en) | Aspergillus oryzae and method for preparing high purity galacto-oligosaccharides by using same | |
| CN114214251B (en) | Bacillus subtilis for producing D-psicose and culture method and application thereof | |
| CN102226159B (en) | Strain of Enterobacter cloacae and its application in the preparation of 2,3-butylene glycol | |
| CN101555503A (en) | Method for separating and extracting L-arabinose from waste wood sugar mother liquid from wood sugar production | |
| CN104805026A (en) | Bacterial strain producing beta-galactosidase and method for preparing high-purity galactooligosaccharide | |
| CN106754411A (en) | One plant height produces the Aspergillus niger strain and its liquid state fermentation enzyme producing method of β D fructofuranosidases | |
| CN107699556A (en) | The method that D psicose epimerases are prepared using bacillus subtilis | |
| CN104130950A (en) | Aspergillus niger and cultivation method and application thereof | |
| CN106148243B (en) | One plant of production saccharase arthrobacterium and its application | |
| CN108949713B (en) | Preparation method of aspergillus oryzae fermentation broth and application of aspergillus oryzae fermentation broth in production of fructo-oligosaccharide | |
| CN104059857B (en) | One strain aspergillosis and the application in preparing transfructosylase thereof | |
| CN101928671A (en) | Method for preparing ginsenoside Rg3 from alternaria alternata YS-2 and fermented ginseng stem-leaf total saponin thereof | |
| CN106191157B (en) | A kind of preparation method of lactosucrose | |
| CN104302758B (en) | Produce turanose bacterial strain and application thereof | |
| CN102533607B (en) | Strain capable of producing beta-galactosidase and method for producing galactooligosaccharides by using beta-galactosidase | |
| CN106282145B (en) | A kind of liquid state fermentation method of adenylic acid deaminase | |
| CN110904171A (en) | Preparation process of low-alcohol-residue xanthan gum product | |
| CN102851220B (en) | Yeast strain capable of high-yield production of beta-galactosidase, and its application | |
| CN102796797B (en) | Method for preparing xylitol and its intermediate D-xylosone by microbial transformation of glucose and strain used in the same | |
| CN104480164A (en) | Enzymic method for preparing lactosucrose |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| CB02 | Change of applicant information |
Address after: 251200 Shandong province Dezhou Dexin Street (Yucheng) National hi tech Industrial Development Zone Applicant after: SHANDONG BAILONG CHUANGYUAN BIO-TECH Co.,Ltd. Address before: Dickson street 251200 Shandong city of Dezhou province Yucheng city high tech Development Zone Applicant before: Shandong Bailong Chuangyuan Biotechnology Co.,Ltd. |
|
| COR | Change of bibliographic data | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A preparation method of lactulose oligosaccharide Effective date of registration: 20221202 Granted publication date: 20190709 Pledgee: China Construction Bank Corporation Yucheng Sub branch Pledgor: SHANDONG BAILONG CHUANGYUAN BIO-TECH Co.,Ltd. Registration number: Y2022980024523 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Granted publication date: 20190709 Pledgee: China Construction Bank Corporation Yucheng Sub branch Pledgor: SHANDONG BAILONG CHUANGYUAN BIO-TECH Co.,Ltd. Registration number: Y2022980024523 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |