CN104745656B - A kind of method that the Portugal's oligosaccharides of β 1,3 is directly produced using curdlan fermentation liquid - Google Patents
A kind of method that the Portugal's oligosaccharides of β 1,3 is directly produced using curdlan fermentation liquid Download PDFInfo
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Abstract
The method that one kind directly produces the Portugal's oligosaccharides of β 1,3 using curdlan fermentation liquid, belongs to technical field of bioengineering.After the present invention carries out thermokalite inactivation treatment to curdlan fermentation liquid, one of nutrient media components as culture Trichoderma harzianum, after adding nitrogen source and micro- auxiliary material, Trichoderma harzianum obtains the Trichoderma harzianum zymotic fluid rich in the endoglucanases of β 1,3 through aerlbic culture；The Trichoderma harzianum zymotic fluid mixes with the fresh curdlan fermentation liquid without thermokalite inactivation treatment and carries out catalytic hydrolysis reaction again, does not carry out separation and Extraction to enzyme liquid and reaction substrate, directly obtains the Portugal's oligosaccharides semifinished products of β 1,3.The inventive method is directly used as enzyme hydrolysis substrate and Trichoderma harzianum fermenting and producing β 1 by the use of curdlan fermentation liquid, the carbon source and derivant of 3 endoglucanases, significantly reduce β 1, operating unit number prepared by 3 Portugal's oligosaccharides, both production efficiency had been improved, it is obvious to reduce three waste discharge and energy consumption, beneficial effect in production process again.
Description
Technical field
β -1 is directly produced using curdlan fermentation liquid the present invention relates to one kind, the method for 3- Portugals oligosaccharides, belongs to bioengineering
Technical field.
Background technology
β -1,3- Portugals oligosaccharides is a kind of bioactive substance with critical function.Its biological function having is：（1）Promote
Enter the growth of intestinal beneficial flora.（2）Adjust organism immune response.Widely using for antibiotic improves pathogen to antigen
Defensive ability/resistance ability, feature β -1,3- Portugals oligosaccharides can improve immunogenicity as the reinforcing agent of antigen.（3）Resist tumour.β-1,
3- Portugals oligosaccharides is strengthening macrophage activity, while producing cytotoxicity to tumour cell, additionally it is possible to it is produced tumour bad
Necrosis factor（TNF-α）, il-1（IL-1）And interferon（IFN-α）Deng so as to resist tumour.（4）Induction plant resists disease
Evil.β -1,3- Portugal oligosaccharides can excite a series of signal to react, so as to promote the synthesis of Buchner's bodies and phytoalexin, so as to
Resist the invasion of pathogenic bacteria.The plant that can be responded β -1,3- Portugals oligosaccharides exciton and produce antibiotic reported at present has greatly
Beans, grape, rice, wheat, tobacco and arabidopsis etc..
The preparation method of β -1,3- Portugals oligosaccharides mainly includes following three kinds at present：Direct extraction method, artificial synthesized method and more
Syrup solution.Direct extraction method includes water extraction, organic solvent extractionprocess and microwave loss mechanisms etc..Extracted by different methods
Afterwards it is generally necessary to the macromolecular substances such as protein and fat therein be removed, then through decolouring, desalination, concentration and drying and other steps
Obtain oligosaccharides crude product.Artificial synthesized method includes enzymatic clarification, chemical synthesis and microwave process for synthesizing.It is used for during enzymatic clarification
The enzyme of oligosaccharides synthesis mainly has glucoside transferase, glycosyl hydrolase and FscMⅢ etc..Abundant raw material sources are more syrup
Solution prepares the most significant advantage of oligosaccharides.Polysaccharide hydrolysis method includes acid-hydrolysis method and enzyme hydrolysis method.The conventional acid reagent of hydrolysis has
HCl, TFA and H2SO4Deng.There is multiple-microorganism to produce β -1 in nature, 3- endoglucanases, be broadly divided into following two
Class：Bacterium and fungi.In bacterium based on bacillus, mainly have bacillus subtilis (Bacillus subtilis), lichens
Bacillus (Bacillus licheniformis) etc.；In fungi based on mould, mainly there is koning trichoderma
(Trichoderma koningii), trichoderma reesei (Trichoderma reesei), Trichoderma viride (Trichoderma viride) and Trichoderma harzianum (Trichoderma harzianum) etc..
Beta-1,3-dextran is the polysaccharide with different branches being formed by connecting by β -1,3- glycosidic bonds.Polysaccharide hydrolysis method is adopted
Preparing raw material mainly has laminarin, schizophan, lentinan, pachymaran and thermal gels etc., in addition to thermal gels, its
He carries different degrees of β -1,6- glycosidic bonds branch by polysaccharide.Compared with other polysaccharide, thermal gels molecular structure is relatively easy,
Without β -1,6- glycosidic bonds branch, can largely it be obtained by microbe fermentation method, lower-price characteristic higher with purity.
Hydrolyzed using thermal gels and prepare β -1,3- Portugals oligosaccharides, can effectively avoided the removal processing step of impurity in other raw materials and its bring
Environmental pollution, substantially reduce oligosaccharides production cost.
The content of the invention
It is an object of the invention to provide one kind β -1, the method for 3- Portugals oligosaccharides, to obtain directly are produced using curdlan fermentation liquid
Obtain β -1,3- Portugals oligosaccharides semifinished product.
Technical scheme：Curdlan fermentation liquid is obtained first with the ventilating fermentations of agrobacterium ATCC 31749
（Applied Biochemistry and Microbiology, 2014,50(1):35–42）, the NaOH for adding 10mol/L is molten
Liquid, the pH of curdlan fermentation liquid is risen to 9 ~ 12, be heated to 40 ~ 55 DEG C, be stirred 30 ~ 50 min, make agrobacterium
Death, then pH is adjusted to slant acidity with 12 mol/L dense HCl（PH is 5 ~ 7）.Curdlan fermentation liquid after processing is as culture
One of nutrient media components of Trichoderma harzianum, add through high-temperature sterilization（121 DEG C, 30min）Concentration nitrogen source solution after processing, addition
10% ~ 20% (V/V) of volume is formulated based on ratio, the composition of concentration nitrogen source solution is tryptone 10% ~ 20% (W/V),
NaNO35% ~ 10% (W/V), pH are 5 ~ 7, are prepared with pure water.InoculationTrichoderma harzianum Rifai ACCC
30371（Food industry science and technology, 2014,35 (12):157-161）Fermentation is aerated, inoculation volume is 5% ~ 15% (V/V),
Ventilation condition is 0.5 ~ 1.0 vvm, and speed change adjustment speed of agitator makes oxygen dissolving value reach more than 30%, and fermentation temperature is 25 ~ 32 DEG C,
Fermentation time is 3 ~ 5d, and acquisition is rich in β -1, the Trichoderma harzianum zymotic fluid of 3- endoglucanases.Without the fresh of inactivation treatment
Curdlan fermentation liquid again with the Trichoderma harzianum zymotic fluid according to a certain volume（10~20:1, V/V）Mixing, using β -1,3- Portugals gather
Reaction is hydrolyzed in sugared restriction endonuclease, and reaction temperature is 40 ~ 60 DEG C, and reaction pH is 4.0 ~ 6.5, obtains being rich in β -1 after reacting 2 ~ 3h,
The mixed liquor of 3- Portugals oligosaccharides, reaction reheat reacting liquid temperature to 70 ~ 90 DEG C, maintain 20 ~ 40min to inactivate enzyme after terminating.Institute
The mixture centrifuged or filtered operation again obtained, centrifuging process are 3000 ~ 8000 r/min, 10 ~ 20 min, collect supernatant
Liquid.Separation of solid and liquid process can also use plate-frame filtering technique, and filter cloth aperture is 200 ~ 400 mesh, and filter pressure is 0.2 ~
0.8MPa, collect filtrate.Finally concentrated and dried, obtain β -1,3- Portugals oligosaccharides semifinished product, concentration and drying temperature do not surpass
Cross 110 DEG C.
Beneficial effects of the present invention：The production of β -1,3- Portugals oligosaccharides is prepared currently with beta-1,3-dextran biological hydrolysis process
Technique is more using beta-1,3-dextran after purification.With the powdered thermal gels of commercialization, laminarin, schizophan, mushroom
Polysaccharide, pachymaran etc. again after dissolving or aquation, add commercialization β -1 after purification through initial gross separation to set out raw material,
3- endoglucanase enzyme solutions, production technology is more complicated, and operating procedure is more, and substrate conversion efficiency is relatively low, particularly using thermal gels as
During substrate, because thermal gels are insoluble in the polysaccharide of water, for the thermal gels of commercialization due to have passed through drying process, easily being formed can not
Inverse triple-helix structure, not only not soluble in water, hydration process is also highly difficult, and thermal gels particle has double-deck tie in hydrolytic process
Structure：I.e. a part of heat setting xanthan molecule is wrapped in the insoluble kernel periphery of densification, its periphery easily quilt with aquation colloidal form
Hydrolysis, but its kernel portion is difficult then to be hydrolyzed, and causes substrate conversion efficiency to be remarkably decreased（Applied Microbiology and
Biotechnology, 2013, 97: 8495– 8503）.Present invention process directly uses fresh curdlan fermentation liquid, heat
Gel particle will not form hud typed double-decker, and substrate can be hydrolyzed thoroughly, and substrate conversion efficiency is high.At the same time, with heat setting
Glue zymotic fluid is one of primary raw material of Trichoderma harzianum ventilating fermentation, and thermal gels are both the carbon source material of Trichoderma harzianum growth, again
It is β -1, the derivant of 3- endoglucanase biosynthesis, is advantageous to efficiently synthesize β -1,3- endoglucanases, finally has
β -1,3- Portugals oligosaccharides is prepared beneficial to efficient.The inventive method is directly used as enzyme hydrolysis substrate and Ha Ci wood by the use of curdlan fermentation liquid
Mould fermenting and producing β -1, the carbon source and derivant of 3- endoglucanases, significantly reduce β -1, operation list prepared by 3- Portugals oligosaccharides
First number, had both improved production efficiency, and it is obvious to reduce three waste discharge and energy consumption, beneficial effect in production process again.
Brief description of the drawings
Fig. 1 β -1,3- Portugals oligosaccharides semifinished product technological process of production schematic diagram.
Embodiment
Embodiment 1：Curdlan fermentation liquid thermokalite inactivation technology
Curdlan fermentation liquid, the same bibliography of fermentation process are obtained using the ventilating fermentations of agrobacterium ATCC 31749
（Applied Biochemistry and Microbiology, 2014,50(1):35–42）.Seed culture based formulas is（g/
L）：Glucose 20, dusty yeast 10, KH2PO41.74 MgSO4⋅7H2O 0.5, initial pH are 7.0.With the ring of transfer needle picking one
The single bacterium colonies of agrobacterium ATCC 31749, it is seeded in the 250mL triangle shaking flasks of the seed culture medium containing 60mL, is placed in shaking table
Under 200 rpm rotating speeds, 30 °C culture 18 h.Basal fermentation medium uses culture medium A, and its composition is（g/L）：Glucose
15, dusty yeast 1.0, NH4Cl 0.5, KH2PO40.5, MgSO4⋅7H2O 0.5, FeCl30.01, MnCl20.01, CaCl2
0.01, NaCl 0.01.By in the 7.0L fermentation tanks of seed liquor transposing to the culture medium A containing 3.5L, inoculation volume ratio is 10%（V/
V）, during thalli growth pH will continuous decrease, use ammoniacal liquor（12.5%, V/V）Regulation zymotic fluid pH value is allowed to constant 7.0,
Fermentation temperature is 30 °C, air flux 1.0vvm, speed of agitator 500rpm.When residual sugar content drops to 1.0 below g/L
When, with 40%（V/V）NaOH solution replaces ammoniacal liquor and adds glucose molten as pH adjusting agent, while with 30mL/h flow constant speed stream
Liquid（50%, V/V）Into fermentation tank, continue 40h, ferment 12 hours and terminate again afterwards.
3.0L zymotic fluids are taken into 7.0L jacketed reactors, stream adds 10mol/L NaOH solution, adjusts zymotic fluid
PH to 11,45 DEG C are warming up to using chuck hot water heating, are stirred 45 min, again with 12 mol/L HCl after inactivation treatment
Solution adjusts pH to slant acidity（pH 6.5）, the curdlan fermentation liquid after being inactivated.
Embodiment 2：Trichoderma harzianum ventilating fermentation technique
After the curdlan fermentation liquid in embodiment 1 after thermokalite inactivation treatment is drained into sterilizing in advance using sterile working
7.0L fermentation tanks in.0.4 L concentration nitrogen source solution is prepared in advance, and its formula composition is：Tryptone 10% (W/V), NaNO3
8% (W/V), pH 6, is prepared, through high-temperature sterilization with pure water（121 DEG C, 30min）Accessed after processing in 7.0L fermentation tanks, on
State after two kinds of materials merge is culture medium B.
The same bibliography of the preparation method of Trichoderma harzianum seed culture medium and seed liquor（Food industry science and technology, 2014,35
(12):157-161）, seed culture based formulas is（g/L）:Glucose 15, dusty yeast 20, ammonium sulfate 2.5, potassium dihydrogen phosphate 6,
Magnesium sulfate 0.8, calcium chloride 1, pH6.0, seed culture condition are：The ring Trichoderma harzianum single bacterium colony of transfer needle picking one, is seeded to and contains
In the 250mL triangle shaking flasks of 40mL seed culture mediums, it is placed in shaking table under 250 rpm rotating speeds, 28 °C of culture 24h.Breathe out thatch wood
Mould ventilating fermentation condition is：Inoculation volume is 5% (V/V), and ventilation condition is 1.0 vvm, and speed change adjustment speed of agitator makes dissolved oxygen
Value reaches more than 30%, and fermentation temperature is 28 DEG C, fermentation time 5d, and acquisition is rich in β -1, the Kazakhstan thatch wood of 3- endoglucanases
Mould zymotic fluid.
Embodiment 3：Enzymatic hydrolysis technique
In Example 1 without flux-calcined curdlan fermentation liquid 3.0L, add in 7.0L jacket reactor, add
Enter the preparation of 300mL embodiments 2 is rich in β -1,3- endoglucanase Trichoderma harzianum zymotic fluids, is stirred（100 r/min）
Reaction is hydrolyzed, reaction temperature is 45 DEG C, and reaction pH is 6.0, and the reaction time is 2.5 h, and reaction is warming up to 80 again after terminating
DEG C, maintain 30min to inactivate enzyme, acquisition is rich in β -1, the mixed liquor of 3- Portugals oligosaccharides.
Embodiment 4：Centrifuge extraction process
The mixed liquor of the gained of embodiment 3 is centrifuged（5000 r/min, 15 min）, collect supernatant.Finally carry out
It is concentrated by evaporation and dries, obtain β -1,3- Portugals oligosaccharides semifinished product, concentration and drying temperature is no more than 110 DEG C.
Embodiment 5：Or it is separated by filtration extraction process
Plate-frame filtering is carried out to the mixed liquor of the gained of embodiment 3, filter cloth aperture is 300 mesh, filter pressure 0.3MPa, is received
Collect filtrate.Finally it is evaporated concentration and dries, obtain β -1,3- Portugals oligosaccharides semifinished product, concentration and drying temperature is no more than 110
℃。
Claims (1)
1. one kind directly produces β -1, the method for 3- Portugals oligosaccharides, it is characterized in that using thermokalite condition to heat using curdlan fermentation liquid
Gel zymotic fluid carries out inactivation treatment；The curdlan fermentation liquid of inactivation treatment as culture Trichoderma harzianum nutrient media components it
One, after adding nitrogen source and micro- auxiliary material, then it is aerated fermentation acquisition and is rich in β -1, the Kazakhstan thatch wood of 3- endoglucanases
Mould zymotic fluid；The Trichoderma harzianum zymotic fluid is mixed and is catalyzed with the fresh curdlan fermentation liquid without thermokalite inactivation treatment again
Hydrolysis, directly obtain β -1,3- Portugals oligosaccharides；Centrifuged or filtered operation, last concentrated and drying process, is obtained
β -1,3- Portugals oligosaccharides semifinished product；Step is：
（1）Inactivation treatment is carried out to curdlan fermentation liquid using thermokalite condition：
10mol/L NaOH solution is added, the pH of curdlan fermentation liquid is risen to 9 ~ 12, is heated to 40 ~ 55 DEG C, stirring is mixed
30 ~ 50 min are closed, pH to slant acidity pH 5 ~ 7 is adjusted with 12 mol/L HCl again after processing；
（2）Prepare the culture medium of Trichoderma harzianum：With through step（1）It is formulated, adds based on the curdlan fermentation liquid of inactivation treatment
Concentration nitrogen source solution through 121 DEG C of high-temperature sterilization 30min processing, 10% ~ 20% V/V of volume is formulated based on adding proportion, it is dense
The composition of contracting nitrogen source solution is tryptone 10% ~ 20% W/V, NaNO35% ~ 10% W/V, pH are 5 ~ 7, are prepared with pure water；
（3）Ventilating fermentation obtains the Trichoderma harzianum zymotic fluid rich in beta-1,3-dextran restriction endonuclease：Using Trichoderma harzianum as kind of setting out
Son, with step（2）That prepares is fermented for culture medium, and Trichoderma harzianum ventilating fermentation condition, inoculation volume is 5% ~ 15% V/V,
Ventilation condition is 0.5 ~ 1.0 vvm, and speed change adjustment speed of agitator makes oxygen dissolving value reach more than 30%, and fermentation temperature is 25 ~ 32 DEG C,
Fermentation time is 3 ~ 5d；
（4）Catalytic hydrolysis reaction：Reaction substrate is the fresh curdlan fermentation liquid without inactivation treatment, and enzyme liquid is step（3）Institute
What is obtained is rich in β -1, the Trichoderma harzianum zymotic fluid of 3- endoglucanases, fresh curdlan fermentation Ye ︰ Trichoderma harzianums is fermented
Reaction is hydrolyzed in liquid 10 ~ 20 ︰ 1 mixing by volume, and reaction temperature is 40 ~ 60 DEG C, and reaction pH is 4.0 ~ 6.5, the reaction time
For 2 ~ 3h, reaction reheats reacting liquid temperature to 70 ~ 90 DEG C, maintains 20 ~ 40min after terminating；
（5）Separation of solid and liquid：Using centrifuging process：3000 ~ 8000 r/min, centrifugation time are 10 ~ 20 min, or plate-frame filtering work
Skill：Filter cloth aperture is 200 ~ 400 mesh, and filter pressure is 0.2 ~ 0.8MPa, collects supernatant or filtrate；
（6）Concentrate and dry：Supernatant or filtrate are concentrated and dried, temperature is no more than 110 DEG C, obtains β -1, and 3- Portugals are few
Sugared semifinished product.
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| CN105566510B (en) * | 2015-12-18 | 2018-01-05 | 南京理工大学 | A kind of Portugal's oligosaccharides and its preparation method and application |
| CN105567779B (en) * | 2016-03-03 | 2018-09-21 | 江南大学 | A kind of fermentation process of low molecular weight thermal gels |
| WO2018086008A1 (en) * | 2016-11-09 | 2018-05-17 | 中国农业大学 | BACTERIAL β-1,3-GLUCANASE AND CODING GENE AND APPLICATION THEREOF |
| CN108467876B (en) * | 2018-03-16 | 2021-12-07 | 华东师范大学 | Fermentation method for increasing yield of curdlan |
| CN110607338B (en) * | 2019-10-12 | 2021-08-24 | 江南大学 | Method for producing branched beta-1, 3-gluco-oligosaccharide by fermentation of mixed fungi |
| CN114601168B (en) * | 2022-03-24 | 2023-02-21 | 江南大学 | Method for preparing prebiotics-containing probiotic microcapsules by spray drying |
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| CN1583801A (en) * | 2004-06-11 | 2005-02-23 | 江南大学 | Extracting process for microbiological polysaccharide-hot gel |
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| CN1583801A (en) * | 2004-06-11 | 2005-02-23 | 江南大学 | Extracting process for microbiological polysaccharide-hot gel |
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| Enzymatic and chemical degradation of curdlan targeting the production of β-(1→3) oligoglucans;C.Grandpierre et al.;《Carbohydrate Polymers》;20070616;第71卷(第2期);第277页摘要、右栏第1段，第278页左栏第4段至第279页左栏第3段，第282页右栏第1段至第285页右栏第3段，图6-10，表2 * |
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