CN104745656A - Method for directly producing beta-1,3-glucooligosaccharides by virtue of thermal gel fermentation liquor - Google Patents
Method for directly producing beta-1,3-glucooligosaccharides by virtue of thermal gel fermentation liquor Download PDFInfo
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Abstract
The invention discloses a method for directly producing beta-1,3-glucooligosaccharides by virtue of thermal gel fermentation liquor, and belongs to the technical field of bioengineering. The method disclosed by the invention comprises the following steps: carrying out hot-alkaline inactivation treatment on the thermal gel fermentation liquor and then using the thermal gel fermentation liquor as one of the components of a culture medium for culturing trichoderma harzianum, adding a nitrogen source and trace element auxiliary materials, and carrying out ventilated culture on trichoderma harzianum to obtain trichoderma harzianum fermentation liquor rich in beta-1,3-endoglucanases; and mixing the trichoderma harzianum fermentation liquor with fresh thermal gel fermentation liquor which is not subjected to the hot-alkaline inactivation treatment so as to carry out a catalytic hydrolysis reaction, thereby directly obtaining beta-1,3-glucooligosaccharide crude products without the need of separating and extracting enzyme liquor and a reaction substrate. According to the method disclosed by the invention, the thermal gel fermentation liquor is directly used as a carbon source and an inducer for producing the beta-1,3-endoglucanases by virtue of the fermentation of the enzyme hydrolysis substrate and trichoderma harzianum, and the operation unit number of the preparation for the beta-1,3-glucooligosaccharides is remarkably reduced, thus increasing the production efficiency of the products, reducing the three-waste emission and energy consumption during the production process, and achieving obvious beneficial effects.
Description
Technical field
The present invention relates to the method that one utilizes curdlan fermentation liquid direct production β-1,3-Portugal oligosaccharides, belong to technical field of bioengineering.
Background technology
β-1,3-Portugal oligosaccharides is the biologically active substance that a class has critical function.Its biological function had is: (1) promotes the growth of intestinal beneficial flora.(2) organism immune response is regulated.Antibiotic widely using improves the defensive ability/resistance ability of pathogenic bacteria to antigen, and functional β-1,3-Portugal oligosaccharides can improve immunogenicity as the toughener of antigen.(3) tumour is resisted.β-1,3-Portugal oligosaccharides, at enhancing macrophage activity, the while of producing Cytotoxic, can also make it produce tumour necrosis factor (TNF-α), il-1 (IL-1) and Interferon, rabbit (IFN-α) etc. to tumour cell, thus opposing tumour.(4) inducing plant resists disease.β-1,3-Portugal oligosaccharides can excite a series of signal to react, thus promotes the synthesis of Buchner's bodies and phytoalexin, thus resists the invasion of pathogenic bacterium.Can responding β-1,3-Portugal oligosaccharides exciton and producing antibiotic plant of current report has soybean, grape, paddy rice, wheat, tobacco and Arabidopis thaliana etc.
The preparation method of current β-1,3-Portugal oligosaccharides mainly comprises following three kinds: direct extraction method, synthetic method and polysaccharide hydrolysis method.Direct extraction method comprises water extraction, organic solvent extractionprocess and microwave loss mechanisms etc.By usually needing removing protein wherein and fat to wait macromolecular substance after diverse ways extracting, then through decolouring, desalination, to concentrate and drying and other steps obtains oligosaccharides crude product.Synthetic method comprises enzymatic clarification, chemical synthesis and microwave process for synthesizing.FscMⅠ, glycosyl hydrolase and FscMⅢ etc. are mainly contained for the enzyme of oligosaccharides synthesis in enzymatic clarification process.Abundant raw material sources are polysaccharide hydrolysis legal systems for the most significant advantage of oligosaccharides.Polysaccharide hydrolysis method comprises acid-hydrolysis method and enzyme hydrolysis method.The sour reagent that hydrolysis is commonly used has HCl, TFA and H
2sO
4deng.Occurring in nature has multiple-microorganism can produce β-1,3-endoglucanase, is mainly divided into following two classes: bacterium and fungi.Based on genus bacillus in bacterium, mainly contain subtilis (
bacillus subtilis), Bacillus licheniformis (
bacillus licheniformis) etc.; Based on mould in fungi, mainly contain koning trichoderma (
trichoderma koningii), Trichodermareesei (
trichoderma reesei), viride (
trichoderma viride) and trichoderma harziarum (
trichoderma harzianum) etc.
β-1,3-dextran is the polysaccharide with different branch be formed by connecting by β-1,3-glycosidic link.The raw materials that polysaccharide hydrolysis method adopts mainly contains laminarin, schizophan, lentinan, Pachymose and heat setting glue etc., and outside heat extraction gel, other polysaccharide are all with β-1,6-glycosidic link branch in various degree.Compared with other polysaccharide, heat setting xanthan molecule structure is relatively simple, is not with β-1,6-glycosidic link branch, obtains in a large number, have purity higher, lower-price characteristic by microbe fermentation method.Utilize hot gel hydrolysis to prepare β-1,3-Portugal oligosaccharides, the removal processing step effectively can avoiding impurity in other raw materials and the environmental pollution brought thereof, greatly reduce oligosaccharides production cost.
Summary of the invention
The object of this invention is to provide the method that one utilizes curdlan fermentation liquid direct production β-1,3-Portugal oligosaccharides, to obtain β-1,3-Portugal oligosaccharides raw product.
Technical scheme of the present invention: first utilize edaphic bacillus ATCC 31749 ventilating fermentation to obtain curdlan fermentation liquid (Applied Biochemistry and Microbiology, 2014,50 (1): 35 – 42), add the NaOH solution of 10mol/L, make the pH of curdlan fermentation liquid rise to 9 ~ 12, be heated to 40 ~ 55 DEG C, be uniformly mixed 30 ~ 50 min, make edaphic bacillus dead, then regulate pH to slant acidity (pH is 5 ~ 7) with the dense HCl of 12 mol/L.Curdlan fermentation liquid after process is as one of nutrient media components cultivating trichoderma harziarum, add through high-temperature sterilization (121 DEG C, concentrated nitrogenous source solution 30min) after process, fill a prescription based on adding proportion 10% ~ 20% (V/V) of volume, what concentrate nitrogenous source solution consists of Tryptones 10% ~ 20% (W/V), NaNO
35% ~ 10% (W/V), pH are 5 ~ 7, prepare with pure water.Inoculation
trichoderma harzianumrifai ACCC 30371(foodstuffs industry science and technology, 2014,35 (12): 157-161) ventilating fermentation is carried out, inoculation volume is 5% ~ 15% (V/V), and ventilation condition is 0.5 ~ 1.0 vvm, and speed change adjustment mixing speed makes oxygen dissolving value reach more than 30%, leavening temperature is 25 ~ 32 DEG C, fermentation time is 3 ~ 5d, obtains the trichoderma harziarum fermented liquid being rich in β-1,3-endoglucanase.Without inactivation treatment fresh curdlan fermentation liquid again with this trichoderma harziarum fermented liquid (10 ~ 20:1 according to a certain volume, V/V) mix, utilize β-1,3-endoglucanase is hydrolyzed reaction, and temperature of reaction is 40 ~ 60 DEG C, and reaction pH is 4.0 ~ 6.5, obtain being rich in β-1 after reaction 2 ~ 3h, the mixed solution of 3-Portugal oligosaccharides, reheats reacting liquid temperature to 70 ~ 90 DEG C after reaction terminates, and maintains 20 ~ 40min and makes enzyme deactivation.The mixture of gained is again through centrifugal or filtering separation operation, and centrifuging process is 3000 ~ 8000 r/min, 10 ~ 20 min, collects supernatant liquor.Solid-liquid separation process also can adopt Plate Filtration technique, and filter cloth aperture is 200 ~ 400 orders, and filter pressure is 0.2 ~ 0.8MPa, collects filtrate.Finally carry out concentrated and dry, obtain β-1,3-Portugal oligosaccharides raw product, concentrated and drying temperature is no more than 110 DEG C.
Beneficial effect of the present invention: β-1, the 3-dextran after utilizing β-1,3-dextran biological hydrolysis process to prepare production technique many employings purifying of β-1,3-Portugal oligosaccharides at present.With commercial Powdered heat setting glue, laminarin, schizophan, lentinan, Pachymoses etc. are the raw material that sets out, again dissolve or aquation after, add the commercialization β-1 after initial gross separation purifying again, 3-endoglucanase enzyme solution, production technique is more complicated, operation steps is many, substrate conversion efficiency is lower, particularly with heat setting glue for substrate time, because heat setting glue is water-fast polysaccharide, commercial heat setting glue is owing to have passed through drying treatment, the irreversible triple-helix structure of easy formation, not only water insoluble, hydration process is also very difficult, in hydrolytic process, hot gel particle has bilayer structure: namely a part of heat setting xanthan molecule is wrapped in fine and close insoluble kernel periphery with aquation colloidal form, its peripheral portion is easily hydrolyzed, but its kernel portion is then difficult to be hydrolyzed, substrate conversion efficiency is caused significantly to decline (Applied Microbiology and Biotechnology, 2013, 97:8495 – 8503).Present invention process directly adopts fresh curdlan fermentation liquid, and hot gel particle can not form hud typed bilayer structure, and substrate can be thoroughly hydrolyzed, and substrate conversion efficiency is high.Meanwhile, one of main raw material being trichoderma harziarum ventilating fermentation with curdlan fermentation liquid, heat setting glue is the carbon source material of trichoderma harziarum growth, again β-1, the biosynthetic inductor of 3-endoglucanase, is conducive to efficiently synthesizing β-1,3-endoglucanase, finally be conducive to efficiently preparing β-1,3-Portugal oligosaccharides.The inventive method directly utilizes curdlan fermentation liquid as enzymic hydrolysis substrate and trichoderma harziarum fermentative production β-1, the carbon source of 3-endoglucanase and inductor, significantly reduce β-1, operating unit number prepared by 3-Portugal oligosaccharides, both improve products production efficiency, additionally reduce three waste discharge and energy consumption in production process, beneficial effect is obvious.
Accompanying drawing explanation
Fig. 1 β-1,3-Portugal oligosaccharides raw product technological process of production schematic diagram.
Embodiment
Embodiment 1: curdlan fermentation liquid thermokalite inactivation technology
Edaphic bacillus ATCC 31749 ventilating fermentation is utilized to obtain curdlan fermentation liquid, the same reference of fermentation process (Applied Biochemistry and Microbiology, 2014,50 (1): 35 – 42).Seed culture based formulas is (g/L): glucose 20, yeast powder 10, KH
2pO
41.74, MgSO
47H
2o 0.5, initial pH are 7.0.With the single bacterium colony of inoculating needle picking one ring edaphic bacillus ATCC 31749, be seeded in the 250mL triangle shaking flask containing 60mL seed culture medium, be placed in shaking table under 200 rpm rotating speeds, 30 ° of C cultivate 18 h.Basal fermentation medium adopts culture medium A, and its composition is (g/L): glucose 15, yeast powder 1.0, NH
4cl 0.5, KH
2pO
40.5, MgSO
47H
2o 0.5, FeCl
30.01, MnCl
20.01, CaCl
20.01, NaCl 0.01.Seed liquor is moved in the 7.0L fermentor tank be connected to containing 3.5L culture medium A, inoculation volume ratio is 10%(V/V), in thalli growth process, pH will continuous decrease, with ammoniacal liquor (12.5%, V/V) fermented liquid pH value is regulated to make it constant in 7.0, leavening temperature is 30 ° of C, and strength ofdraft is 1.0vvm, and mixing speed is 500rpm.When residual sugar content drops to 1.0 below g/L, with 40%(V/V) NaOH solution replacement ammoniacal liquor is as pH adjusting agent, and add glucose solution (50%, V/V) in fermentor tank with the flow constant speed stream of 30mL/h, lasting 40h, ferments afterwards again and terminates for 12 hours simultaneously.
Get 3.0L fermented liquid in 7.0L jacketed reactor, stream adds the NaOH solution of 10mol/L, regulate the pH to 11 of fermented liquid, chuck hot water heating is adopted to be warming up to 45 DEG C, be uniformly mixed 45 min, HCl solution again with 12 mol/L after inactivation treatment regulates pH to slant acidity (pH 6.5), obtains the curdlan fermentation liquid after deactivation.
Embodiment 2: trichoderma harziarum ventilating fermentation technique
Adopt aseptic technique by the 7.0L fermentor tank after the curdlan fermentation liquid drainage in embodiment 1 after thermokalite inactivation treatment to sterilizing in advance.Prepare 0.4 L in advance and concentrate nitrogenous source solution, its formula consists of: Tryptones 10% (W/V), NaNO
38% (W/V), pH are 6, with pure water preparation, after high-temperature sterilization (121 DEG C, 30min) process in access 7.0L fermentor tank, are substratum B after above-mentioned two kinds of materials merge.
The same reference of preparation method (the foodstuffs industry science and technology of trichoderma harziarum seed culture medium and seed liquor, 2014,35 (12): 157-161), seed culture based formulas is (g/L): glucose 15, yeast powder 20, ammonium sulfate 2.5, potassium primary phosphate 6, magnesium sulfate 0.8, calcium chloride 1, pH6.0, seed culture condition is: inoculating needle picking one ring trichoderma harziarum list bacterium colony, be seeded in the 250mL triangle shaking flask containing 40mL seed culture medium, be placed in shaking table under 250 rpm rotating speeds, 28 ° of C cultivate 24h.Trichoderma harziarum ventilating fermentation condition is: inoculation volume is 5% (V/V), and ventilation condition is 1.0 vvm, and speed change adjustment mixing speed makes oxygen dissolving value reach more than 30%, leavening temperature is 28 DEG C, fermentation time is 5d, obtains the trichoderma harziarum fermented liquid being rich in β-1,3-endoglucanase.
Embodiment 3: enzymatic hydrolysis technique
In Example 1 without flux-calcined curdlan fermentation liquid 3.0L, add in the jacket reactor of 7.0L, what add 300mL embodiment 2 preparation is rich in β-1,3-endoglucanase trichoderma harziarum fermented liquid, be uniformly mixed (100 r/min) to be hydrolyzed reaction, temperature of reaction is 45 DEG C, and reaction pH is 6.0, and the reaction times is 2.5 h, 80 DEG C are warming up to again after reaction terminates, maintain 30min and make enzyme deactivation, obtain the mixed solution being rich in β-1,3-Portugal oligosaccharides.
Embodiment 4: centrifugation extraction process
The mixed solution of embodiment 3 gained is carried out centrifugal (5000 r/min, 15 min), collects supernatant liquor.Finally carry out evaporation concentration and drying, obtain β-1,3-Portugal oligosaccharides raw product, concentrated and drying temperature is no more than 110 DEG C.
Embodiment 5: or filtering separation extraction process
Carry out Plate Filtration to the mixed solution of embodiment 3 gained, filter cloth aperture is 300 orders, and filter pressure is 0.3MPa, collects filtrate.Finally carry out evaporation concentration and drying, obtain β-1,3-Portugal oligosaccharides raw product, concentrated and drying temperature is no more than 110 DEG C.
Claims (1)
1. one kind utilizes the method for curdlan fermentation liquid direct production β-1,3-Portugal oligosaccharides, it is characterized in that utilizing thermokalite condition to carry out inactivation treatment to curdlan fermentation liquid; The curdlan fermentation liquid of inactivation treatment, as one of nutrient media components cultivating trichoderma harziarum, after adding nitrogenous source and micro-auxiliary material, then carries out the trichoderma harziarum fermented liquid that β-1,3-endoglucanase is rich in ventilating fermentation acquisition; This trichoderma harziarum fermented liquid mixes with the fresh curdlan fermentation liquid without thermokalite inactivation treatment and carries out catalytic hydrolysis reaction again, directly obtains β-1,3-Portugal oligosaccharides; Through centrifugal or filtering separation operation, finally by concentrated and drying treatment, obtain β-1,3-Portugal oligosaccharides raw product; Step is:
(1) thermokalite condition is utilized to carry out inactivation treatment to curdlan fermentation liquid:
Add the NaOH solution of 10mol/L, make the pH of curdlan fermentation liquid rise to 9 ~ 12, be heated to 40 ~ 55 DEG C, be uniformly mixed 30 ~ 50 min, the HCl again with 12 mol/L after process regulates pH to slant acidity pH 5 ~ 7;
(2) substratum of trichoderma harziarum is prepared: fill a prescription based on the curdlan fermentation liquid through step (1) inactivation treatment, add the concentrated nitrogenous source solution through 121 DEG C of high-temperature sterilization 30min process, fill a prescription based on adding proportion 10% ~ 20% V/V of volume, what concentrate nitrogenous source solution consists of Tryptones 10% ~ 20% W/V, NaNO
35% ~ 10% W/V, pH are 5 ~ 7, prepare with pure water;
(3) ventilating fermentation obtains and is rich in β-1, the trichoderma harziarum fermented liquid of 3-endoglucanase: with trichoderma harziarum for the seed that sets out, that prepares with step (2) ferments for substratum, trichoderma harziarum ventilating fermentation condition, inoculation volume is 5% ~ 15% V/V, and ventilation condition is 0.5 ~ 1.0 vvm, and speed change adjustment mixing speed makes oxygen dissolving value reach more than 30%, leavening temperature is 25 ~ 32 DEG C, and fermentation time is 3 ~ 5d;
(4) catalytic hydrolysis reaction: reaction substrate is the fresh curdlan fermentation liquid without inactivation treatment, enzyme liquid be step (3) gained be rich in β-1, the trichoderma harziarum fermented liquid of 3-endoglucanase, hydrolysis reaction is carried out in 10 ~ 20 ︰ 1 mixing by volume of fresh curdlan fermentation liquid ︰ trichoderma harziarum fermented liquid, temperature of reaction is 40 ~ 60 DEG C, and reaction pH is 4.0 ~ 6.5, and the reaction times is 2 ~ 3h, reheat reacting liquid temperature to 70 ~ 90 DEG C after reaction terminates, maintain 20 ~ 40min;
(5) solid-liquid separation: adopt centrifuging process: 3000 ~ 8000 r/min, centrifugation time is 10 ~ 20 min, or Plate Filtration technique: filter cloth aperture is 200 ~ 400 orders, and filter pressure is 0.2 ~ 0.8MPa, collects supernatant or filtrate;
(6) concentrated and dry: carry out concentrated and dry to supernatant liquor or filtrate, temperature is no more than 110 DEG C, obtains β-1,3-Portugal oligosaccharides raw product.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105566510A (en) * | 2015-12-18 | 2016-05-11 | 南京理工大学 | Glucooligosaccharide and preparation method and application thereof |
| CN105567779A (en) * | 2016-03-03 | 2016-05-11 | 江南大学 | Fermentation method of high-yield and low-molecular-weight thermal gel |
| 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 |
| CN108467876A (en) * | 2018-03-16 | 2018-08-31 | 华东师范大学 | A kind of fermentation process improving curdlan yield |
| CN110607338A (en) * | 2019-10-12 | 2019-12-24 | 江南大学 | Method for producing branched beta-1, 3-gluco-oligosaccharide by fermentation of mixed fungi |
| CN114601168A (en) * | 2022-03-24 | 2022-06-10 | 江南大学 | Method for preparing prebiotics-containing probiotic microcapsules by spray drying |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105566510A (en) * | 2015-12-18 | 2016-05-11 | 南京理工大学 | Glucooligosaccharide and preparation method and application thereof |
| CN105567779A (en) * | 2016-03-03 | 2016-05-11 | 江南大学 | Fermentation method of high-yield and low-molecular-weight thermal gel |
| 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 |
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| CN110036108B (en) * | 2016-11-09 | 2022-05-17 | 中国农业大学 | Bacterial beta-1, 3-glucanase, coding gene and application thereof |
| CN108467876A (en) * | 2018-03-16 | 2018-08-31 | 华东师范大学 | A kind of fermentation process improving curdlan yield |
| CN108467876B (en) * | 2018-03-16 | 2021-12-07 | 华东师范大学 | Fermentation method for increasing yield of curdlan |
| CN110607338A (en) * | 2019-10-12 | 2019-12-24 | 江南大学 | Method for producing branched beta-1, 3-gluco-oligosaccharide by fermentation of mixed fungi |
| 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 |
| CN114601168A (en) * | 2022-03-24 | 2022-06-10 | 江南大学 | Method for preparing prebiotics-containing probiotic microcapsules by spray drying |
| 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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