CN1370779A - Nucleotide extracting process from waste gourmet powder material - Google Patents
Nucleotide extracting process from waste gourmet powder material Download PDFInfo
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- CN1370779A CN1370779A CN 01106511 CN01106511A CN1370779A CN 1370779 A CN1370779 A CN 1370779A CN 01106511 CN01106511 CN 01106511 CN 01106511 A CN01106511 A CN 01106511A CN 1370779 A CN1370779 A CN 1370779A
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Abstract
The nucleotide extracting process for waste gourmet powder material includes the steps of: separation of nucleic acid from glutamic acid corynebacterial contained in waste gourmet powder material, deposition of the nucleic acid, orientated enzymolysis of deposited nucleic acid with phosphodiesterase, and final purification of the enzymolysis product with active carbon column. Compared with available extraction process, the present invention has high yield, high purity and low cost and is suitable for industrial production.
Description
The present invention relates to utilize industrial waste to produce the method for Nucleotide, refer to a kind of method of from waste gourmet powder material, extracting Nucleotide particularly.
Waste gourmet powder material is meant fermented gournet powder liquid after having extracted L-glutamic acid, and the waste water that is produced is through coarse filtration and remaining residue.This residue generally is scattered paste shape, and its solid content accounts for 30～40%, and major ingredient is a Corynebacterium glutamicum, and all the other are water.At present, the rejectable waste that this production monosodium glutamate is produced is mostly thrown aside not usefulness, has not only greatly wasted efficient resource wherein, and the discharging of the waste material environment of having gone back severe contamination.Day by day enhanced is current in environmental consciousness, how to make full use of the Corynebacterium glutamicum in the waste gourmet powder material, makes it be converted into the nutritive substance that HUMAN HEALTH is had good benefit, has become the focus that people pay close attention to.People find by the further investigation to bacterium, many bacteriums are under certain temperature range and specific alkaline environment, enzyme digestion reaction product nucleus thuja acid can take place in its intravital nucleic acid material and multiple nuclease, the Nucleotide that is generated comprise a part 5 '-Nucleotide, also contain some 3 '-Nucleotide, 2 '-composition such as Nucleotide and nucleosides, this phenomenon promptly is the self-dissolving of thalline.Corynebacterium glutamicum is no exception, utilize the self-dissolving of Corynebacterium glutamicum also can extract Nucleotide, its production technique mainly is the phosphodiesterase that utilizes Corynebacterium glutamicum thalline self under the condition that the pH value is 9～10 alkaline environment, temperature is 50～70 ℃, enzymolysis nucleic acid material wherein, make its major part be converted into Nucleotide, utilize the anionite-exchange resin Nucleotide of purifying again.But the Corynebacterium glutamicum cell concentration required owing to this autolysis method is very low, only needs about 2%, otherwise its pH value is difficult to control, thereby its extraction yield is very low; And under the condition of alkalescence, there is the nuclease of a lot of different performances in Corynebacterium glutamicum inside, and the product component of its self-dissolving is very complicated, and the enzymolysis transformation efficiency is also very low, be unsuitable for directed making 5 '-Nucleotide; Simultaneously because this autolysis method has adopted the anionite-exchange resin Nucleotide of purifying, and the adsorptive capacity of anionite-exchange resin is lower, is subject to the influence of salt concn, and required equipment is huge, and usage ratio of equipment is very low, causes product production also low excessively; In sum, this autolysis method production technique required equipment investment is too big, and the Nucleotide extraction yield is too low, is not suitable for big industrial production.
Purpose of the present invention will overcome present Corynebacterium glutamicum autolysis method exactly and extract the existing disadvantage of Nucleotide technology, make full use of the useful resources in the existing waste gourmet powder material, a kind of product yield height is provided, purity is good, cost is low and is suitable for the method for from waste gourmet powder material, extracting Nucleotide of suitability for industrialized production.
For realizing this purpose, the method of from waste gourmet powder material, extracting Nucleotide that the present invention developed, be earlier in the contained Corynebacterium glutamicum of waste gourmet powder material, nucleic acid to be decomposed out, utilize phosphodiesterase that the nucleic acid that is decomposited is carried out directional enzymatic again, utilize activated carbon column that the product of institute's enzymolysis is purified at last.This method may further comprise the steps:
1) extraction of nucleic acid: get solid content and account for 30～40% the waste gourmet powder material and the water mixing and stirring of 2～3 times of weight, regulate its pH value to 3～4 and kept 25～35 minutes; Then the pH value is transferred to 7～8, adds the salt of above-mentioned mixeding liquid volume amount 4～6%, treat that salt dissolving finishes after, be warming up to 90～100 ℃, kept 1～2 hour; Its temperature is reduced to 40 ℃～50 ℃ again, the pH value transfers to 4～6, adds an amount of anionic flocculant simultaneously, after thalline in mixed solution flocculates into piece, it is separated, and isolated clear liquid is the extracting solution that contains nucleic acid, with this nucleic acid extraction liquid be refrigerated to 8～12 ℃ stand-by;
2) precipitation of nucleic acid: the concentration of the made nucleic acid extraction liquid of the step 1) that is prepared in advance volume 16～25% is that the hydrochloric acid of 3N places pretipitatin jar, adding step 1) gained nucleic acid extraction liquid again mixes with it, the pH value of this nucleic acid mixed solution is between 1～2, just reach the iso-electric point of nucleic acid, this moment, nucleic acid promptly precipitated in a large number; After the nucleic acid precipitation, supernatant is taken out light, the gained precipitation is dissolved in water into the nucleic acid magma of 1～2% concentration, its pH value to 4.8～5.2 of re-adjustment, be warming up to 80～90 ℃ stand-by;
3) preparation of phosphodiesterase enzyme liquid: getting weight is step 2) wheat bran that utilizes Penicillium citrinum bacterial classification fermentative production of 2.8～3.2 times of pure nucleic acid amounts is mould in the gained nucleic acid magma, under 40～50 ℃ temperature, soaked 1～2 hour with the water of 9～10 times of weight, be cooled to 20～30 ℃ then, the filtering solid is mould, heating up is cooled to about 30 ℃ clear liquid rapidly again after keeping 4～6 minutes about 70 ℃, subsequent removal spore, gained clear liquid are phosphodiesterase enzyme liquid;
4) the phosphodiesterase enzyme liquid-phase mixing of the nucleic acid magma of gained and step 3) gained mixed enzymolysis: with step 2), hydrolysis is 1～2 hour under 65～75 ℃, the condition of pH value 4.8～5.5, again its pH value is transferred to 7.2～7.8, and be warming up to about 90 ℃, keep after 12～15 minutes, be cooled to 30～40 ℃ rapidly, remove insolubles wherein, the gained clear liquid is the thick liquid of Nucleotide that needs purification;
5) activated carbon column is purified: the pH value of the thick liquid of step 4) gained Nucleotide is adjusted to upward activated carbon column of 1.5～3.0 backs, flow velocity upper prop with 0.03～0.05 milliliter of effluent liquid of every milliliter of charcoal post per minute finishes, then with the soft water wash-out impurity elimination of same flow velocity, use the elutriant of forming by the ammoniacal liquor of 40～60% alcohol and 0.5～1% instead with same flow velocity again and carry out wash-out, keep charcoal post wash-out under 50～70 ℃ of constant temperature, till alcohol detection did not have white opacity, the gained elutriant was concentration and is about 3～5% Nucleotide seminal fluid until effluent liquid; The temperature that keeps this Nucleotide seminal fluid is 50～70 ℃, concentrates the alcohol that reclaims wherein under vacuum condition, and making wherein, the concentration of Nucleotide is increased to 9～12%; PH value to 2.5～3.5 of this high density nucleosides acid solution of re-adjustment, the alcohol more than 95% that adds 3～5 times of its volumes promptly gets the Nucleotide precipitation; This precipitation vacuum-drying is promptly got the Nucleotide finished product.
The invention has the advantages that: adopted elder generation that the nucleic acid extraction in the Corynebacterium glutamicum is come out in the method, utilized phosphodiesterase the nucleic acid that is extracted to be carried out the operational path of directional enzymatic again.And the cell concentration that participates in the Corynebacterium glutamicum of nucleic acid extraction can reach about 10%, be about 5 times of cell concentration in the autolysis method production technique, so significantly increased the consumption of Corynebacterium glutamicum in each production process, both improved the utilization ratio of conversion unit, laid a good foundation for the raising of final Nucleotide recovery rate again.The phosphodiesterase that is adopted has very strong directional property to the enzymolysis of nucleic acid, and the product overwhelming majority that its enzymolysis generates is 5 '-Nucleotide, and can not the autolyzate that includes various complicated ingredients, products obtained therefrom composition unanimity like this, based on very high purity.Used the activated carbon column Nucleotide of purifying in the while method, the amount of charcoal absorption Nucleotide is big, and is not subjected to the influence of salt concn, makes the rule film of equipment used significantly to reduce, plant factor further improves, and the Nucleotide output of being extracted also is greatly improved.Thereby present method can adapt to the needs of mass industrialized production.In addition,, make production cost decline to a great extent, not only turn waste into wealth, and alleviated the problem of environmental pollution that the waste gourmet powder material discharging is brought because the present invention has used zymic alternative materials---waste gourmet powder material.
Below be a kind of specific embodiment that extracts the method for Nucleotide from waste gourmet powder material proposed by the invention, this method may further comprise the steps:
1) extraction of nucleic acid: get solid content and account for the waste gourmet powder material about 35% and the water mixing and stirring of 2 times of weight, the sodium hydroxide solution that adds concentration and be 6N is regulated its pH value to 3.5 and was kept 30 minutes.Then the pH value is transferred to 7.2, adds the salt of above-mentioned mixeding liquid volume amount 4%, treat that salt dissolving finishes after, squeeze into extracting cylinder.It is warming up to 94～96 ℃ in extracting cylinder, mixed solution reacted and began to separate out nucleic acid this moment, after keeping 40 minutes under this temperature, began the OD of sampling monitoring mixed solution with spectrophotometer
260Index stops to extract after it no longer raises, and this process needs 1～1.5 hour approximately.By interchanger its temperature is reduced to 40 ℃～50 ℃ again, the pH value transfers to 4.2～4.5, adds the anionic flocculant solution of being dissolved into 0.1% concentration in right amount simultaneously, after the thalline in mixed solution flocculates into piece, utilizes D
424High speed separator separates it, and isolated clear liquid is the extracting solution that contains nucleic acid, with this nucleic acid extraction liquid place freezing jar be refrigerated to 8～12 ℃ stand-by;
2) precipitation of nucleic acid: the concentration of the made nucleic acid extraction liquid of the step 1) that is prepared in advance volume 20% is that the hydrochloric acid of 3N places pretipitatin jar, vacuumizing suction step 1) gained nucleic acid extraction liquid again mixes with it, make the pH value of this nucleic acid mixed solution be between 1～2 immediately, just reach the iso-electric point of nucleic acid, this moment, nucleic acid promptly precipitated in a large number.Here it should be noted that the hybrid mode of nucleic acid extraction liquid and hydrochloric acid soln, nucleic acid extraction liquid be drawn in the hydrochloric acid soln that it is high that its pH value will be hanged down the back earlier, just in time impels the nucleic acid precipitation; If hydrochloric acid soln is drawn in the nucleic acid extraction liquid, the height back is low earlier for its pH value, and some protein precipitations are got off, and influences the extraction yield and the purification degree of nucleic acid.After the nucleic acid precipitation, supernatant is taken out light, the gained precipitation is dissolved in water into the nucleic acid magma of 1～2% concentration, its pH value to 5.0 of re-adjustment, and it is stand-by to be warming up to 85 ℃ of left and right sides;
3) preparation of phosphodiesterase enzyme liquid: the label that utilizes Institute of Microorganism, Academia Sinica to produce is mould for the wheat bran of the Penicillium citrinum bacterial classification fermentative production of AS3.2788, weight is step 2) 3 times of pure nucleic acid amount in the gained nucleic acid magma, soaked 1 hour under 45 ℃ temperature with the water of 10 times of weight, be cooled to 20 ℃ then, mould with 80 order silk screen filtering solids, heat up again clear liquid is cooled to rapidly about 30 ℃ after keeping 5 minutes about 70 ℃, with after channel separator is removed spore, the gained clear liquid is phosphodiesterase enzyme liquid;
4) the phosphodiesterase enzyme liquid-phase mixing of the nucleic acid magma of gained and step 3) gained mixed enzymolysis: with step 2), hydrolysis about 70 ℃, under the condition of pH value 5.0～5.2 2 hours, again its pH value is transferred to 7.2～7.4, and be warming up to about 90 ℃, keep after 15 minutes, be cooled to rapidly about 30 ℃, with Plate Filtration removal insolubles wherein, the gained clear liquid is the thick liquid of Nucleotide that needs purification;
5) activated carbon column is purified: the pH value of the thick liquid of step 4) gained Nucleotide is adjusted to upward activated carbon column of 1.5 backs, flow velocity upper prop with 0.03～0.05 milliliter of effluent liquid of every milliliter of charcoal post per minute finishes, then with the soft water wash-out impurity elimination in about 6 hours of same flow velocity, use the elutriant of forming by the ammoniacal liquor of 50% alcohol and 1% instead with same flow velocity again and carry out wash-out, keep charcoal post wash-out under 60 ℃ constant temperature, till alcohol detection did not have white opacity, the gained elutriant was concentration and is about 4% Nucleotide seminal fluid until effluent liquid.The temperature that keeps this Nucleotide seminal fluid is 50 ℃, concentrates the alcohol that reclaims wherein under vacuum condition, and making wherein, the concentration of Nucleotide is increased to 9～12%.The pH value to 3.0 of this high density nucleosides acid solution of re-adjustment, the alcohol more than 95% that adds 5 times of its volumes promptly gets the Nucleotide precipitation, this precipitation is placed to dry in the vacuum shelf dryer at last promptly to get the Nucleotide finished product.
Claims (1)
- A kind of method of extracting Nucleotide from waste gourmet powder material may further comprise the steps:1) extraction of nucleic acid: get solid content and account for 30～40% the waste gourmet powder material and the water mixing and stirring of 2～3 times of weight, regulate its pH value to 3～4 and kept 25～35 minutes; Then the pH value is transferred to 7～8, adds the salt of above-mentioned mixeding liquid volume amount 4～6%, treat that salt dissolving finishes after, be warming up to 90～100 ℃, kept 1～2 hour; Its temperature is reduced to 40 ℃～50 ℃ again, the pH value transfers to 4～6, adds an amount of anionic flocculant simultaneously, after thalline in mixed solution flocculates into piece, it is separated, and isolated clear liquid is the extracting solution that contains nucleic acid, with this nucleic acid extraction liquid be refrigerated to 8～12 ℃ stand-by;2) precipitation of nucleic acid: the concentration of the made nucleic acid extraction liquid of the step 1) that is prepared in advance volume 16～25% is that the hydrochloric acid of 3N places pretipitatin jar, adding step 1) gained nucleic acid extraction liquid again mixes with it, the pH value of this nucleic acid mixed solution is between 1～2, just reach the iso-electric point of nucleic acid, this moment, nucleic acid promptly precipitated in a large number; After the nucleic acid precipitation, supernatant is taken out light, the gained precipitation is dissolved in water into the nucleic acid magma of 1～2% concentration, its pH value to 4.8～5.2 of re-adjustment, be warming up to 80～90 ℃ stand-by;3) preparation of phosphodiesterase enzyme liquid: getting weight is step 2) wheat bran that utilizes Penicillium citrinum bacterial classification fermentative production of 2.8～3.2 times of pure nucleic acid amounts is mould in the gained nucleic acid magma, under 40～50 ℃ temperature, soaked 1～2 hour with the water of 9～10 times of weight, be cooled to 20～30 ℃ then, the filtering solid is mould, heating up is cooled to about 30 ℃ clear liquid rapidly again after keeping 4～6 minutes about 70 ℃, subsequent removal spore, gained clear liquid are phosphodiesterase enzyme liquid;4) the phosphodiesterase enzyme liquid-phase mixing of the nucleic acid magma of gained and step 3) gained mixed enzymolysis: with step 2), hydrolysis is 1～2 hour under 65～75 ℃, the condition of pH value 4.8～5.5, again its pH value is transferred to 7.2～7.8, and be warming up to about 90 ℃, keep after 12～15 minutes, be cooled to 30～40 ℃ rapidly, remove insolubles wherein, the gained clear liquid is the thick liquid of Nucleotide that needs purification;5) activated carbon column is purified: the pH value of the thick liquid of step 4) gained Nucleotide is adjusted to upward activated carbon column of 1.5～3.0 backs, flow velocity upper prop with 0.03～0.05 milliliter of effluent liquid of every milliliter of charcoal post per minute finishes, then with the soft water wash-out impurity elimination of same flow velocity, use the elutriant of forming by the ammoniacal liquor of 40～60% alcohol and 0.5～1% instead with same flow velocity again and carry out wash-out, keep charcoal post wash-out under 50～70 ℃ of constant temperature, till alcohol detection did not have white opacity, the gained elutriant was concentration and is about 3～5% Nucleotide seminal fluid until effluent liquid; The temperature that keeps this Nucleotide seminal fluid is 50～70 ℃, concentrates the alcohol that reclaims wherein under vacuum condition, and making wherein, the concentration of Nucleotide is increased to 9～12%; PH value to 2.5～3.5 of this high density nucleosides acid solution of re-adjustment, the alcohol more than 95% that adds 3～5 times of its volumes promptly gets the Nucleotide precipitation; This precipitation vacuum-drying is promptly got the Nucleotide finished product.
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| CNB011065117A CN1152043C (en) | 2001-02-27 | 2001-02-27 | Nucleotide extracting process from waste gourmet powder material |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942486A (en) * | 2010-09-07 | 2011-01-12 | 南京工业大学 | Method for producing organic acid by monosodium glutamate fermentation waste thalli |
| CN101148637B (en) * | 2007-08-10 | 2011-05-11 | 华南理工大学 | Hydrolysate enriched with biological bioactive peptide for accelerating beer yeast fermenting and its preparation method and application |
| CN102732509A (en) * | 2012-07-19 | 2012-10-17 | 中粮生物化学（安徽）股份有限公司 | Method for extracting ribonucleic acid from fermentation thalli |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3227453A4 (en) * | 2014-12-02 | 2018-06-27 | Lakeview Nutrition LLC | Extracts of whole stillage and other biomass and methods thereof |
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2001
- 2001-02-27 CN CNB011065117A patent/CN1152043C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101148637B (en) * | 2007-08-10 | 2011-05-11 | 华南理工大学 | Hydrolysate enriched with biological bioactive peptide for accelerating beer yeast fermenting and its preparation method and application |
| CN101942486A (en) * | 2010-09-07 | 2011-01-12 | 南京工业大学 | Method for producing organic acid by monosodium glutamate fermentation waste thalli |
| CN101942486B (en) * | 2010-09-07 | 2012-09-05 | 南京工业大学 | Method for producing organic acid by monosodium glutamate fermentation waste thalli |
| CN102732509A (en) * | 2012-07-19 | 2012-10-17 | 中粮生物化学（安徽）股份有限公司 | Method for extracting ribonucleic acid from fermentation thalli |
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| CN1152043C (en) | 2004-06-02 |
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