CN1053697C - Fermentation method for producing D-ribose novel strain, and method for prepn. of D-ribose using said strain - Google Patents
Fermentation method for producing D-ribose novel strain, and method for prepn. of D-ribose using said strain Download PDFInfo
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- CN1053697C CN1053697C CN95112736A CN95112736A CN1053697C CN 1053697 C CN1053697 C CN 1053697C CN 95112736 A CN95112736 A CN 95112736A CN 95112736 A CN95112736 A CN 95112736A CN 1053697 C CN1053697 C CN 1053697C
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Abstract
The present invention relates to a microorganism bacterial strain for producing D-ribose by microorganism fermentation and a method for preparing the D-ribose by the microorganism bacterial strain, which belongs to the field of microorganism technology. The microorganism bacterial strain for producing D-ribose by microorganism fermentation is characterized in that B. subtilis is used as a starting bacterial strain; cells are processed by mutagenic agents of chemistry, physics, etc. to obtain a nutrition-deficient mutational strain of shikimic acid; the nutrition-deficient mutational strain of shikimic acid is fermented to produce the D-ribose. The present invention uses a fermentation method to produce the D-ribose at normal temperature and pressure. The present invention has the advantages of wide raw material source, low cost and no strict requirement for the content of aromatic amino acid in a raw material and a manufacture method of corn steep liquor, and can improve the pollution of chemical synthesis for the environment.
Description
The present invention relates to microbial strains and D-ribose production method, belong to microbial technology field with Production by Microorganism Fermentation D-ribose.
D-ribose is the composition of Yeast Nucleic Acid in the microbe, is again the reduced form inductor, is crucial material on physiology.D-ribose industrial be the important synthesis material of Wei ShengsuB2 always.Recently, D-ribose is as seasonings, and synthesis materials such as seasonings spices have broad application prospects in foodstuffs industry.Make cheap D-ribose, extensive profound significance arranged industrial.
The manufacture method of D-ribose has the isolating method of extracting from natural goods, and the method manufacturing of using chemosynthesis is also arranged.When the organic chemistry synthesis method is made ribose, be to adopt a kind of mercury utmost point to carry out the electrolytic reduction reaction to make the earliest, this method can cause the pollution of mercury.Chemical synthesis after updating, from glucose through oxidation, displacement, transform, lactonize, seven steps reaction back acquisition D-ribose such as reduction.This chemical synthesis long reaction time, the operation steps complexity, equipment used is many, the manufacturing cost height, yield is low, and can cause environmental pollution.
Before the present invention finishes in the disclosed a technology, D-ribose with bacillus pumilus (Bacilluspumilus) sudden change pearl is produced bacterium, on its fermentation raw material, to adopt the specific method for preparing corn steep liquor, because the amount of die aromatischen Aminosaeuren is being controlled the fermentation of bacterial strain.This fermentation process complex process, the cost height.
The objective of the invention is to adopt chemistry, the physical mutagenesis factors such as ultraviolet ray, ethylmethane sulfonate as mutation source, parental plant is carried out the directed mutagenesis seed selection, obtain the shikimic acid auxotrophy mutant strain, changed the carbohydrate metabolism approach.
The fermentation process that another object of the present invention provides that a kind of technology is simple, low cost of manufacture, employing above-mentioned bacterial strains prepare D-ribose.
The present invention realizes by following scheme:
The present invention at first relates to subtilis (Bacillus subtilis) JSIM-1018 mutant strain bacterial classification, it is characterized in that with Bacillus subtilis be starting strain, handles cell with mutagenic compound such as chemistry, physics, obtains shikimic acid defective type mutant strain.This bacterial strain thalline in culturing process is flexure type, numeric type and lock shape structure.Subtilis described in the invention (Bacillus subtilis) JSIM-1018 mutant strain bacterial classification has been protected the numbering that the root of Dahurian angelica is protected root of Dahurian angelica management committee common micro-organisms center, registered on the books at Chinese microorganism strain: CGMCC NO.0238 September 19 nineteen ninety-five
One, D-ribose produces the acquisition of bacterium subtilis (Bacillus subtilis) JSIM-1018 mutant strain.
1, bacterial classification:
From the dissimilar bacterium of 28 strains, Bacillus, Brevibacterium, Cory-nebacterium, Psendmonas is among the Arthrobacter, there are three strain Bac-illus sp. bacterial strains to have the ability of accumulation D-ribose through screening, a highest strain SM-18 bacterial strain, D-ribose accumulation volume 6.35mg/ml, all the other are all below 3mg/ml.
2, induction mutation of bacterium process:
From the B.subtilis bacterial strain of three strains accumulation D-ribose, selection B.subtilisSM-18 is a parental plant, is under 25-32 ℃ with bacterial strain in temperature on the inclined-plane, cultivates and makes bacteria suspension in 12-24 hour, and cell count is 1 * 10
9Individual/ml, mutafacient system becomes the alternate or continuous processing of agent thoroughly with physics such as ultraviolet ray, ethylmethane sulfonate, chemistry routinely.Time of ultraviolet irradiation 0.5-3.0 minute, the ethylmethane sulfonate ultimate density is 0.5-2.0%, treatment time 20-45 minute, treated cell respectively culture transferring to containing and not containing on the minimum medium of shikimic acid, being 25-32 ℃ in temperature cultivated 2-5 days down, detect on the minimum medium that contains shikimic acid and grow, and do not have the bacterium colony of not growing on the minimum medium of shikimic acid, the preservation of numbering B.subtilis JSIM-1018 refrigerator.
Minimum medium is formed (%):
Glucose: 0.1-0.5; Ammonium sulfate 0.2; Dipotassium hydrogen phosphate 1.4; Potassium primary phosphate 0.6; Sal epsom 0.02; Manganous sulfate 0.001; Ferrous sulfate 0.001; VITAMIN B4 40-60ug/ml; Histidine 40-60ug/ml; The minimum medium shikimic acid content 40-60ug/ml that contains shikimic acid.
3, the shikimic acid auxotrophy mutant strain of mutagenic and breeding acquisition, D-ribose accumulation volume has greatly raising than parental plant.Then through repeatedly ultraviolet ray and ethylmethane sulfonate mutagenic treatment repeatedly, from mutant strain, constantly select excellently, through the nutritional requirement of bacterial strain, the research of culture condition many aspects, obtained B.subtilis JSIM-1018 bacterial strain again.
Two, the new bacterial strain of B.subtilis prepares the method for D-ribose
B.subtilis fermentative production D-ribose method mainly realizes through the following steps:
(1), slant medium (%)
Get glucose 0.1-0.5; Peptone 0.5-1.0; Yeast extract paste 0.1-0.4; Sodium-chlor 0.1-0.4; Agar 1.5-2.5; PH is 7.0-7.5, and this substratum can be made into test tube slant or the big inclined-plane of eggplant bottle by different needs.The bacterial classification culture transferring of refrigerator preservation to the inclined-plane, was cultivated 24-48 hour down at temperature 30-34 ℃.
(2), seed
Substratum (%)
Get glucose 0.5-2.0; Corn steep liquor 0.5-2.0; Dipotassium hydrogen phosphate 0.1-0.5; Potassium primary phosphate 0.1-0.5; Sal epsom 0.1-0.5; Manganous sulfate 0.005-0.01; PH is 7.0-7.5.
Seeding tank can be carbon steel or stainless steel, is preferably standard type.300 liters of capacity, liquid amount 150-210 liter.Rotating speed 250-350 rev/min.Conventional sterilization, fermentor tank is cooled to 32-35 ℃, inserts the bacterial classification that washes with sterilized water from the big inclined-plane, and used big inclined-plane eggplant bottle 4-10 is under 28-36 ℃ in temperature only, cultivates 16-24 hour.Air quantity 1: 0.25-0.5.
(3) fermentation
Substratum (%)
Take the sugared 18-20 that W-Gum hydrolysis sugar or Semen Maydis powder make through amylase, saccharifying enzyme double-enzyme method; Corn steep liquor 0.5-2.0; Ammonium sulfate 0.5-1.0; Yeast powder 0.1-0.5; Sal epsom 0.05-0.1; Manganous sulfate 0.05-0.1; Lime carbonate 1-2.5; PH is 7.5-8.0, and fermentor tank can be stainless steel or carbon steel system, is preferably standard type.Mixing speed 160-240 rev/min, 3000 liters of capacity, liquid amount are the 1500-2100 liter.
Medium sterilization can separately sterilization back and other composition merge separately saccharine material.Directly sterilization also can mix.Time 4-8 minute, be cooled to the whole seeds in the 32-35 ℃ of access seeding tank, inoculum size 5-10%.In temperature is 34-38 ℃ of following ventilation stir culture 60-80 hour, air quantity 1: 0.4-0.8.The project of measuring in the fermenting process: D-ribose, glucose, PH, O.D, CO2.Measured every 6 hours.Glucose exhausts substantially, and D-ribose no longer increases, and CO2 reduces to below 1, can put jar.
(4), extract
Putting fermented liquid behind the jar, to transfer PH be 7.5-10.5, and add flocculation agent: chitosan or chitin, flocculation agent consumption are the 100-600PPM of fermented liquid total amount, and at this moment, the thalli granule in the fermented liquid, impurity become big, connect to floss, easily separate with clear liquid.Through the post precipitation press filtration, suction filtration or the centrifugal thalline impurity of removing obtain fermentation clear liquid.Positively charged ion and anion-exchange resin column on the clear liquid are removed the salt in the fermented liquid.Use activated carbon decolorizing, destainer concentrating under reduced pressure, thickening temperature are not higher than 55 ℃, and the D-ribose syrup that is concentrated into more than 30% is standby.Perhaps obtain the crystallization of D-ribose at the ethanol that concentrates 4 times of amounts of back interpolation concentrated solution.
The following examples elaborate to the present invention:
Example 1:
1, substratum preparation:
(1), slant medium (%)
Get glucose 0.5, peptone 1.0, yeast extract paste 0.4, sodium-chlor 0.1, agar 2.0, constant volume to 100 milliliter, PH7.2.
(2), seed culture medium (%)
Semen Maydis powder enzymolysis sugar 2.0; Corn steep liquor 1.0; Dipotassium hydrogen phosphate 0.5, potassium primary phosphate 0.2; Sal epsom 0.1; Manganous sulfate 0.005, constant volume to 200 liter, PH7.2.
(3), fermention medium (%)
Semen Maydis powder enzymolysis sugar 18; Corn steep liquor 0.6; Ammonium sulfate 1.0; Yeast powder 0.2; Sal epsom 0.1; Manganous sulfate 0.01; Lime carbonate 2.0; Constant volume to 2000 liter, PH7.5.
(4), fermentation
On Bacillus subtilis JSIM-1018 inoculation to the 5 eggplant bottle inclined-plane with preservation, cultivated 48 hours for 30 ℃, use stroke-physiological saline solution, lawn on the inclined-plane is washed, under aseptic condition, integrate with in the serum bottle, insert in the seeding tank with pressure differential method.300 liters of seeding tank volumes, 200 liters of liquid amounts.250 rev/mins of rotating speeds, air quantity 1: 0.5, in temperature is under 35 ℃ ± 1 ℃, cultivated 22 hours, and inserted and fill in the fermentor tank of 2000 liters of fermention mediums 160 rev/mins of rotating speeds, air quantity 1: 0.8, inoculum size 10% is 38 ± 1 ℃ of fermentations 70 hours in temperature, D-ribose content 81.75 grams per liters in the fermented liquid.
Fermented liquid is transferred PH8.0, add chitosan 300PPM.Fermented liquid separates supernatant liquor and precipitation through post precipitation, and the precipitation part is removed precipitation with filter press technique, merges supernatant liquor.Supernatant liquor is through positive post, cloudy post desalination, activated carbon decolorizing, and 50 ℃ are evaporated to 35% D-ribose slurry, 124.5 kilograms in actual pure D-ribose.
Example 2
(1), slant medium is with example 1
(2), seed culture medium (%)
Get glucose 2.0; Corn steep liquor 2.0; Dipotassium hydrogen phosphate 0.5; Potassium primary phosphate 0.5; Sal epsom 0.2; Manganous sulfate 0.005, constant volume to 200 liter, PH7.5.
(3), fermention medium (%)
Get industrial grape 20; Corn steep liquor 2.0; Ammonium sulfate 0.6, yeast powder 0.5; Sal epsom 0.05; Manganous sulfate 0.05; Lime carbonate 1.5; 2000 liters of constant volumes, the PH7.5. fermentation is all identical with example 1 with extraction conditions, ferments 78 hours, and D-ribose content 70.75 grams per liters in the fermented liquid are through extracting real 107 kilograms in the pure D-ribose that gets.
Example 3
In fermentation, remove with Fen, shallow lake hydrolysis sugar 18%; Beyond the corn steep liquor 1.5%, all the other are all identical with example 1, ferment 78 hours, and D-ribose content 60.04 grams per liters in the fermentation extract real 90 kilograms in the pure D-ribose that gets in back.
Compared with the prior art the present invention has the following advantages:
1, adopt D-Ribose by Fermentation to carry out at normal temperatures and pressures, the raw material source is wide, Cheap.
2, the used bacterial classification of the present invention is bacillus subtilis mutant strain, and nutritional requirement is extensive, Do not have strict to aromatic amino acid amount contained in the raw material, corn steep liquor preparation method Requirement.
3, can directly carry out the D-ribose fermenting and producing with coarse raw materials such as corn flour, produce This reduction.
4, the method for producing D-ribose by microbial fermentation has been improved chemical synthesis to environment The pollution that causes, radical change working condition.
Claims (8)
1. a fermentative production D-ribose novel strain is characterized in that with the subtilis being starting strain, adopts mutagenic compound such as chemistry, physics to handle cell, obtains the shikimic acid auxotrophy mutant strain.
2. the new bacterial strain of fermentative production D-ribose according to claim 1, it is characterized in that described mutagenic compound for ultraviolet ray, ethylmethane sulfonate is continuous or alternate starting strain is carried out selection by mutation, ultraviolet irradiation time is 0.5-3 minute, the concentration of ethylmethane sulfonate is 0.5-2%, and the treatment time is 20-45 minute.
3. the new bacterial strain of fermentative production D-ribose according to claim 1 is characterized in that described mutant strain, and morphological features is flexure type in culturing process, numeric type, lock shape type etc.
4. the new bacterial strain of fermentative production D-ribose according to claim 1 is characterized in that this bacterial strain is subtilis ISIM-1018, and preserving number is CGM4CC NO.0238
5. method for preparing D-ribose, it is characterized in that the described new bacterial strain of aforementioned 1-4 claim, culture transferring is to slant medium, in temperature is under 30-34 ℃, cultivate after 24-48 hour, be transferred in the seed culture medium, in seeding tank, cultivate, and cultivation 16-24 hour of under temperature is 28-36 ℃, ventilating, air quantity is 1: 0.25-0.5, with cultured seed liquid, with the inoculum size of 5-10%, be transferred in the fermention medium, be under 34-38 ℃ in temperature, the stir culture of in fermentor tank, ventilating 60-80 hour, ventilation is 1: 0.4-0.8, and mixing speed is 160-240 rev/min, obtains D-ribose fermented liquid, fermented liquid after the fermentation ends is transferred PH7.5-10.5, add flocculation agent chitosan 100-600PPM, fermented liquid is through flocculation, precipitation, obtain clear liquid behind the suction filtration, positively charged ion and anionite-exchange resin desalination on the fermentation clear liquid, use activated carbon decolorizing, destainer gets D-ribose syrup through concentrating under reduced pressure.
6. the method for preparing D-ribose according to claim 5 is characterized in that described slant medium comprises glucose 0.1-0.5%, peptone 0.5-1.0; Yeast extract paste 0.1-0.4%; Sodium-chlor 0.1-0.4%; Agar 1.5-2.5%; PH is 7.0-7.5.
7. the method for preparing D-ribose according to claim 5 is characterized in that described seed bacterium culture medium comprises: glucose 0.5-2.0%, corn steep liquor 0.5-2.0%; Dipotassium hydrogen phosphate 0.1-0.5%; Manganous sulfate 0.005-0.01%; Sal epsom 0.1-0.5%; PH is 7.0-7.5.
8. the method for preparing D-ribose according to claim 5 is characterized in that described fermention medium comprises the sugared 18-20% that Semen Maydis powder (starch) makes, corn steep liquor 0.5-2.0%; Ammonium sulfate 0.5-1.0%; Yeast powder 0.1-0.5%; Sal epsom 0.05-0.1%; Manganous sulfate 0.05-0.1%, lime carbonate 1-2.5%; PH is 7.5-8.0.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95112736A CN1053697C (en) | 1995-10-25 | 1995-10-25 | Fermentation method for producing D-ribose novel strain, and method for prepn. of D-ribose using said strain |
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| CN95112736A CN1053697C (en) | 1995-10-25 | 1995-10-25 | Fermentation method for producing D-ribose novel strain, and method for prepn. of D-ribose using said strain |
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| CN1122833A CN1122833A (en) | 1996-05-22 |
| CN1053697C true CN1053697C (en) | 2000-06-21 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1100147C (en) * | 2000-12-16 | 2003-01-29 | 王树庆 | Two-step fermentation process of producing D-ribose |
| CN101475970B (en) * | 2008-01-04 | 2013-06-12 | 上海希迪制药有限公司 | Method for producing crystal D-ribose |
| CN101555504B (en) * | 2009-05-26 | 2012-01-11 | 郑州拓洋生物工程有限公司 | Method for producing D-ribose by utilizing fermentation of bacillus pumilus transketolase variant |
| CN113142542A (en) * | 2021-02-25 | 2021-07-23 | 仇俊鹏 | Fermented seasoning sauce using grains as raw materials, preparation method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3607648A (en) * | 1968-02-01 | 1971-09-21 | Takeda Chemical Industries Ltd | Method for the production of d-ribose |
| JPS5191388A (en) * | 1975-02-04 | 1976-08-10 | dd riboosuno seizoho |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3607648A (en) * | 1968-02-01 | 1971-09-21 | Takeda Chemical Industries Ltd | Method for the production of d-ribose |
| JPS5191388A (en) * | 1975-02-04 | 1976-08-10 | dd riboosuno seizoho |
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