CN102936615A - Method for promoting L-sorbose to convert to generate 2-keto-L-gulonic acid - Google Patents
Method for promoting L-sorbose to convert to generate 2-keto-L-gulonic acid Download PDFInfo
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Abstract
The invention discloses a method for promoting L-sorbose to convert to generate 2-keto-L-gulonic acid, belonging to the technical field of microbial fermentation. The method comprises two steps of seed cultivation and fermentative cultivation; according to the method, astragalus extract in an additive amount of 0.75-2.00g/L or gastrodia elata extract in an additive amount of 1.50-3.00g/L is added in a fermentation medium, or the astragalus extract and the astrodia elata extract in additive amounts of 1.00-1.50g/L and 1.50-2.00g/L are added in the fermentation medium at the same time, so as to promote the common ketogulonigenium vulgare (small bacterium) and huge bacillus (large bacterium) to coordinately grow to realize the efficient synthesis of the 2-keto-L-gulonic acid; and through coordinating the mutual functions of the two bacteria, the growth of the small bacterium is promoted, the output of the 2-keto-L-gulonic acid is improved and the fermentation period is shortened. According to the method, a 10L tank is used for the fermentation experiment and a predicted effect is obtained.
Description
Technical field
The present invention relates to a kind of L-of promotion sorbose and transform the method that generates KGA, particularly a kind of extract promotion ordinary student ketone group 2-KLG bacterium (little bacterium) and efficiently synthetic method of bacillus megaterium (large bacterium) harmonic growth realization KGA by in fermention medium, adding the Radix Astragali, rhizoma Gastrodiae or the Radix Astragali and rhizoma Gastrodiae.The microorganism belonging to genus fermentation technical field.
Background technology
Vitamins C (Vitamin C is called for short Vc) claims again L-AA (L-Ascorbic acid), is the VITAMIN of needed by human, and physiological action is extensive, all occupies critical role in medicine and foodstuffs industry.At present domestic generally adopt " two-step fermenting " of China's independent research produced, and the first step of " two-step fermenting " is single bacterium fermentation, and D-glucitol is changed into the L-sorbose; Second step is mixed fermentation, is commonly called as little bacterium by ordinary student ketone group 2-KLG bacterium Ketogulonogenium vulgarum() and bacillus megaterium Bacillus megaterium(be commonly called as large bacterium) acting in conjunction the L-sorbose is changed into KGA (2-KGA); KGA is again through extracting and transform the generation vitamins C.
Produce in the ascorbic second step mixed fermentation process at " two-step fermenting ", little bacterium is acid-producing bacteria, but single culture poor growth, acid producing ability is low, large bacterium self does not produce acid, and can promote its growth during as concomitance bacterium and little bacterium co-cultivation and produce sour, therefore, promote the harmonic growth between two bacterial strains, be very important to improving acid producing ability.
The Radix Astragali has multiple effect as herbal medicine and is widely used, its chemical ingredients mainly is flavonoid, saponins and polysaccharide compound, astragalus polysaccharides and Radix Astragali saponin are the higher main components of Radix Astragali content, in addition, the Radix Astragali also contains the various trace elements such as monose, amino acid, protein, riboflavin, folic acid, nicotinic acid, vitamin P, linolic acid, linolenic acid, selenium and silicon.At present, the Radix Astragali is existing the application in the middle of microbial fermentation, and it can play to a great extent microbial growth and promote or restraining effect.
Rhizoma Gastrodiae is just well-known before more than 2,000 years as famous herbal medicine, the main component that its content is higher is Gastrodine, in addition, rhizoma Gastrodiae also contains VANILLYL ALCOHOL MIN 98, Citric Acid, methyl citrate, succsinic acid, palmitinic acid, β-sitosterol, daucosterol, sucrose, gastrodia elata polysaccharide, and the various trace elements such as iron, fluorine, manganese, zinc, strontium, iodine, copper.Nowadays acting on of rhizoma Gastrodiae obtained development and application in the food and medicine, and the effect that the Multiple components that it contains is brought into play in microorganism culturing is also affirmed.
The extract of the Radix Astragali and rhizoma Gastrodiae has certain influence to microbial growth, but there is not yet so far two kinds of Chinese herbal medicine extracts to the efficiently synthetic research report that affects of KGA.
Summary of the invention
Technical problem to be solved by this invention provides a kind of L-of promotion sorbose of " two-step fermenting " Progresses of Vitamin C Productive Technology and transforms the method that generates KGA, i.e. a kind of extract promotion ordinary student ketone group 2-KLG bacterium (little bacterium) and efficiently synthetic method of bacillus megaterium (large bacterium) harmonic growth realization KGA by add the Radix Astragali, rhizoma Gastrodiae or the Radix Astragali and rhizoma Gastrodiae in fermention medium.
The present invention solves the technical scheme that its technical problem takes: a kind of L-of promotion sorbose transforms the method that generates KGA, be a kind of extract promotion ordinary student ketone group 2-KLG bacterium (little bacterium) and efficiently synthetic method of bacillus megaterium (large bacterium) harmonic growth realization KGA by add the Radix Astragali, rhizoma Gastrodiae or the Radix Astragali and rhizoma Gastrodiae in fermention medium, it is as follows:
One, seed culture
The mixed strains liquid of preparation ordinary student ketone group 2-KLG bacterium and bacillus megaterium;
Two, fermentation culture
1, takes by weighing each composition of fermention medium by prescription and intend in fermention medium the Radix Astragali extract that the addition with 0.75～2.00g/L adds, or the Rhizoma Gastrodiae extract that adds with the addition of 1.50～3.00g/L, or the Radix Astragali extract and the Rhizoma Gastrodiae extract that add simultaneously with the addition of 1.00～1.50g/L, 1.50～2.00g/L respectively;
2, preparation fermention medium;
3, the mixed strains liquid of preparation is inoculated in 10% inoculum size (v/v) has added Radix Astragali extract, or Rhizoma Gastrodiae extract, or added simultaneously in the fermention medium of Radix Astragali extract and Rhizoma Gastrodiae extract, condition bottom fermentation at 28～32 ℃ of temperature, pH6.0～7.2, mixing speed 150～500rpm/min, air flow 3～15L/min, tank pressure 0.02～0.05MPa, to terminal, produce KGA.
Beneficial effect of the present invention: in fermention medium, add the Radix Astragali extract of 0.75～2.00g/L, Rhizoma Gastrodiae extract or the Radix Astragali extract of 1.00～1.50g/L and the Rhizoma Gastrodiae extract of 1.50～2.00g/L of 1.50～3.00g/L, by coordinating the interaction of two kinds of bacterium, not only promoted little bacteria growing, improved the output of KGA, but also shortened fermentation period, realized the purpose of efficient synthetic KGA.
Embodiment
The present invention enumerate following instance in order to illustrate better.But scope of the present invention is not limited only to this, and its claimed scope is recorded in the claim of claim.
Embodiment 1～9, by in fermention medium respectively with the addition M(g/L of 0.00g/L, 0.50g/L, 0.75g/L, 1.00g/L, 1.25g/L, 1.50g/L, 1.75g/L, 2.00g/L, 2.50 g/L) add Radix Astragali extract, produce KGA with following " promoting the L-sorbose to transform the method that generates KGA ", step is as follows:
One, seed culture
Take by weighing each composition of seed culture medium by prescription, preparation seed culture medium (g/L): L-sorbose 20, yeast extract paste 3, extractum carnis 3, peptone 10, corn steep liquor 1.5, urea 1, KH
2PO
41, MgSO
40.2, CaCO
31, regulating pH is 6.5～7.0,121 ℃ of sterilization 20min;
Lawn is inoculated in the seed culture medium of preparation on the fresh inclined-plane of the scraping one big or small bacterium mixed bacterium of ring, under the condition of 28～30 ℃ of temperature, mixing speed 200rmp/min, cultivate 20～22h, the mixed strains liquid of preparation ordinary student ketone group 2-KLG bacterium (little bacterium) and bacillus megaterium (large bacterium);
Two, fermentation culture
1, the Radix Astragali extract that takes by weighing each composition of fermention medium and intend in fermention medium, adding by prescription;
2, preparation fermention medium (g/L): L-sorbose 80, corn steep liquor 10, urea 2, KH
2PO
41, MgSO
40.2, Radix Astragali extract M, regulating pH is 7.0～7.2,121 ℃ of sterilization 20min in the 10L fermentor tank, wherein L-sorbose, urea are sterilized separately;
3, the mixed strains liquid with preparation is inoculated in the fermention medium that has added Radix Astragali extract with 10% inoculum size (v/v), the liquid amount of 10L fermentation cylinder for fermentation substratum is 7L, condition bottom fermentation at temperature 28～32 ℃ of (automatic control), pH6.0～7.2, mixing speed 200～500rpm/min, air flow 5～12L/min, tank pressure 0.03～0.04MPa, to terminal, produce KGA.
The effect that obtains is as shown in table 1.
Table 1:
Table 1 reflection Radix Astragali extract produces acid to the mixed bacterium growth of ordinary student ketone group 2-KLG bacterium (little bacterium) and bacillus megaterium (large bacterium) certain promoter action, compared with the control, in fermention medium respectively with 0.75g/L, 1.00g/L, 1.25g/L, 1.50g/L, 1.75g/L, 2.00g/L addition add Radix Astragali extract, to fermentation termination: the 2-KGA accumulation volume improves 1.15mg/mL at least, fermentation period shortens 2h at least, the 2-KGA generating rate improves 6.08% at least, and wherein in fermention medium, adding the Radix Astragali extract best results with the addition of 1.50g/L, to fermentation termination: the 2-KGA accumulation volume has improved 3.08mg/mL, fermentation period has shortened 4h, the 2-KGA generating rate has improved 13.26%.
Embodiment 10～16, by in fermention medium respectively with the addition N(g/L of 0.00g/L, 1.00g/L, 1.50g/L, 2.00g/L, 2.50g/L, 3.00g/L, 3.50g/L) add Rhizoma Gastrodiae extract, produce KGA with following " promoting the L-sorbose to transform the method that generates KGA ", step is as follows:
One, seed culture
With embodiment 1～9;
Two, fermentation culture
1, the Rhizoma Gastrodiae extract that takes by weighing each composition of fermention medium and intend in fermention medium, adding by prescription;
2, preparation fermention medium (g/L): L-sorbose 80, corn steep liquor 10, urea 2, KH
2PO
41, MgSO
40.2, Rhizoma Gastrodiae extract N, regulating pH is 7.0～7.2,121 ℃ of sterilization 20min in the 10L fermentor tank, wherein L-sorbose, urea are sterilized separately;
3, the mixed strains liquid with preparation is inoculated in the fermention medium that has added Rhizoma Gastrodiae extract with 10% inoculum size (v/v), the liquid amount of 10L fermentation cylinder for fermentation substratum is 7L, condition bottom fermentation at temperature 28～32 ℃ of (automatic control), pH6.0～7.2, mixing speed 200～500rpm/min, air flow 5～12L/min, tank pressure 0.03～0.04MPa, to terminal, produce KGA.
The effect that obtains is as shown in table 2.
Table 2:
Table 2 reflection Rhizoma Gastrodiae extract produces acid to the mixed bacterium growth of ordinary student ketone group 2-KLG bacterium (little bacterium) and bacillus megaterium (large bacterium) certain promoter action, compared with the control, in fermention medium respectively with 1.50g/L, 2.00g/L, 2.50g/L, 3.00g/L addition add Rhizoma Gastrodiae extract, to fermentation termination: the 2-KGA accumulation volume improves 1.93mg/mL at least, fermentation period shortens 1.5h at least, the 2-KGA generating rate improves 5.49% at least, and wherein in fermention medium, adding the Rhizoma Gastrodiae extract best results with the addition of 2.50g/L, to fermentation termination: the 2-KGA accumulation volume has improved 3.85mg/mL, fermentation period has shortened 4.5h, the 2-KGA generating rate has improved 15.38%.
Embodiment 17～26 is orthogonal experimental design, by in fermention medium respectively with 0.00g/L, 1.00g/L, 1.00g/L, 1.00g/L, 1.25g/L, 1.25g/L, 1.25g/L, 1.50g/L, 1.50g/L, 1.50g/L addition M(g/L) add Radix Astragali extract+in fermention medium respectively with 0.00g/L, 1.50g/L, 1.75g/L, 2.00g/L, 1.50g/L, 1.75g/L, 2.00g/L, 1.50g/L, 1.75g/L, 2.00g/L addition N(g/L) add Rhizoma Gastrodiae extract, produce KGA with following " promoting the L-sorbose to transform the method that generates KGA ", step is as follows:
One, seed culture
With embodiment 1～9;
Two, fermentation culture
1, the Radix Astragali extract and the Rhizoma Gastrodiae extract that take by weighing each composition of fermention medium and intend in fermention medium, adding simultaneously by prescription;
2, preparation fermention medium (g/L): L-sorbose 80, corn steep liquor 10, urea 2, KH
2PO
41, MgSO
40.2, Radix Astragali extract M, Rhizoma Gastrodiae extract N, regulating pH is 7.0～7.2,121 ℃ of sterilization 20min in the 10L fermentor tank, wherein L-sorbose, urea are sterilized separately;
3, the mixed strains liquid with preparation is inoculated in the fermention medium that has added simultaneously Radix Astragali extract and Rhizoma Gastrodiae extract with 10% inoculum size (v/v), the liquid amount of 10L fermentation cylinder for fermentation substratum is 7L, condition bottom fermentation at temperature 28～32 ℃ of (automatic control), pH6.0～7.2, mixing speed 200～500rpm/min, air flow 5～12L/min, tank pressure 0.03～0.04MPa, to terminal, produce KGA.
The effect that obtains is as shown in table 3.
Table 3:
1, table 3 has reflected that Radix Astragali extract+Rhizoma Gastrodiae extract produces the promoter action of acid to the mixed bacterium growth of ordinary student ketone group 2-KLG bacterium (little bacterium) and bacillus megaterium (large bacterium), compared with the control, in fermention medium respectively with 1.00g/L, 1.00g/L, 1.00g/L, 1.25g/L, 1.25g/L, 1.25g/L, 1.50g/L, 1.50g/L, 1.50g/L addition add Radix Astragali extract+in fermention medium respectively with 1.50g/L, 1.75g/L, 2.00g/L, 1.50g/L, 1.75g/L, 2.00g/L, 1.50g/L, 1.75g/L, 2.00g/L addition add Rhizoma Gastrodiae extract, to fermentation termination: the 2-KGA accumulation volume improves 2.31mg/mL at least, fermentation period shortens 1.5h at least, the 2-KGA generating rate improves 6.04% at least;
2, table 3 reflects that also the best collocation addition that Radix Astragali extract and Rhizoma Gastrodiae extract add simultaneously is in fermention medium: Radix Astragali extract 1.25g/L, Rhizoma Gastrodiae extract 1.75g/L, to fermentation termination: the 2-KGA accumulation volume has improved that 4.24mg/mL, fermentation period have shortened 5h, the 2-KGA generating rate has improved 17.58%.
Appendix:
The measuring method of 2-KGA content
2-KGA adopts improved iodometric determination, concrete grammar is as follows: accurately draw sample 2mL in test tube, add 7mol/L sulfuric acid 2mL, in boiling water bath, heat 25min after shaking up, wash in the 250mL triangular flask with the purified water gradation after taking out cooling, add 0.5% starch solution 3mL, be titrated to blue 30s with 0.1mol/L iodine liquid and do not take off, be titration end point.
The measuring method of fermented liquid residual sugar content
Adopt anthrone method to measure residual sugar in the fermented liquid, concrete grammar is: get fermentation broth sample 1mL and add the purified water constant volume in the 100mL volumetric flask, draw 1mL constant volume diluent in the test tube of clean dried again, adding 6mL anthrone solution shakes up and leaves standstill 10min; Blank substitutes fermentation broth sample with purified water and carries out aforesaid operations; Utilize spectrophotometer to survey the OD value in wavelength 620nm place colorimetric, then be scaled residual sugar content.When fermented liquid residual sugar content≤0.42mg/mL, be fermentation termination.
Claims (4)
1. one kind promotes the L-sorbose to transform the method that generates KGA, it is characterized in that it is as follows:
(1), seed culture
The mixed strains liquid of preparation ordinary student ketone group 2-KLG bacterium and bacillus megaterium;
(2), fermentation culture
(1), takes by weighing each composition of fermention medium by prescription and intend in fermention medium the Radix Astragali extract that the addition with 0.75～2.00g/L adds, or the Rhizoma Gastrodiae extract that adds with the addition of 1.50～3.00g/L, or the Radix Astragali extract and the Rhizoma Gastrodiae extract that add simultaneously with the addition of 1.00～1.50g/L, 1.50～2.00g/L respectively;
(2), preparation fermention medium;
(3), the mixed strains liquid of preparation is inoculated in 10% inoculum size (v/v) has added Radix Astragali extract, or Rhizoma Gastrodiae extract, or added simultaneously in the fermention medium of Radix Astragali extract and Rhizoma Gastrodiae extract, condition bottom fermentation at 28～32 ℃ of temperature, pH6.0～7.2, mixing speed 150～500rpm/min, air flow 3～15L/min, tank pressure 0.02～0.05MPa, to terminal, produce KGA.
2. promotion L-sorbose according to claim 1 transforms the method that generates KGA, it is characterized in that: its addition with 1.50g/L in fermention medium adds Radix Astragali extract.
3. promotion L-sorbose according to claim 1 transforms the method that generates KGA, it is characterized in that: its addition with 2.50g/L in fermention medium adds Rhizoma Gastrodiae extract.
4. promotion according to claim 1 L-sorbose transforms the method that generates KGA, it is characterized in that: it in fermention medium respectively the addition with 1.25g/L, 1.75g/L add simultaneously Radix Astragali extract and Rhizoma Gastrodiae extract.
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Cited By (4)
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| CN108070622A (en) * | 2016-11-11 | 2018-05-25 | 湖南尔康制药股份有限公司 | A kind of method that biological fermentation process prepares 1,3- propylene glycol |
| CN110760468A (en) * | 2019-11-29 | 2020-02-07 | 宁夏启元药业有限公司 | Method for improving efficiency of vitamin C precursor 2-keto-L-gulonic acid |
| CN110938564A (en) * | 2019-12-05 | 2020-03-31 | 石药集团维生药业（石家庄）有限公司 | Method for promoting growth and metabolism of ketogenic gulonospora |
| CN111893073A (en) * | 2020-08-14 | 2020-11-06 | 山东鲁维制药有限公司 | Method for promoting growth and acid production of ketogulonic acid bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108070622A (en) * | 2016-11-11 | 2018-05-25 | 湖南尔康制药股份有限公司 | A kind of method that biological fermentation process prepares 1,3- propylene glycol |
| CN110760468A (en) * | 2019-11-29 | 2020-02-07 | 宁夏启元药业有限公司 | Method for improving efficiency of vitamin C precursor 2-keto-L-gulonic acid |
| CN110938564A (en) * | 2019-12-05 | 2020-03-31 | 石药集团维生药业（石家庄）有限公司 | Method for promoting growth and metabolism of ketogenic gulonospora |
| CN110938564B (en) * | 2019-12-05 | 2023-04-14 | 石药集团维生药业（石家庄）有限公司 | Method for promoting growth and metabolism of ketogenic gulonospora |
| CN111893073A (en) * | 2020-08-14 | 2020-11-06 | 山东鲁维制药有限公司 | Method for promoting growth and acid production of ketogulonic acid bacteria |
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