CN102978273B - Method using three-bacterium mixed fermentation to convert sorbose into 2-keto-L-gulonic acid - Google Patents
Method using three-bacterium mixed fermentation to convert sorbose into 2-keto-L-gulonic acid Download PDFInfo
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Abstract
The invention belongs to the technical field of vitamin C biological fermentation, and particularly relates to a method using three-bacterium mixed fermentation to convert sorbose into 2-keto-L-gulonic acid which is in a second step of the vitamin C biological fermentation. Seed solution of manufacturing technique of the second step of the vitamin C biological fermentation is enlarged step by step, when in fermental cultivation, with total inoculation amount with volume percent of 15-20%, seed solution of A isolates and B isolates are introduced into a fermentation tank to be fermented and cultivated to a fermentation ending point (namely when the concentration of remained sorbose in fermentation liquor is smaller than or equal to 1mg/ml), and the purpose that the mixed bacteria fermentation is adopted for converting the sorbose to the 2-keto-L-gulonic is achieved. The A isolates is composed of companion fungus bacillus cereus/ companion fungus bacillus megatherium and acid-forming bacteria ketogenic base ketogulonigenium vulgare. The B isolates is composed of companion fungus bacillus thuringiensis and acid-forming bacteria ketogenic base ketogulonigenium vulgare. According to the novel method using the three-bacterium mixed fermentation, respective advantages of the two kinds of the companion funguses are made full use, fermentation period can be shortened by 6-8 hours, energy consumption is reduced by 10-15%, and fermentation conversion percent is improved by 2-4%.
Description
Technical field
The invention belongs to the vitamin C bio fermentation technical field, specifically a kind of method of utilizing the mixed bacterium biological fermentation to transform the sorbose KGA in Vc second step biological fermentation.
Background technology
The suitability for industrialized production of Vc two stage fermentation, its second step fermentation is the mixed fermentation of two kinds of bacterium, realizes the bio-transformation from the L-sorbose to KGA.Two kinds of bacterium comprise bacterium with acid producing ability-ordinary student ketone group 2-KLG bacterium (being commonly called as acid-producing bacteria or little bacterium) and do not have and transform acid producing ability but can promote little bacteria growing and produce sour concomitance bacterium (being commonly called as large bacterium).Existing result of study shows, ordinary student ketone group 2-KLG bacterium single culture go down to posterity survival rate and produce the KGA ability all a little less than, could Fast Growth while only having with the concomitance bacterium mixed culture and produce sour.But the kind of concomitance bacterium, quantity and growth characteristics have material impact to growth and the product acid of little bacterium, and then are changing the efficiency of vitamins C second step fermentation.
At present, in production, the concomitance bacterium of application mainly contains bacillus cereus (Bacills cereus), bacillus megaterium (Bacillus megaterium) and bacillus thuringiensis (Bacillsthuringiensis) etc., and only with wherein the mixed fermentation product acid of two kinds of bacterium is carried out in a kind of concomitance bacterium and acid-producing bacteria collocation.Because the efficient association ability time length of a strain concomitance bacterium is shorter, be difficult to continue in all stage of fermentation the effector that provides sufficient, so, the acid producing ability of acid-producing bacteria can't remain on optimum regime during the fermentation for a long time, makes the efficiency of second step mixed fungus fermentation be difficult to effectively improve.In addition, due to the hysteresis of detected result and the complexity of the multistage expansion process of seed liquor, the large tank of two step mixed fungus fermentations microbiological contamination often occurs and dyes the phenomenon of phage, phage kills concomitance bacterium, make the association factor can not meet acid-producing bacteria growth and fermentation and acid, cause fermenting speed obviously to reduce and even put the tank situation, had a strong impact on the output of 2-KLG, and caused a large amount of energy consumptions and material unaccounted-for (MUF).
Summary of the invention
The object of the invention is to provide a kind of method of utilizing three kinds of bacterium mixed fermentations to transform the sorbose KGA.
For achieving the above object, the present invention adopts technical scheme to be:
A kind of seed liquor of utilizing three kinds of bacterium mixed fermentations to transform the method Vc two stage fermentation production technique of sorbose KGA enlarges step by step; during fermentation culture by the seed liquor of A fungus strain and B fungus strain; be cultured to fermentation termination (to fermented liquid in remaining sorbose concentration≤1mg/ml) with the total inoculum size access fermentation cylinder for fermentation of volume percent 15-20%, realizing utilizing mixed fermentation to transform sorbose is KGA;
Wherein the A fungus strain is comprised of concomitance bacterium bacillus cereus/concomitance bacterium bacillus megaterium and the living ketone group 2-KLG of acid-producing bacteria bacterium; The B fungus strain is given birth to ketone group 2-KLG bacterium by concomitance bacterium bacillus thuringiensis and acid-producing bacteria and is formed.
The seed liquor of described A fungus strain and the B fungus strain ratio of 2:1～10:1 is by volume mixed the access fermentor tank.
Described two kinds of fungus strains are joined respectively in fermentor tank, and the fungus strain that two kinds of priorities add makes total inoculum size reach 15-20% be inoculated into fermentor tank in 20 hours in.
Described in Vc two stage fermentation process, A fungus strain and B fungus strain are through continuous three grades of seed enlarged culturing, and three grades of seed liquor that then obtain are inoculated in fermentor tank; Wherein the continuous three grades of seed enlarged culturing of A fungus strain and B fungus strain are respectively and using the inoculum size of volume ratio 1-5%, 5-10% and 5-10% respectively as the inoculum sizes at different levels of three grades of seed enlarged culturing, culture temperature at different levels are 27-31 ℃ simultaneously, fermentation time is at 6-14 hour, and ventilation is that the 1:0.8-1.0(dissolved oxygen is than being 25-30%); Every grade of seed reaches kind of liquid dragon in a middle ancient times acid content and reaches 3.0-10.0mg/ml, and transferred species is to next stage.
Three grades of seed liquor of the A fungus strain of continuous enlarged culturing and B fungus strain are inoculated in fermentor tank, at 27-31 ℃, tank pressure 0.005-0.02Mpa, carry out fermentation culture under pH 6.0-8.0, in fermenting process, stream adds the L-sorbose, until remaining sorbose concentration≤1mg/ml in fermented liquid is KGA thereby realize utilizing mixed fermentation to transform sorbose.
In described fermenting process, be interrupted or continuous current adding substrate L-sorbose, the stream rate of acceleration is 2-10m
3/ hour, reach 7-10mg/ml to sorbose final concentration in fermented liquid.
The present invention has following advantage
1. the present invention realizes that it is KGA that three bacterium mixed fermentations transform sorbose, can shorten fermentation period 6-8 hour, reduces energy consumption 10-15%, improves fermentation conversion rate 2-4%.
2. simple process of the present invention, do not need to add main equipment, do not need existing installation is too much transformed yet.
3. the present invention realizes three bacterium mixed culture of Vc two stage fermentation, two kinds of concomitance bacteriums can provide the association effector of continuous and effective, thereby make little bacteria growing and produce acid in optimum regime, stopped to occur that because effector is not enough little bacterium produces the phenomenon that sour efficiency is not high in the two stage fermentation process, its fermentation has been transformed and continued efficiently to carry out.
4. three bacterium mixed culture of the present invention, the Growth and reproduction characteristic difference of two kinds of concomitance bacteriums wherein, the early growth of A fungus strain is fast, and B fungus strain late growing stage is fast, two kinds of concomitance bacterium co-cultivation, can effectively make up during the fermentation the fermentation efficiency caused because of a kind of concomitance bacterium quantity not sufficient low.
5. three bacterium mixed fermentations of the present invention, two kinds of concomitance bacteriums are present in fermented liquid simultaneously, have strengthened in the fermenting process dying the resistance of phage, have reduced the phenomenon of putting tank because dying phage, have improved fermentation efficiency.
6. the interaction mechanism that the present invention is based on Vc two stage fermentation concomitance bacterium and acid-producing bacteria reaches the difference of metabolism and growth characteristic separately, adopts different fermentations fungus strain kind liquid jointly to access in proportion the pattern of bulk fermentation, completes the high-efficiency fermenting of KGA.
Embodiment
Embodiment 1
Get respectively the A fungus strain formed by bacillus cereus and living ketone group 2-KLG bacterium and the B fungus strain formed by bacillus thuringiensis and living ketone group 2-KLG bacterium from inclined-plane kind liquid, using respectively the 5%(volume ratio) as three grades of inoculum sizes of the kind liquid of amplification continuously, be inoculated in respectively (seed culture medium in the seed culture medium of different grades, peptone 0.3% by weight percentage, extractum carnis 0.3%, yeast extract paste 0.3%, corn steep liquor 0.3%, MgSO
40.01%, FeSO
40.01%, L-sorbose 2%, pH 7.0, agar 2.5%, surplus is water; 121 ℃ of high-temperature sterilization 20min; Stand-by.), respectively at 8-10mg/ml, transferred species is to next stage to different grades fermented liquid dragon in middle ancient times acid yield for shake-flask culture 20 hours;
After three grades of enlarged culturing, get respectively A seed liquor 2.4ml and B seed liquor 0.8ml in above-mentioned seed liquor, altogether 20ml fermention medium (culture medium prescription: corn steep liquor by weight percentage, 1.5%, urea 1.2%, sorbose 8%, MgSO is equipped with in the 3.2ml access
40.01%, distilled water is settled to 1000ml, regulate pH to 7.0,121 ℃ of high-temperature sterilization 20min) in triangular flask, with 29 ℃, tank pressure 0.005-0.02Mpa, 150 rev/mins of lower shaking culture 38 hours, in fermenting process, stream adds the L-sorbose, until remaining sorbose concentration≤1mg/ml in fermented liquid is KGA thereby realize utilizing mixed fermentation to transform sorbose.Final fermented liquid dragon in middle ancient times acid number reaches 68.0mg/ml, and transformation efficiency reaches 94.4%.
Embodiment 2-4
Application examples 1
Select 0.5 ton of fermentor tank, add containing 300 liters of the conventional Vc two stage fermentation substratum of 8%L-sorbose, inoculate respectively through continuous three grades of enlarged culturing by bacillus cereus and give birth to 15 liters of 45 liters of the A fungus strain seed liquor that ketone group 2-KLG bacterium forms and the B fungus strain seed liquor formed by bacillus thuringiensis and life ketone group 2-KLG bacterium, in 29 ℃, ventilation 1:0.8-1.0(liquid V: gas V/ divides, dissolved oxygen is than being 25-30%) ferment 36 hours, in final fermented liquid, KGA content is 67.89mg/ml, and transformation efficiency reaches 94.3%.
Application examples 2
In 4 tons of fermentor tanks, add containing 2.2 tons of the conventional Vc two stage fermentation substratum of L-sorbose 8%, inoculate respectively 0.3 ton of continuous three grades of enlarged culturing of warp by bacillus cereus and give birth to A fungus strain seed liquor that ketone group 2-KLG bacterium forms and 0.1 ton of continuous three grades of enlarged culturing of warp form B fungus strain seed liquor by bacillus thuringiensis and life ketone group 2-KLG bacterium, at 29 ℃, ventilation 1:0.8-1.0, ferment 34 hours, in final fermented liquid in fermented liquid KGA content be 68.27mg/ml, transformation efficiency reaches 94.8%.
Claims (4)
1. a method of utilizing three kinds of bacterium mixed fermentations to transform the sorbose KGAs; it is characterized in that: the seed liquor of Vc two stage fermentation production technique enlarges step by step; during fermentation culture by the seed liquor of A fungus strain and B fungus strain; total inoculum size access fermentation cylinder for fermentation with volume percent 15-20% is cultured to fermentation termination, and realizing utilizing mixed fermentation to transform sorbose is KGA;
Wherein the A fungus strain is comprised of concomitance bacterium bacillus cereus/concomitance bacterium bacillus megaterium and the living ketone group 2-KLG of acid-producing bacteria bacterium; The B fungus strain is given birth to ketone group 2-KLG bacterium by concomitance bacterium bacillus thuringiensis and acid-producing bacteria and is formed;
The seed liquor of described A fungus strain and the B fungus strain ratio of 2:1～10:1 is by volume mixed the access fermentor tank;
Described two kinds of fungus strains are joined respectively in fermentor tank, and the fungus strain that two kinds of priorities add makes total inoculum size reach 15-20% be inoculated into fermentor tank in 20 hours in.
2. by the method for utilizing three kinds of bacterium mixed fermentations to transform the sorbose KGA claimed in claim 1, it is characterized in that: described in Vc two stage fermentation process, A fungus strain and B fungus strain are through continuous three grades of seed enlarged culturing, and three grades of seed liquor that then obtain are inoculated in fermentor tank; Wherein the continuous three grades of seed enlarged culturing of A fungus strain and B fungus strain are respectively and using the inoculum size of volume ratio 1-5%, 5-10% and 5-10% respectively as the inoculum sizes at different levels of three grades of seed enlarged culturing, culture temperature at different levels are 27-31 ℃ simultaneously, fermentation time is at 6-14 hour, and ventilation is 1:0.8-1.0; Every grade of seed reaches kind of liquid dragon in a middle ancient times acid content and reaches 3.0-10.0mg/ml, and transferred species is to next stage.
3. by the method for utilizing three kinds of bacterium mixed fermentations to transform the sorbose KGA claimed in claim 2, it is characterized in that: three grades of seed liquor of the A fungus strain of continuous enlarged culturing and B fungus strain are inoculated in fermentor tank, at 27-31 ℃, tank pressure 0.005-0.02Mpa, carry out fermentation culture under pH6.0-8.0, in fermenting process, stream adds the L-sorbose, until remaining sorbose concentration≤1mg/ml in fermented liquid is KGA thereby realize utilizing mixed fermentation to transform sorbose.
4. by the method for utilizing three kinds of bacterium mixed fermentations to transform the sorbose KGA claimed in claim 3, it is characterized in that: in described fermenting process, be interrupted or continuous current adding substrate L-sorbose, the stream rate of acceleration is 2-10m
3/ hour, reach 7-10mg/ml to sorbose final concentration in fermented liquid.
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| CN1360024A (en) * | 2000-12-21 | 2002-07-24 | 张忠泽 | Consortive process for culturing bacillus thuringiensis |
| CN101575588A (en) * | 2009-06-22 | 2009-11-11 | 江苏江山制药有限公司 | Preparation technology for 2-keto -L-gulonic acid ferment strain |
| WO2010062707A1 (en) * | 2008-10-30 | 2010-06-03 | Joule Unlimited, Inc. | Methods and compositions for producing carbon-based products of interest in micro-organisms |
| CN102181504A (en) * | 2011-03-15 | 2011-09-14 | 中国科学院沈阳应用生态研究所 | Applications of companion fungus activity extracellular fluid preparation in two-step fermentation of Vc |
| CN102465166A (en) * | 2010-11-04 | 2012-05-23 | 江苏江山制药有限公司 | Method for improving 2-keto-L-gulonic acid fermentation production strength |
| CN102586381A (en) * | 2011-11-01 | 2012-07-18 | 江苏江山制药有限公司 | Production process for improving fermentative strength of 2-keto-L-gulonic acid |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1360024A (en) * | 2000-12-21 | 2002-07-24 | 张忠泽 | Consortive process for culturing bacillus thuringiensis |
| WO2010062707A1 (en) * | 2008-10-30 | 2010-06-03 | Joule Unlimited, Inc. | Methods and compositions for producing carbon-based products of interest in micro-organisms |
| CN101575588A (en) * | 2009-06-22 | 2009-11-11 | 江苏江山制药有限公司 | Preparation technology for 2-keto -L-gulonic acid ferment strain |
| CN102465166A (en) * | 2010-11-04 | 2012-05-23 | 江苏江山制药有限公司 | Method for improving 2-keto-L-gulonic acid fermentation production strength |
| CN102181504A (en) * | 2011-03-15 | 2011-09-14 | 中国科学院沈阳应用生态研究所 | Applications of companion fungus activity extracellular fluid preparation in two-step fermentation of Vc |
| CN102586381A (en) * | 2011-11-01 | 2012-07-18 | 江苏江山制药有限公司 | Production process for improving fermentative strength of 2-keto-L-gulonic acid |
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