CN101418272A - Bacterial strain producing L-lactic acid and method for producing L-lactic acid by using the same through synchronous diastatic fermentation - Google Patents
Bacterial strain producing L-lactic acid and method for producing L-lactic acid by using the same through synchronous diastatic fermentation Download PDFInfo
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- CN101418272A CN101418272A CNA2008102353657A CN200810235365A CN101418272A CN 101418272 A CN101418272 A CN 101418272A CN A2008102353657 A CNA2008102353657 A CN A2008102353657A CN 200810235365 A CN200810235365 A CN 200810235365A CN 101418272 A CN101418272 A CN 101418272A
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Abstract
The invention relates to a strain for producing L-lactic acid and a method for producing the L-lactic acid through saccharifying and fermenting Jerusalem artichoke simultaneously with the strain, which belongs to the technical fields of biological energy source and conversion technology, enzymes and metabolic regulation and control technology, as well as related fermentation engineering. The method uses aspergillus niger and lactobacillus to convert the Jerusalem artichoke into the L-lactic acid through a novel process of synchronous saccharification and fermentation. An aspergillus niger strain SL-09 with high inulinase vitality and a lactobacillus G-02 for producing the L-lactic acid with high optical purity are obtained from the natural world; when the two strains are mixed for culture, the inulinase vitality of the aspergillus niger is improved significantly due to the obvious synergy; in a lactic acid fermentation process, the Jerusalem artichoke powder is used as a substrate; and with a feed-batch fermentation process and after 36 hours of fermentation, the concentration of the L-lactic acid is 120.5 grams per liter and the conversion rate is improved to 94.5 percent. The invention provides a basis for the industrialization of the transformation of the Jerusalem artichoke to the lactic acid.
Description
Technical field
A kind of bacterial strain of L-lactic acid and method of producing L-lactic acid with this bacterium simultaneous saccharification and fermentation jerusalem artichoke of producing belongs to bioenergy and transformation technology, enzyme and metabolic regulation technology, and relevant fermentation engineering field.
Background technology
Lactic acid is widely used in food, makeup, medicine, weaving and the chemical industry as a kind of basic material commonly used; In order to improve lactic acid production, to reduce cost, the research worker has done a large amount of work on production bacterial strain and zymotechnique both at home and abroad.In recent years, the heavy demand of biodegradable polymerization lactic acid has promoted the development of fermentative Production L-lactic acid significantly.
Jerusalem artichoke (having another name called Jerusalem artichoke, Jerusalem artichoke, Westerner ginger) is the annual or per nnial herb of composite family Helianthus.Now all there is plantation various places, China north and south, and cultivated area just progressively enlarges.This plant is not only nutritious, and is cold-resistant, anti-desert, and salt tolerant alkali, disease and insect resistance and once plant lifelong income, reproductivity is strong and can be grown in preferably extremely on the barren soil, and production process need not managed.The stem tuber of jerusalem artichoke is rich in amino acid, sugar, VITAMIN etc.The well developed root system cauline leaf of jerusalem artichoke is luxuriant, can check winds and fix drifting sand, and prevents erosion, and beautifies, environment purification maintaining ecological balance.Its adaptability is strong, and production cost is low, and end-use is wide, and society, economy, ecological benefits are remarkable.Plantation and comprehensive development and utilization jerusalem artichoke, its development prospect is very considerable with suiting measures to local conditions.So be subjected to common attention in recent years at home and abroad.The moisture content of bright jerusalem artichoke stem tuber is about 70%, and total sugar content is about 20%, and this crop is used as always and produces the good raw material of alcoholic acid for many years.And the research of exploitation jerusalem artichoke production lactic acid does not appear in the newspapers as yet.
The main component of jerusalem artichoke is an inulin, is the unavailable polyfructosan of milk-acid bacteria, is exactly the synthetic of inulinase so the exploitation Jerusalem artichoke raw material is produced the most important condition of lactic acid.The microorganism that can synthesize inulinase has many kinds because himself extensive growth demand and widely lytic enzyme be that aspergillus niger is widely used in Glucoamylase preperation industry.Studies show that inulinase is a kind of inducible enzyme, and the synthetic inhibition that is subjected to glucose of this enzyme, therefore, must reduce the concentration of glucose in the fermented liquid as much as possible in order further to improve the activity of inulinase.
Summary of the invention
The purpose of this invention is to provide a kind of bacterial strain of the L-of producing lactic acid and the method for producing L-lactic acid with this bacterium simultaneous saccharification and fermentation jerusalem artichoke.In order farthest to reduce the cost of jerusalem artichoke fermenting lactic acid, improve production intensity and transformation efficiency, improve the final lactic acid concn of karusen simultaneously, the high L-lactic acid optical purity junket milk-acid bacteria that the present invention adopts this laboratory screening to obtain, aspergillus niger SL-09[World J Microbiol Biotechnol (2008) the 24:133-138DOI 10.1007/s1274-007-9450-3 that this bacterium and this laboratory is possessed independent intellectual property rights] carry out mixed culture, improved the inulinase activity significantly; In zymotechnique of lactic acid, use synchronous saccharification and zymotechnique then, and this technology fermentation parameter is optimized, and in whole fermentation process, need not to add inorganic salt.Because the concentration of sugar remains at lower level during the fermentation, make enzyme work and buttermilk acid bacteria fermentation vigor all obtain fully playing, through the 36h fermentation, the L-lactic acid concn reaches 120.5g/L, has reached 94.5% transformation efficiency simultaneously.
Technical scheme of the present invention: a kind of bacterial strain that produces L-lactic acid, its classification called after junket milk-acid bacteria (Lactobacillus sp.) G-02 is preserved in Chinese typical culture collection center the sixth of the twelve Earthly Branches, and deposit number is CCTCC NO:M 208232.
With the method that described bacterial strain simultaneous saccharification and fermentation jerusalem artichoke produces L-lactic acid, use the aspergillus niger SL-09 that possesses higher inulinase vigor, after 12h is carried out in fermentation, insert junket milk-acid bacteria G-02, adopt the technology of synchronous saccharification and fermentation then, step is:
(1) preparation of jerusalem artichoke powder:
Fresh jerusalem artichoke rhizome is cleaned, cut into slices, and 40 ℃ of air seasoning 48h, air-dry jerusalem artichoke sheet adopt the pulverizer polishing, and fine powder is crossed 50 mesh sieves; It is 56% that dry powder contains the total reducing sugar amount;
(2) preparation of buttermilk acid bacterial classification:
The sub-substratum composition of buttermilk acid bacterial classification is counted with g/L: glucose 30, peptone 10, yeast extract paste 10, (NH
4)
2HPO
4, 2, MnSO
4H
2O 0.2, CaCO
3, 20, initial pH 7.0; One ring junket milk-acid bacteria G-02 seed is inserted in the substratum of 100mL sterilization, behind 30 ℃, 140r/min one-level shake-flask culture 18h, 6 one-level nutrient solutions are inserted respectively in 6 flat round flasks of 5L that the 3L substratum is housed, continue secondary and cultivate 30h;
(3) the product enzyme of aspergillus niger:
Producing enzyme substratum composition counts with g/L: jerusalem artichoke powder 50, soybean cake powder 40, sucrose ester 6; Aspergillus niger strain SL-09 is inserted in the 50L fermentor tank that 20L product enzyme substratum is housed, 30 ℃, ventilation 0.8L/min, rotating speed 140r/min, after carrying out 12h, fermentation inserts the junket milk-acid bacteria, being about to 6 secondary nutrient solutions of gained inserts, produce in the enzyme process and need not to regulate pH, behind total incubation time 60h, produce inulinase restriction endonuclease and excision enzyme and reach 275.6U/ml and 571.8U/mL respectively, gained inulinase liquid is used to produce L-lactic acid, this inulinase liquid not only disposes conveniently, and has the stronger buffer system from farm crop itself;
(4) synchronous saccharification produces L-lactic acid with fermentation:
Add water by step (3) gained inulinase liquid, add substrate jerusalem artichoke powder and form fermented liquid, synchronous saccharification and fermentation jerusalem artichoke produce the inulinase inoculum size of lactic acid and enzyme work and are respectively 35% and 50U/mL, in order farthest to reduce the restraining effect of high concentration sugar to junket milk-acid bacteria vigor, improve the transformation efficiency of sugar simultaneously to lactic acid, adopt the feed supplement strategy of portion-wise addition substrate, beginning in fermentation, when 12h and 24h, respectively with 40Kg, the jerusalem artichoke powder of 25Kg and 25Kg adds in the 400L fermented liquid, 30 ℃ of leavening temperatures, total fermentation time 36h secondary fermentation stops, lactic acid concn is 120.5g/L, and transformation efficiency is 94.5% of a theoretical yield.
Beneficial effect of the present invention: commercially available price that jerusalem artichoke is present and potato butt this suitable (bright jerusalem artichoke is 300～500 yuan/ton), its cultivated area is just with nearly 20% speed cumulative year after year, so the conversion jerusalem artichoke is a lactic acid, resolving three rural isssues is safeguarded that still ecological environment problem all has far-reaching society and economic implications.
Obtain a strain homofermentation junket milk-acid bacteria from occurring in nature, its optical purity of producing L (+)-lactic acid reaches 95%.This strain junket milk-acid bacteria is in the technology of cultivating altogether with aspergillus niger, because there is synergy in two strain bacterium, makes aspergillus niger produce enzyme level and is improved by a relatively large margin.Studies show that the junket milk-acid bacteria joining day when 12h, it is the highest that aspergillus niger produces the inulinase vigor, cultivates through 60h, and inulinase restriction endonuclease and excision enzyme vigor reach 275.6 and 571.8U/mL respectively.
Then the synchronous saccharification and the zymotechnique that adopt jerusalem artichoke powder direct production lactic acid are optimized, junket milk-acid bacteria inoculum size, the enzyme amount of living is remarkable to the lactic fermentation intensity effect, and jerusalem artichoke powder concentration is remarkable to lactic acid concn and transformation efficiency influence.Studies show that optimum inoculation amount, enzyme amount alive and jerusalem artichoke powder concentration are respectively 35%, 50U/mL and 230g/L.Employing is the feed supplement synchronous saccharification and the zymotechnique of substrate with the jerusalem artichoke powder, and the substrate of having avoided being present in the zymotechnique suppresses phenomenon, has improved whole lactic acid-producing intensity and transformation efficiency significantly.Through the 36h fermentation, the fermented liquid lactic acid concn reaches 120.5g/L, and transformation efficiency reaches 94.5%.
Characteristics of the present invention are:
1. obtain L (+)-lactic acid optical purity from row filter and reach 95% junket milk-acid bacteria G-02, this bacterial strain is in the technology of cultivating altogether with aspergillus niger SL-09, have significant synergy, the substratum total reducing sugar is descended rapidly, aspergillus niger produces the inulinase restriction endonuclease and the excision enzyme vigor all is significantly improved.
2. first synchronous saccharification and fermentation jerusalem artichoke being produced junket milk-acid bacteria inoculum size in the lactic acid technology, enzyme amount alive and concentration of substrate explores the influence of ferment strength and transformation efficiency, discovery is utilizing with footwork in the jerusalem artichoke fermenting lactic acid, and too high enzyme amount alive and concentration of substrate have significantly reduced effect to lactic acid fermented transformation efficiency.
3. adopting feed supplement synchronous saccharification and zymotechnique first, is that substrate is produced L-lactic acid with the jerusalem artichoke powder, has avoided the restraining effect of sugar in the karusen.Fermentation is terminated at 36h, and final karusen lactic acid content is 120.5g/L, and transformation efficiency is 94.5%.
The biological material specimens preservation
A kind of bacterial strain that produces L-lactic acid, its classification called after junket milk-acid bacteria (Lactobacillus sp.) G-02 is preserved in Chinese typical culture collection center the sixth of the twelve Earthly Branches, is called for short CCTCC, and preservation date on November 19th, 2008, deposit number is CCTCC NO:M 208232.
Embodiment
Embodiment 1:
Fresh jerusalem artichoke rhizome is cleaned, cut into slices, and 40 ℃ of air seasoning 48h, air-dry jerusalem artichoke sheet adopt the pulverizer polishing, and fine powder is crossed 50 mesh sieves; It is 56% that dry powder contains the total reducing sugar amount.
The sub-substratum of buttermilk acid bacterial classification is formed in g/L: glucose 30, peptone 10, yeast extract paste 10, (NH
4)
2HPO
4, 2, MnSO
4H
2O 0.2, CaCO
3, 20, initial pH 7.0; One ring junket milk-acid bacteria G-02 seed is inserted in the substratum of 100mL sterilization, behind 30 ℃, 140r/min one-level shake-flask culture 18h, 6 one-level nutrient solutions are inserted respectively in 6 flat round flasks of 5L that the 3L substratum is housed, secondary is cultivated 30h.
Producing enzyme substratum composition counts with g/L: jerusalem artichoke powder 50, soybean cake powder 40, sucrose ester 6.
Aspergillus niger strain SL-09 is inserted in the 50L fermentor tank that 20L product enzyme substratum is housed, 30 ℃, ventilation 0.8L/min, rotating speed 140r/min, after carrying out 12h, fermentation inserts the junket milk-acid bacteria, being about to 6 secondary nutrient solutions of gained inserts, produce in the enzyme process and need not to regulate pH, behind total incubation time 60h, produce inulinase restriction endonuclease and excision enzyme and reach 275.6U/ml and 571.8U/mL respectively, gained inulinase liquid is used to produce L-lactic acid.
Gained inulinase liquid Jia Shui, add substrate jerusalem artichoke powder and form fermented liquid, synchronous saccharification and fermentation jerusalem artichoke produce the inulinase inoculum size of lactic acid and enzyme work and are respectively 35% and 50U/mL, when the beginning of fermenting, 12h and 24h, jerusalem artichoke powder with 40Kg, 25Kg and 25Kg adds in the 400L fermented liquid respectively, 30 ℃ of leavening temperatures, total fermentation time 36h secondary fermentation stops, and lactic acid concn is 120.5g/L, and transformation efficiency is 94.5% of a theoretical yield.
Claims (2)
1, a kind of bacterial strain that produces L-lactic acid, its classification called after junket milk-acid bacteria (Lactobacillus sp.) G-02 is preserved in Chinese typical culture collection center the sixth of the twelve Earthly Branches, and deposit number is CCTCC NO:M 208232.
2, the method for producing L-lactic acid with the described bacterial strain simultaneous saccharification and fermentation of claim 1 jerusalem artichoke, it is characterized in that using the aspergillus niger SL-09 that possesses higher inulinase vigor, after 12h is carried out in fermentation, insert junket junket milk-acid bacteria G-02, adopt the technology of synchronous saccharification and fermentation then, step is:
(1) preparation of jerusalem artichoke powder:
Fresh jerusalem artichoke rhizome is cleaned, cut into slices, and 40 ℃ of air seasoning 48h, air-dry jerusalem artichoke sheet adopt the pulverizer polishing, and fine powder is crossed 50 mesh sieves; It is 56% that dry powder contains the total reducing sugar amount;
(2) preparation of buttermilk acid bacterial classification:
The sub-substratum composition of buttermilk acid bacterial classification is counted with g/L: glucose 30, peptone 10, yeast extract paste 10, (NH
4)
2HPO
4, 2, MnSO
4H
2O 0.2, CaCO
3, 20, initial pH 7.0; One ring junket milk-acid bacteria G-02 seed is inserted in the substratum of 100mL sterilization, behind 30 ℃, 140r/min one-level shake-flask culture 18h, 6 one-level nutrient solutions are inserted respectively in 6 flat round flasks of 5L that the 3L substratum is housed, continue secondary and cultivate 30h;
(3) the product enzyme of aspergillus niger:
Producing enzyme substratum composition counts with g/L: jerusalem artichoke powder 50, soybean cake powder 40, sucrose ester 6;
Aspergillus niger strain SL-09 is inserted in the 50L fermentor tank that 20L product enzyme substratum is housed, 30 ℃, ventilation 0.8L/min, rotating speed 140r/min, after carrying out 12h, fermentation inserts the junket milk-acid bacteria, being about to 6 secondary nutrient solutions of gained inserts, produce in the enzyme process and need not to regulate pH, behind total incubation time 60h, produce inulinase restriction endonuclease and excision enzyme and reach 275.6U/ml and 571.8U/mL respectively, gained inulinase liquid is used to produce L-lactic acid;
(4) synchronous saccharification produces L-lactic acid with fermentation:
By step (3) gained inulinase liquid Jia Shui, add substrate jerusalem artichoke powder and form fermented liquid, synchronous saccharification and fermentation jerusalem artichoke produce the inulinase inoculum size of lactic acid and enzyme work and are respectively 35% and 50U/mL, when the beginning of fermenting, 12h and 24h, jerusalem artichoke powder with 40Kg, 25Kg and 25Kg adds in the 400L fermented liquid respectively, 30 ℃ of leavening temperatures, total fermentation time 36h secondary fermentation stops, and lactic acid concn is 120.5g/L, and transformation efficiency is 94.5% of a theoretical yield.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087680A (en) * | 2015-08-19 | 2015-11-25 | 沈阳科纳提克生物科技有限公司 | Lactobacillus fermentation culture medium and process for producing lactic acid at high yield |
| CN107022493A (en) * | 2017-03-24 | 2017-08-08 | 江苏天种牧业股份有限公司 | A kind of aspergillus oryzae strain of high yield complex enzyme for feed and its application |
| CN107022578A (en) * | 2017-04-01 | 2017-08-08 | 北京理工大学 | A kind of method that production inulinase, enzymic hydrolysate of jerusalem artichoke and the coupling of fermentation by saccharomyces cerevisiae three prepare ethanol |
| CN110964756A (en) * | 2019-11-11 | 2020-04-07 | 盐城工学院 | Method for preparing L-lactic acid by using jerusalem artichoke in full value |
-
2008
- 2008-12-08 CN CNA2008102353657A patent/CN101418272A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087680A (en) * | 2015-08-19 | 2015-11-25 | 沈阳科纳提克生物科技有限公司 | Lactobacillus fermentation culture medium and process for producing lactic acid at high yield |
| CN107022493A (en) * | 2017-03-24 | 2017-08-08 | 江苏天种牧业股份有限公司 | A kind of aspergillus oryzae strain of high yield complex enzyme for feed and its application |
| CN107022578A (en) * | 2017-04-01 | 2017-08-08 | 北京理工大学 | A kind of method that production inulinase, enzymic hydrolysate of jerusalem artichoke and the coupling of fermentation by saccharomyces cerevisiae three prepare ethanol |
| CN110964756A (en) * | 2019-11-11 | 2020-04-07 | 盐城工学院 | Method for preparing L-lactic acid by using jerusalem artichoke in full value |
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Open date: 20090429 |