CN105177067A - Method for producing L-lactic acid through double-enzyme hydrolyzed crop leftover double-strain fermentation - Google Patents
Method for producing L-lactic acid through double-enzyme hydrolyzed crop leftover double-strain fermentation Download PDFInfo
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- CN105177067A CN105177067A CN201510666773.8A CN201510666773A CN105177067A CN 105177067 A CN105177067 A CN 105177067A CN 201510666773 A CN201510666773 A CN 201510666773A CN 105177067 A CN105177067 A CN 105177067A
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- lactic acid
- pfansteihl
- bagasse
- saccharified liquid
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Abstract
The invention discloses a method for producing L-lactic acid through double-enzyme hydrolyzed crop leftover double-strain fermentation. Firstly two enzymes are added into lignocellulose for enzyme hydrolysis to obtain saccharification liquid, two strains which can produce L-lactic acid by respectively using pentose and hexose are successively added, the efficiency of fermentation through a single strain is higher and the yield of the L-lactic acid is improved. The method for producing L-lactic acid through double-enzyme hydrolyzed crop leftover double-strain fermentation has the advantages that the enzyme hydrolysis extent of the lignocellulose is improved, the utilization of the saccharification liquid is improved, the yield of the L-lactic acid is increased and the economic value and social significance are greater.
Description
Technical field
The present invention relates to Pfansteihl preparation field, be specifically related to a kind of method that double enzymolysis crops leftovers mixed fermentation produces Pfansteihl.
Background technology
Pfansteihl is a kind of important organic acid, has a wide range of applications in industries such as chemical industry, food, medicine. in recent years, the exploitation of polylactic acid-based biodegradable plastic makes the demand of Pfansteihl surge.Pfansteihl illustrates its application prospect widely with its unique structure advantage that it has.Along with exhaustion and the Novel environment-friendlymaterial material of petroleum resources---increasing considerably of poly(lactic acid) demand, people are striving to find the new forms of energy of petroleum replacing fuel and are reducing the new raw material of Pfansteihl production cost
Be that raw material production Pfansteihl receives increasing concern and research, the material that lignocellulose is mainly mixed by Mierocrystalline cellulose, hemicellulose and xylogen with lignocellulose.In plant cellulose, Mierocrystalline cellulose, hemicellulose account for 70% of lignocellulose quality, and Mierocrystalline cellulose and hemicellulose have to pass through after hydrolysis forms hexose and five-carbon sugar respectively, could be utilized and fermentation production of L-lactic acid by microorganism.85% ~ 90% of natural hemicellulose hydrolysate is wood sugar.Therefore, for reducing the production cost of Pfansteihl, develop more efficient, economic production technique, the industrialization realizing utilizing Mierocrystalline cellulose to produce Pfansteihl becomes the task of top priority.
The development of enzyme engineering makes people have understanding to cellulase hydrolysis technology, and cellulosic zymolysis technique starts there has been further development.Cellulase hydrolysis can carry out at normal temperatures, simple to the requirement of equipment, does not produce the material harmful to subsequent fermentation, and this can save energy and minimizing facility investment greatly.Many industrial countries and scientific research institution are all at active development in the world for cellulosic zymolysis technique, and Progress in industrialization carries out.But enzymolysis cellulose conversion rate is low, enzymic activity instability significantly limit the Commercial cultivation of cellulase hydrolysis.In recent years, multiple cellulase is used in combination, and the ultra-filtration membrane of enzyme reclaims and gene cloning of cellulase and be expressed as to address these problems to provide and use for reference well.
Summary of the invention
For solving the problem, the invention provides a kind of method that double enzymolysis crops leftovers mixed fermentation produces Pfansteihl, adding the utilization ratio of enzymatic saccharification liquid, improve Pfansteihl output.
For achieving the above object, the technical scheme that the present invention takes is:
Double enzymolysis crops leftovers mixed fermentation produces a method for Pfansteihl, comprises the steps:
S1, bagasse is smashed after, be that the NaOH solution of 0.5% is sprayed bagasse powder with massfraction, spray evenly and rub 5min, be placed in the environment 12h of 45 DEG C, spray every 4h and rub once, each 5min, obtains bagasse;
After S2, the bagasse getting 2g gained adjust pH to 4.8 after adding 30ml distilled water, add cellulase and each 1mL of zytase, be placed in enzymatic saccharification 8h on 50 DEG C of shaking tables, filter, obtain saccharified liquid;
S3, by the saccharified liquid of step S2 gained under 0.09 atmospheric pressure, heating in water bath to 53 DEG C, rotating speed 60-80 is concentrated to the saccharified liquid of certain sugared concentration;
S4, get peptone 10, yeast extract paste 5g, MgSO
4.7H
2o0.5, (NH)
4sO
44, KH
2pO
40.4, NaCl0.1g, CaCO
4the saccharified liquid 50ml of 3g, step S3 gained is equipped with fermention medium;
S5, the seed liquor of getting 5mL lactobacterium casei are inoculated in the fermention medium that step S4 is equipped with, and are placed in shaking table culture temperature 35 DEG C, speed 150r/min; After cultivating 24h, take out fermentation flask, the seed liquor of inoculation 5mL Rhizopus oryzae, is placed in shaking table and cultivates 60h, temperature 32 DEG C, speed 200r/min.
Wherein, the seed culture fluid of lactobacterium casei comprises following composition (g/L): glucose 50, peptone 10, extractum carnis 5, yeast extract paste 5, anhydrous sodium acetate 5, MgSO
4.7H
2o20mL, MnSO
4.7H
2o20mL, calcium carbonate 20g, pH6.8, tween 1mL.
Wherein, the seed culture fluid of Rhizopus oryzae comprises following composition (g/L): glucose 80, (NH)
4sO
44, KH
2pO
40.3, ZnSO
4.7H
2o0.44, MgSO
4.7H
2o0.25.
Wherein, being compressed into certain sugared concentration in the application is that as fructose concentration is too low, then lactic acid production is too low in order to the sugared concentration needed for fermentable.
The present invention has following beneficial effect:
By rub process cellulase and lignocellulose substrate are directly contacted more thorough, improve the reactivity of cellulase to lignocellulose on the one hand, improve the vigor of cellulase on the other hand, add cellulase and zytase that bagasse is hydrolyzed is more complete simultaneously, by the reasonable interpolation of lactobacterium casei and Rhizopus oryzae, improve the utilization ratio of saccharified liquid, improve the output of Pfansteihl.
Accompanying drawing explanation
Fig. 1 is the schema that a kind of double enzymolysis crops leftovers of embodiment of the present invention mixed fermentation produces the method for Pfansteihl.
Fig. 2 is the result schematic diagram that embodiment of the present invention HPLC detects lactic acid purity.
Embodiment
In order to make objects and advantages of the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The bacterium lactobacterium casei CICC6028 (LactobacilluscaseiCICC6028) that the embodiment of the present invention uses, is purchased from Chinese industrial micro-non-hibernating eggs preservation center (CICC); Rhizopus oryzae M12 (RhizopusoryzaeM12) is obtained by laboratory mutagenic and breeding.Storage medium is PDA inclined-plane, the suitableeest culture temperature 28 DEG C; The seed culture fluid composition of the lactobacterium casei used is (g/L): glucose 50, peptone 10, extractum carnis 5, yeast extract paste 5, anhydrous sodium acetate 5, MgSO
4.7H
2o20mL, MnSO
4.7H
2o20mL, calcium carbonate 20g, pH6.8, tween 1mL; The seed culture fluid composition of the Rhizopus oryzae used is (g/L): glucose 80, (NH)
4sO
44, KH
2pO
40.3, ZnSO
4.7H
2o0.44, MgSO
4.7H
2o0.25, Rhizopus oryzae utilize be saccharified liquid five-carbon sugar produce Pfansteihl; Lactobacterium casei utilizes the hexose of saccharified liquid to produce Pfansteihl.
Embodiment
As shown in Figure 1, S1, bagasse is smashed after, be that the NaOH solution of 0.5% is sprayed bagasse powder with massfraction, spray evenly and rub 5min, be placed in the environment 12h of 45 DEG C, spray every 4h and rub once, each 5min, obtains bagasse;
After S2, the bagasse getting 2g gained adjust pH to 4.8 after adding 30ml distilled water, add cellulase and each 1mL of zytase, be placed in enzymatic saccharification 8h on 50 DEG C of shaking tables, filter, obtain saccharified liquid;
S3, by the saccharified liquid of step S2 gained under 0.09 atmospheric pressure, heating in water bath to 53 DEG C, rotating speed 60-80 is concentrated to the saccharified liquid of certain sugared concentration;
S4, get peptone 10, yeast extract paste 5g, MgSO
4.7H
2o0.5, (NH)
4sO
44, KH
2pO
40.4, NaCl0.1g, CaCO
4the saccharified liquid 50ml of 3g, step S3 gained is equipped with fermention medium;
S5, the seed liquor of getting 5mL lactobacterium casei are inoculated in the fermention medium that step S4 is equipped with, and are placed in shaking table culture temperature 35 DEG C, speed 150r/min; After cultivating 24h, take out fermentation flask, the seed liquor of inoculation 5mL Rhizopus oryzae, is placed in shaking table and cultivates 60h, temperature 32 DEG C, speed 200r/min.
The product of embodiment gained is taken out and carries out the detection of lactic acid content, result as shown in Figure 2, efficient liquid phase chromatographic analysis, chromatographic column is the C18 of Di Ma company anti-phase (4.6mm × 250mm, 5 μm); Moving phase is the ammonium dibasic phosphate solution of 0.01mol/L, with phosphoric acid adjust pH to 2.6, after 0.45 μm of aperture synthetic cellulose ester membrane filtration, and ultrasonic degas 1h; Flow velocity 0.8mL/min, determined wavelength 210nm, sample size 20 μ L, column temperature is room temperature.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. double enzymolysis crops leftovers mixed fermentation produces a method for Pfansteihl, it is characterized in that, comprises the steps:
S1, bagasse is smashed after, be that the NaOH solution of 0.5% is sprayed bagasse powder with massfraction, spray evenly and rub 5min, be placed in the environment 12h of 45 DEG C, spray every 4h and rub once, each 5min, obtains bagasse;
After S2, the bagasse getting 2g gained adjust pH to 4.8 after adding 30ml distilled water, add cellulase and each 1mL of zytase, be placed in enzymatic saccharification 8h on 50 DEG C of shaking tables, filter, obtain saccharified liquid;
S3, by the saccharified liquid of step S2 gained under 0.09 atmospheric pressure, heating in water bath to 53 DEG C, rotating speed 60-80 is concentrated to the saccharified liquid of certain sugared concentration;
S4, get peptone 10, yeast extract paste 5g, MgSO
4.7H
2o0.5, (NH)
4sO
44, KH
2pO
40.4, NaCl0.1g, CaCO
4the saccharified liquid 50ml of 3g, step S3 gained is equipped with fermention medium;
S5, the seed liquor of getting 5mL lactobacterium casei are inoculated in the fermention medium that step S4 is equipped with, and are placed in shaking table culture temperature 35 DEG C, speed 150r/min; After cultivating 24h, take out fermentation flask, the seed liquor of inoculation 5mL Rhizopus oryzae, is placed in shaking table and cultivates 60h, temperature 32 DEG C, speed 200r/min.
2. a kind of double enzymolysis crops leftovers mixed fermentation according to claim 1 produces the method for Pfansteihl, and it is characterized in that, the seed culture fluid of lactobacterium casei comprises following composition (g/L): glucose 50, peptone 10, extractum carnis 5, yeast extract paste 5, anhydrous sodium acetate 5, MgSO
4.7H
2o20mL, MnSO
4.7H
2o20mL, calcium carbonate 20g, pH6.8, tween 1mL.
3. a kind of double enzymolysis crops leftovers mixed fermentation according to claim 1 produces the method for Pfansteihl, and it is characterized in that, the seed culture fluid of Rhizopus oryzae comprises following composition (g/L): glucose 80, (NH)
4sO
44, KH
2pO
40.3, ZnSO
4.7H
2o0.44, MgSO
4.7H
2o0.25.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497901A (en) * | 2009-03-03 | 2009-08-05 | 合肥工业大学 | Novel technological process for producing high optical purity L-lactic acid by semi-continuous high-density fermentation of Rhizopus oryzae |
| CN101724663A (en) * | 2008-10-24 | 2010-06-09 | 中国农业大学 | Method for producing L-lactic acid by utilizing corn cob and special rhizopus oryzae thereof |
| CN103627739A (en) * | 2013-12-12 | 2014-03-12 | 广西大学 | Method for producing L-lactic acid by fermenting bagasse cellulose |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724663A (en) * | 2008-10-24 | 2010-06-09 | 中国农业大学 | Method for producing L-lactic acid by utilizing corn cob and special rhizopus oryzae thereof |
| CN101497901A (en) * | 2009-03-03 | 2009-08-05 | 合肥工业大学 | Novel technological process for producing high optical purity L-lactic acid by semi-continuous high-density fermentation of Rhizopus oryzae |
| CN103627739A (en) * | 2013-12-12 | 2014-03-12 | 广西大学 | Method for producing L-lactic acid by fermenting bagasse cellulose |
Non-Patent Citations (2)
| Title |
|---|
| 刘雅: "纤维质原料二步发酵生产 L-乳酸", 《中国优秀硕士学位论文全文数据库工程科技J辑》 * |
| 王冬冬: "酶解纤维质原料发酵生产L-乳酸", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Inventor after: Li Shichang Inventor after: Gu Shaobin Inventor after: Wang Yankai Inventor after: Li Xiaolin Inventor after: Wang Dahong Inventor after: Lu Jiaqi Inventor before: Gu Shaobin Inventor before: Wang Yankai Inventor before: Li Xiaolin Inventor before: Wang Dahong Inventor before: Lu Jiaqi |
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Application publication date: 20151223 |