CN101886095B - Method for producing high-concentration D-lactic acid by adopting synchronous enzymolysis and fermentation on peanut meal and special culture medium thereof - Google Patents
Method for producing high-concentration D-lactic acid by adopting synchronous enzymolysis and fermentation on peanut meal and special culture medium thereof Download PDFInfo
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Abstract
The invention discloses a method for producing high-concentration D-lactic acid by adopting synchronous enzymolysis and fermentation on peanut meal and a special culture medium thereof. The special culture medium adopts peanut meal as a fermenting culture medium of an organic nitrogen source. In addition, protease is added into the special culture medium. In the method for producing the high-concentration D-lactic acid, Sporolactobacillus inulinus CASD CGMCC (Computer Aided System Design China General Microbiological Culture Collection center) No.2185 is inoculated to the special culture medium for fermenting to obtain the high-concentration D-lactic acid. The method for producing the high-concentration D-lactic acid of the invention is obviously better than the traditional method for producing the high-concentration D-lactic acid, has the advantages of high purity, high production efficiency, low requirement on instrument and equipment, low cost, and the like and is suitable for large-area popularization and application.
Description
Technical field
The invention belongs to the fermentation engineering field, particularly relate to a kind of method and special culture media thereof that adopts the synchronous enzymatic hydrolysis and fermentation of peanut meal to produce high-concentration D-lactic acid.
Background technology
Lactic acid (lactic acid), formal name used at school are alpha-hydroxypropionic acid (d-hydroxy-propionic acid), and the chemical molecular formula of lactic acid is C
2H
5OCOOH is a kind of simple alcohol acid.An asymmetric carbon atom is arranged in the lactic acid molecules, so lactic acid has opticity.Pfansteihl is levorotation, and D-ALPHA-Hydroxypropionic acid is dextrorotatory, and DL-LACTIC ACID is racemism.In recent years, the derivative poly(lactic acid) of lactic acid has caused people's extensive concern, its physicals and polystyrene are closely similar, it is except the excellent specific property with the plastic material take petroleum chemicals as raw material, also having maximum characteristics is biodegradabilities, but namely at the occurring in nature natural degradation, do not cause environmental pollution, solved the white pollution problems that perplexs for world's environmental protection.But many unstable properties of pure poly (l-lactic acid) (PLLA), and the poly-D-ALPHA-Hydroxypropionic acid of adding suitable proportion carries out polymerization, can improve performance, has widened the range of application of poly(lactic acid).D-ALPHA-Hydroxypropionic acid is the precursor of synthetic multiple chiral material as a chiral centre, has important application in the chiral material in the fields such as low-toxin farm chemicals, weedicide and makeup is synthetic.
(the Ding Zijian etc. such as Ding Zijian, biological processing, the 2nd volume, the 3rd phase, the 30-36 page or leaf, 2004) reported first employing lactobacillus (Sporolactobacillus sp.) prepare D-ALPHA-Hydroxypropionic acid from glucose fermentation, fermentation can be produced the D-ALPHA-Hydroxypropionic acid of 40.7 grams per liters, inversion rate of glucose 67.8% in 72 hours.Chinese patent CN200610097453.6 discloses a kind of technique of combined fermentation production of D-lactic acid, produces acid 7.5%～13.1%.Chinese patent CN 200710176056.2 has reported and has utilized synanthrin lactobacillus (Sporolactobacillusinulinus) semicontinuous fermentation to produce D-ALPHA-Hydroxypropionic acid that the concentration of the D-ALPHA-Hydroxypropionic acid that this inventive method obtains reaches as high as 162 grams per liters.Chinese patent CN200810098908.5 has reported the technique of lactobacillus Sporolactobacillus sp.Y2-8 bacterial strain and fermentation production of D-lactic acid thereof, and D-ALPHA-Hydroxypropionic acid content reaches 9.1%～16.5% in the fermented liquid, and the D-ALPHA-Hydroxypropionic acid optical purity reaches 99.1%.Chinese patent CN200810116194.6 has reported plant lactobacillus production D-ALPHA-Hydroxypropionic acid, and total lactic acid concn is 18% in the fermented liquid, D-ALPHA-Hydroxypropionic acid optical purity 84～86%.The fermentative Production Pfansteihl concentration of report can reach 190 grams per liters at present, and optical purity can reach (Chinese patent CN200710176060.9) more than 99%.Compare with Pfansteihl, the concentration of fermentative Production D-ALPHA-Hydroxypropionic acid and optical purity also need further raising.
Usually adopt yeast extract as nitrogenous source because lactic fermentation is produced, improved to a certain extent the production cost of lactic acid.For this problem, at present domestic have the comparatively cheap corn steep liquor of employing, soybean meal hydrolysate etc. to substitute yeast extract as nitrogenous source.The cheap high molecular weight protein such as dregs of beans can not directly be used as lactic acid fermented nitrogenous source usually, and needs to make high molecular weight protein be decomposed into the small molecules such as small peptide and amino acid through pre-treatment such as acidolysis, is beneficial to the utilization of lactic acid-producing bacterial strain.The acidolysis process not only needs acidproof equipment, also need to carry out lot of energy under comparatively high temps.High temperature and sour environment have certain destruction to nutritive substances such as amino acid simultaneously.Proteolytic enzyme can be degraded to high molecular weight protein small-molecular peptides and amino acid.Compare with the acidolysis process, the enzymolysis process condition is gentleer, is conducive to preserve the effective constituent in the protein hydrolyte.Generally, separate with fermenting process as the proteolysis preprocessing process of fermentation nitrogen source, hydrolytic process needs independent place, equipment and operation, has increased equipment investment and the complicated operation degree of whole fermentation production process.If proteolysis process and fermenting process can be integrated mutually, proteolysis and lactic fermentation are carried out in fermentor tank simultaneously, just can save independent proteolysis operation, thereby save space and facility investment, be conducive to reduce the D-ALPHA-Hydroxypropionic acid production cost.
Peanut meal is from the product of Semen arachidis hypogaeae after oil plant is refined in squeezing, is rich in vegetable-protein.The distribution of China's peanut is very extensive, and China's peanut yield ranks among the best in the world.The cheap nitrogenous source that peanut meal is widely distributed as another kind, high yield, nitrogen content are abundant also is not used for the report of D-ALPHA-Hydroxypropionic acid fermentative production.
Summary of the invention
The invention provides the special culture media that utilizes the synchronous enzymatic hydrolysis and fermentation of peanut meal to produce high-concentration D-lactic acid.
Special culture media provided by the present invention is with the fermention medium of peanut meal as organic nitrogen source.
In described special culture media, also be added with proteolytic enzyme, take with the proteolysis in the peanut meal as small-molecular peptides and amino acid, and peanut meal enzymolysis process and fermentation production of D-lactic acid are carried out synchronously.
Specifically, described special culture media comprises: cerelose 135～150 grams per liters, and peanut meal 20～40 grams per liters, neutral protease or flavor protease or compound protease 0～0.5 grams per liter, surplus is water; The pH of described substratum is 5.5～6.5.
Also can be added with pH adjusting agent in the described special culture media, be preferably calcium carbonate, its content in fermention medium is 70 ± 2 grams per liters.
Second purpose of the present invention provides a kind of easy and simple to handle, method that purity is higher utilizes the synchronous enzymatic hydrolysis and fermentation of peanut meal to produce high-concentration D-lactic acid.
The production method of high-concentration D-lactic acid provided by the present invention is synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC No.2185 to be inoculated in the above-mentioned fermention medium ferment, and obtains high-concentration D-lactic acid.
Produce in the method for D-ALPHA-Hydroxypropionic acid at the synchronous enzymatic hydrolysis and fermentation of above-mentioned employing peanut meal, described inoculum size is 5～10% volume ratios; Fermentation condition is: cultivated 42～70 hours for 37～47 ℃; Being preferably 42 ℃ cultivated 48 hours.
Proceed to 30～36 hours in fermentation culture, glucose concn is down to about 50 grams per liters, also need add glucose, makes that glucose concn reaches 120～130 grams per liters in the fermented liquid.
For improving fermentation efficiency, described fermenting process adopts intermittent stirring, ferments in the triangular flask, stirs once every vibration in 8～12 hours; Fermentation cylinder for fermentation stirred once in per 6～8 hours, stirred 5～10 minutes at every turn, and revolution is preferably 100 rev/mins.
In addition, for obtaining better ferment effect, before above-mentioned fermentation process was implemented, described synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC No.2185 bacterial classification also need carry out slant culture and seed culture before fermentation.
Described slant culture is with in synanthrin lactobacillus (Sporolactobacillus inulinus) the CASDCGMCC No.2185 access slant medium, 37～47 ℃ of lower cultivations 48～72 hours; Described slant culture based formulas is: glucose 10 grams per liters, and yeast powder 5 grams per liters, calcium carbonate 1 grams per liter, agar 20 grams per liters, solvent are water, and the pH of described slant medium is 6.5, and initial pH is 6～7.
Described seed culture is to access in the seed culture medium through synanthrin lactobacillus (Sporolactobacillusinulinus) CASD of slant culture CGMCC No.2185 again, 37～47 ℃ of lower cultivations 30～36 hours; Described seed culture based formulas is: described seed culture based formulas is: glucose 40 grams per liters, and yeast powder 5 grams per liters, calcium carbonate 3 grams per liters, surplus is water; Described seed culture medium pH is 6～7.
The high-concentration D-lactic acid and the application of peanut meal in synchronously enzymatic hydrolysis and fermentation production high-concentration D-lactic acid that obtain with aforesaid method also belong to protection scope of the present invention.
The present invention has realized peanut meal is produced D-ALPHA-Hydroxypropionic acid as the synchronous enzymatic hydrolysis and fermentation of cheap nitrogenous source first, and the inventive method compared with prior art has following advantage:
1. the fermentation culture based component is simple, the make a living by product of yield peanut oil of selected nitrogenous source peanut meal, and cheap, aboundresources greatly reduces production cost.
2. enzymolysis peanut meal and fermenting lactic acid carry out simultaneously, have saved the pretreated step of organic nitrogen source, have saved effectively that the place takies and equipment investment.
3. employing the inventive method, can significantly improve the output of D-ALPHA-Hydroxypropionic acid, compare with present report (Chinese patent CN200810116194.6 report Lactic Acid from Fermentation Broth concentration is 180 grams per liters), D-ALPHA-Hydroxypropionic acid output is significantly improved, can reach more than 200 grams per liters, glucose acid invert ratio reaches 98.5% (report such as Ding Zijian adopts lactobacillus fermentation preparation D-ALPHA-Hydroxypropionic acid, glucose acid invert ratio 67.8%), and optical purity reaches 99.3%.
In sum, the production method of high-concentration D-lactic acid of the present invention obviously is better than existing high-concentration D-lactic acid production method, have purity height, production efficiency high, to advantages such as the plant and instrument requirement are low and with low cost, suit large area to popularize and use.
Below in conjunction with specific embodiment the present invention is described in further details.
Embodiment
Synanthrin lactobacillus used in the present invention (Sporolactobacillus inulinus) CASD derives from China Committee for Culture Collection of Microorganisms common micro-organisms center and (is called for short CGMCC, the address is: the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), the applicant is preservation before, and preservation registration number is CGMCC No.2185; This bacterial strain is open in the patent of paper that the applicant delivers and application.
Employed cerelose is available from the sugared company limited of Zibo rainbow space industry in the fermention medium of the present invention, peanut meal is available from Beijing Kang Mingwei substratum technology limited liability company, neutral protease is available from letter Bioisystech Co., Ltd of Beijing Novi (50,000 unit of activity/grams, add after the sterilization), flavor protease is available from letter Bioisystech Co., Ltd of Beijing Novi (20,000 unit of activity/grams, add after the sterilization), compound protease is available from letter Bioisystech Co., Ltd of Beijing Novi (20,000 unit of activity/grams add after the sterilization).
The basic step of utilizing the synchronous enzymatic hydrolysis and fermentation of peanut meal to produce D-ALPHA-Hydroxypropionic acid of the present invention is as follows:
(1) slant culture: synanthrin lactobacillus (Sporolactobacillus inulinus) CASDCGMCC No.2185 is accessed slant medium, the inclined-plane is placed in the incubator cultivates, 37～47 ℃ of culture temperature, incubation time 48～72 hours, the initial pH value of slant medium are 6～7.
(2) seed culture: change cultured bacterial classification over to seed culture medium, in incubator, leave standstill cultivation, 37～47 ℃ of culture temperature, incubation time 30～36 hours, the seed culture medium initial pH value is 6～7.
(3) fermentation culture: with the inoculum size of 5～10% volume ratios, change cultured seed liquor over to fermention medium and carry out fermentation culture.37～47 ℃ of leavening temperatures, fermentation time 42～70 hours can carry out intermittence and stir in the fermentation cylinder for fermentation process, stirred once, and stirred preferred 100 rev/mins of revolution at every turn 5～10 minutes in per 6～8 hours.Fermentation is stirred once every vibration in 8～12 hours in the triangular flask.
(4) fermentor tank glucose feed supplement: fermentation culture proceeds to 30～36 hours in the above-mentioned steps (3), and glucose concn is down to about 50 grams per liters, adds glucose, makes that glucose concn reaches 120～130 grams per liters in the fermented liquid.
(5) sample determination: get fermented liquid with 6,000 rev/mins speed centrifugal 5 minutes, get supernatant liquor dilution suitable multiple, detect glucose content, D-ALPHA-Hydroxypropionic acid content and Pfansteihl content.Glucose concn is measured with bio-sensing analyser SBA-40C (Shandong Province academy sciences Biology Research Institute).Pfansteihl and D-ALPHA-Hydroxypropionic acid content adopt Agilent 1100 liquid chromatographs (Anjelen Sci. ﹠ Tech. Inc) to measure, chiral separation post (Mitsubishi chemical company, MCI GEL-CRS10 W (3 μ) 4.6ID * 50mm, the optics allosome separates to be used), 0.002mol/L copper sulfate is moving phase, flow 0.5mL/min, sample size 20 μ L, UV-detector detects wavelength 254nm, 25 ℃ of service temperatures.The D-ALPHA-Hydroxypropionic acid standard substance are Sigma-Aldrich company product, and article No. is L0625, and the Pfansteihl standard substance are Sigma-Aldrich company product, and article No. is L1750.Under this chromatographic condition, the retention time of D-ALPHA-Hydroxypropionic acid standard substance is 9.724 minutes.
D-ALPHA-Hydroxypropionic acid optical purity method of calculation: D-ALPHA-Hydroxypropionic acid optical purity (%)=(D-ALPHA-Hydroxypropionic acid concentration (grams per liter) ÷ [D-ALPHA-Hydroxypropionic acid concentration (grams per liter)+Pfansteihl concentration (grams per liter)]) * 100%[Bioresource Technology 101 (2010) 6494～6498]
Glucose acid invert ratio method of calculation: transformation efficiency (%)=(D-ALPHA-Hydroxypropionic acid concentration (grams per liter) ÷ glucose utilization (grams per liter)) * 100%
Method therefor is ordinary method if no special instructions among the following embodiment.Its objective is content for a better understanding of the present invention, therefore, the example of giving an example does not limit protection scope of the present invention.
Embodiment 1 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-ALPHA-Hydroxypropionic acid, 50mL fermented liquid/100mL triangular flask, fermentation time 42 hours at 37 ℃ of bottom fermentations.
Employed each substratum is composed as follows in the present embodiment:
Slant medium: glucose 10 grams per liters, yeast powder 5 grams per liters, calcium carbonate 1 grams per liter, agar 20 grams per liters, solvent are water, the pH of described slant medium is 6.5,121 ℃ of sterilizations 20 minutes.
Seed culture medium: glucose 40 grams per liters, yeast powder 5 grams per liters, calcium carbonate 3 grams per liters, solvent are water, the pH of described seed culture medium is 6.5,121 ℃ of sterilizations 20 minutes.
Fermention medium: cerelose 135 grams per liters, peanut meal 40 grams per liters, neutral protease 0.1 grams per liter, calcium carbonate 70 grams per liters, surplus is water, the pH of described fermention medium is 6.5,115 ℃ of sterilizations 20 minutes.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: synanthrin lactobacillus (Sporolactobacillus inulinus) CASDCGMCC No 2185 is inoculated on the slant medium, cultivated 72 hours for 42 ℃;
(2) seed culture: the bacterial strain that step (1) is cultivated is encircling in the 100mL triangular flask that the 30mL seed culture medium is housed with inoculation articulating 2 under the aseptic condition, 42 ℃ leave standstill cultivation 36 hours, then the inoculum size with 10% volume ratio is inoculated in the new seed culture medium, 42 ℃ of static cultivations 36 hours, be activated seed once, make seed culture fluid;
(3) fermentation culture: the inoculum size of above-mentioned seed culture medium with 10% volume ratio is inoculated in the fermention medium (50mL fermented liquid/100mL triangular flask), be placed on 37 ℃ of static cultivations, stir once every vibration in 12 hours, cultivated 42 hours, finish fermentation.
After the fermentation ends, according to detection and the method for calculation described in the above-mentioned embodiment, detect D-ALPHA-Hydroxypropionic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-ALPHA-Hydroxypropionic acid optical purity.3 repetitions are established in this experiment altogether.D-ALPHA-Hydroxypropionic acid content 112 ± 3 grams per liters, glucose content 20 ± 1 grams per liters, glucose acid invert ratio 97.4%, the D-ALPHA-Hydroxypropionic acid optical purity reaches 99.2%.
Embodiment 2 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-ALPHA-Hydroxypropionic acid, 50mL fermented liquid/100mL triangular flask, fermentation time 42 hours at 42 ℃ of bottom fermentations.
Employed each substratum is composed as follows in the present embodiment:
Slant medium, seed culture medium and fermention medium are with embodiment 1.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 1;
(2) seed culture: with embodiment 1;
(3) fermentation culture: the inoculum size of above-mentioned seed culture medium with 10% volume ratio is inoculated in the fermention medium (50mL fermented liquid/100mL triangular flask), be placed on 42 ℃ and leave standstill cultivation, stir once every vibration in 12 hours, cultivated 42 hours, finish fermentation.
After the fermentation ends, according to detection and the method for calculation described in the above-mentioned embodiment, detect D-ALPHA-Hydroxypropionic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-ALPHA-Hydroxypropionic acid optical purity.3 repetitions are established in this experiment altogether.D-ALPHA-Hydroxypropionic acid content 129 ± 2 grams per liters, glucose content 0.3 ± 0.2 grams per liter, glucose acid invert ratio 96.0%, the D-ALPHA-Hydroxypropionic acid optical purity reaches 99.1%.
Embodiment 3 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-ALPHA-Hydroxypropionic acid, 50mL fermented liquid/100mL triangular flask, fermentation time 42 hours at 47 ℃ of bottom fermentations.
Employed each substratum is composed as follows in the present embodiment:
Slant medium, seed culture medium and fermention medium are with embodiment 1.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 1;
(2) seed culture: with embodiment 1;
(3) fermentation culture: the inoculum size of above-mentioned seed culture medium with 10% volume ratio is inoculated in the fermention medium (50mL fermented liquid/100mL triangular flask), be placed on 47 ℃ and leave standstill cultivation, stir once every vibration in 12 hours, cultivated 42 hours, finish fermentation.
After the fermentation ends, according to detection and the method for calculation described in the above-mentioned embodiment, detect D-ALPHA-Hydroxypropionic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-ALPHA-Hydroxypropionic acid optical purity.3 repetitions are established in this experiment altogether.D-ALPHA-Hydroxypropionic acid content 128 ± 2 grams per liters, glucose content 3.7 ± 0.6 grams per liters, glucose acid invert ratio 97.5%, the D-ALPHA-Hydroxypropionic acid optical purity reaches 99.3%.
Embodiment 4 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-ALPHA-Hydroxypropionic acid at 20 grams per liter peanut meal concentration bottom fermentations, 50mL fermented liquid/100mL triangular flask, 42 ℃ of leavening temperatures, fermentation time 48 hours.
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 1;
Fermention medium: peanut meal concentration is 20 grams per liters, adds simultaneously cerelose 135 grams per liters, neutral protease 0.5 grams per liter, and calcium carbonate 70 grams per liters, surplus is water, the pH of described fermention medium is 6.5,115 ℃ of sterilizations 20 minutes.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 1;
(2) seed culture: with embodiment 1;
(3) fermentation culture: the inoculum size of above-mentioned seed culture medium with 10% volume ratio is inoculated in the fermention medium (50mL fermented liquid/100mL triangular flask), and 42 ℃ leave standstill cultivation, stir once every vibration in 12 hours, cultivate 48 hours, finish fermentation.
After the fermentation ends, according to detection and the method for calculation described in the above-mentioned embodiment, detect D-ALPHA-Hydroxypropionic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-ALPHA-Hydroxypropionic acid optical purity.3 repetitions are established in this experiment altogether, and the result shows, D-ALPHA-Hydroxypropionic acid content 102.0 ± 4 grams per liters, and glucose content 30 ± 2 grams per liters, glucose acid invert ratio 97.0%, the D-ALPHA-Hydroxypropionic acid optical purity reaches 99.4%.
Embodiment 5 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-ALPHA-Hydroxypropionic acid at 30 grams per liter peanut meal concentration bottom fermentations, 50mL fermented liquid/100mL triangular flask, 42 ℃ of leavening temperatures, fermentation time 48 hours.
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: peanut meal concentration is 30 grams per liters, adds simultaneously cerelose 135 grams per liters, neutral protease 0.5 grams per liter, and calcium carbonate 70 grams per liters, surplus is water, the pH of described fermention medium is 6.5,115 ℃ of sterilizations 20 minutes.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 1;
(2) seed culture: with embodiment 1;
(3) fermentation culture: with embodiment 4.
After the fermentation ends, according to detection and the method for calculation described in the above-mentioned embodiment, detect D-ALPHA-Hydroxypropionic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-ALPHA-Hydroxypropionic acid optical purity.3 repetitions are established in this experiment altogether, and the result shows, D-ALPHA-Hydroxypropionic acid content 128 ± 5 grams per liters, and glucose content 3 ± 0.5 grams per liters, glucose acid invert ratio 96.5%, the D-ALPHA-Hydroxypropionic acid optical purity reaches 99.2%.
Embodiment 6 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-ALPHA-Hydroxypropionic acid at 40 grams per liter peanut meal concentration bottom fermentations, 50mL fermented liquid/100mL triangular flask, 42 ℃ of leavening temperatures, fermentation time 48 hours.
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: peanut meal concentration is 40 grams per liters, adds simultaneously cerelose 135 grams per liters, neutral protease 0.5 grams per liter, and calcium carbonate 70 grams per liters, surplus is water, the pH of described fermention medium is 6.5,115 ℃ of sterilizations 20 minutes.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 1;
(2) seed culture: with embodiment 1;
(3) fermentation culture: with embodiment 4.
After the fermentation ends, according to detection and the method for calculation described in the above-mentioned embodiment, detect D-ALPHA-Hydroxypropionic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-ALPHA-Hydroxypropionic acid optical purity.3 repetitions are established in this experiment altogether, and the result shows, D-ALPHA-Hydroxypropionic acid content 129 ± 5 grams per liters, and glucose content 4 ± 3 grams per liters, glucose acid invert ratio 97.8%, optical purity reaches 99.2%.
Embodiment 7 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to add different types of proteolytic enzyme fermentation production of D-lactic acid, 50mL fermented liquid/100mL triangular flask, 42 ℃ of standing for fermentation of temperature 60 hours.
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: design 3 kinds of substratum, add respectively neutral protease 0.5 grams per liter, flavor protease 0.5 grams per liter, compound protease 0.5 grams per liter adds cerelose 135 grams per liters simultaneously, peanut meal 40 grams per liters, calcium carbonate 70 grams per liters, surplus is water, and the pH of described fermention medium is 6.5,115 ℃ of sterilizations 20 minutes.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 1;
(2) seed culture: with embodiment 1;
(3) fermentation culture: the inoculum size of above-mentioned seed culture medium with 5% volume ratio is inoculated in the fermention medium (50mL fermented liquid/100mL triangular flask), and 42 ℃ leave standstill cultivation, stir once every vibration in 12 hours, cultivate 60 hours, finish fermentation.
After the fermentation ends, according to detection and the method for calculation described in the above-mentioned embodiment, detect D-ALPHA-Hydroxypropionic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-ALPHA-Hydroxypropionic acid optical purity.3 repetitions are established in this experiment altogether, and the result shows, adds flavor protease, compound protease, the substratum of neutral protease produce respectively D-ALPHA-Hydroxypropionic acid 108 ± 1 grams per liters, 112 ± 1 grams per liters, 129 ± 3 grams per liters, glucose acid invert ratio is up to 98.0%, and the D-ALPHA-Hydroxypropionic acid optical purity is up to 99.2%.
Embodiment 8 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-ALPHA-Hydroxypropionic acid at the situation bottom fermentation that does not add neutral protease, 50mL fermented liquid/100mL triangular flask, 42 ℃ of temperature, fermentation time 48 hours.
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: cerelose 135 grams per liters, peanut meal 40 grams per liters, calcium carbonate 70 grams per liters, surplus is water, the pH of described fermention medium is 6.5,115 ℃ of sterilizations 20 minutes.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 1;
(2) seed culture: with embodiment 1;
(3) fermentation culture: the inoculum size of above-mentioned seed culture medium with 10% volume ratio is inoculated in the fermention medium (50mL fermented liquid/100mL triangular flask), and 42 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate and finish fermentation in 48 hours.
After the fermentation ends, according to detection and the method for calculation described in the above-mentioned embodiment, detect D-ALPHA-Hydroxypropionic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-ALPHA-Hydroxypropionic acid optical purity.3 repetitions are established in this experiment altogether, and the result shows, D-ALPHA-Hydroxypropionic acid content 58 ± 3 grams per liters, glucose content 75 ± 3 grams per liters, glucose acid invert ratio 96.6%, the optical purity 99.3% of D-ALPHA-Hydroxypropionic acid.
Embodiment 9 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 fermentation production of D-lactic acid in 5 liters of automatic fermenters, 42 ℃ of leavening temperatures, fermentation time 66 hours.
Employed each substratum is composed as follows in the present embodiment:
(1) slant medium is with embodiment 1;
(2) seed culture medium is with embodiment 1;
(3) fermention medium: cerelose 150 grams per liters, peanut meal 40 grams per liters, neutral protease 0.5 grams per liter, calcium carbonate 70 grams per liters, surplus is water, the pH of described fermention medium is 6.5,115 ℃ of sterilizations 20 minutes.Prepare again sugared mother liquor and calcium carbonate powders, sterilized 20 minutes for 115 ℃.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 1;
(2) seed culture: with the bacterial strain of step 1 cultivation, under aseptic condition, encircle in the 100mL triangular flask that the 30mL seed culture medium is housed with inoculation articulating 2,42 ℃ leave standstill cultivation 36 hours, then the 30mL seed culture fluid is inoculated in the new seed culture medium of 300mL, 42 ℃ leave standstill cultivation 36 hours, make seed culture fluid;
(3) fermentation culture: the seed culture fluid that 300mL step (2) is made accesses under aseptic condition in 5 liters of automatic fermenters (emerging BIOTECH is protected in Shanghai) that 3.7 liters of fermention mediums are housed, 42 ℃ leave standstill cultivation, stirred once every 6 hours, each rotating speed with 100 rev/mins stirred 5 minutes, cultivated 30 hours, glucose concn is about 50 grams per liters in the fermented liquid at this moment, add glucose 280 grams, add calcium carbonate 160 gram, make that glucose concn reaches 120～130 grams per liters in the fermented liquid.Finished fermentation in 66 hours.
After the fermentation ends, according to detection and the method for calculation described in the above-mentioned embodiment, detect D-ALPHA-Hydroxypropionic acid content, glucose content in the fermented liquid, calculate transformation efficiency and D-ALPHA-Hydroxypropionic acid optical purity.The result shows: D-ALPHA-Hydroxypropionic acid content 205 ± 3 grams per liters, glucose content 1.0 ± 0.6 grams per liters, glucose acid invert ratio 98.5%, the optical purity 99.3% of D-ALPHA-Hydroxypropionic acid.
Embodiment 10 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 fermentation production of D-lactic acid in 50 liters of automatic fermenters, 42 ℃ of leavening temperatures, fermentation time 70 hours.
Employed each substratum is composed as follows in the present embodiment:
(1) slant medium is with embodiment 1;
(2) seed culture medium is with embodiment 1;
(3) fermention medium: with embodiment 9;
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 1;
(2) seed culture: with the bacterial strain of step 1 cultivation, under aseptic condition, encircle in the 100mL triangular flask that the 30mL seed culture medium is housed with inoculation articulating 2,42 ℃ leave standstill cultivation 36 hours, then the 30mL seed culture fluid is inoculated in the new seed culture medium of 300mL, 42 ℃ leave standstill cultivation 36 hours, then the 300mL seed culture fluid is inoculated in the new seed culture medium of 3000mL, 42 ℃ leave standstill cultivation 36 hours, make seed culture fluid;
(3) fermentation culture: the seed culture fluid that 3000mL step 2 is made accesses under aseptic condition in 50 liters of automatic fermenters (emerging BIOTECH is protected in Shanghai) that 37 liters of fermention mediums are housed, 42 ℃ leave standstill cultivation, stirred once every 8 hours, each rotating speed with 100 rev/mins stirred 5 minutes, cultivated 36 hours, glucose concn is about 50 grams per liters in this moment fermented liquid, adds glucose 2800 gram and 1600 and restrains calcium carbonate, makes that glucose concn reaches 120～130 grams per liters in the fermented liquid.Finished fermentation in 70 hours.
After the fermentation ends, according to detection and the method for calculation described in the above-mentioned embodiment, detect D-ALPHA-Hydroxypropionic acid content, glucose content in the fermented liquid, calculate transformation efficiency and D-ALPHA-Hydroxypropionic acid optical purity.The result shows: D-ALPHA-Hydroxypropionic acid content 203 ± 3 grams per liters, glucose content 2 ± 1 grams per liters, glucose acid invert ratio 97.8%, the optical purity 99.2% of D-ALPHA-Hydroxypropionic acid.
Claims (7)
1. method of utilizing peanut meal fermentative production high-concentration D-lactic acid, be synanthrin lactobacillus (Sporo1actobacillus inulinus) CASD CGMCC No.2185 to be carried out inoculating in the special culture media after slant culture and the seed culture ferment, obtain high-concentration D-lactic acid;
Described slant culture is with in synanthrin lactobacillus (Sporolactobacillus inulinus) the CASD CGMCC No.2185 access slant medium, 37～47 ℃ of lower cultivations 48～72 hours; Described slant culture based formulas is: glucose 10 grams per liters, and yeast powder 5 grams per liters, calcium carbonate 1 grams per liter, agar 20 grams per liters, solvent are water, and the pH of described slant medium is 6.5, and initial pH is 6～7;
Described seed culture is to access in the seed culture medium through synanthrin lactobacillus (Sporolactobacillus inulinus) the CASD CGMCC of slant culture No.2185 again, 37～47 ℃ of lower cultivations 30～36 hours; Described seed culture based formulas is: glucose 40 grams per liters, and yeast powder 5 grams per liters, calcium carbonate 3 grams per liters, surplus is water; Described seed culture medium pH is 6～7;
Described special culture media, with the fermention medium of peanut meal as organic nitrogen source, described substratum comprises: cerelose 135～150 grams per liters, peanut meal 20～40 grams per liters, neutral protease or flavor protease or compound protease 0～0.5 grams per liter, pH adjusting agent calcium carbonate 70 ± 2 grams per liters, surplus are water; The pH of described substratum is 5.5～6.5.
2. method according to claim 1, it is characterized in that: the described inoculum size that is inoculated in the special culture media is 5～10% volume ratios; Described fermentation condition is: cultivated 42～70 hours for 37～47 ℃.
3. method according to claim 2 is characterized in that: described fermentation condition is 42 ℃ and cultivated 48 hours.
4. according to claim 1 and 2 or 3 described methods, it is characterized in that: proceed to 30～36 hours in fermentation culture, glucose concn is down to about 50 grams per liters, also need add glucose, makes that glucose concn reaches 120～130 grams per liters in the fermented liquid.
5. according to claim 1 and 2 or 3 described methods, it is characterized in that: described fermenting process adopts intermittent stirring, ferments in the triangular flask, stirs once every vibration in 8～12 hours; Fermentation cylinder for fermentation stirred once in per 6～8 hours, stirred 5～10 minutes at every turn, and revolution is 100 rev/mins.
6. method according to claim 4 is characterized in that: described fermenting process adopts intermittent stirring, ferments in the triangular flask, stirs once every vibration in 8～12 hours; Fermentation cylinder for fermentation stirred once in per 6～8 hours, stirred 5～10 minutes at every turn, and revolution is 100 rev/mins.
Peanut meal in the described fermentative production high-concentration D-lactic acid of claim 1 method as the application of special culture media nitrogenous source.
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| PCT/CN2010/002182 WO2011156945A1 (en) | 2010-06-13 | 2010-12-28 | Method for fermentation production of high concentration of d-lactic acid with synchronous enzymolysis by using peanut meal and dedicated medium thereof |
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| CN101886095B (en) * | 2010-06-13 | 2013-10-30 | 天津工业生物技术研究所 | Method for producing high-concentration D-lactic acid by adopting synchronous enzymolysis and fermentation on peanut meal and special culture medium thereof |
| CN102643875B (en) * | 2012-04-24 | 2013-10-23 | 中国科学院微生物研究所 | Method for producing D-lactic acid by utilizing jerusalem artichoke hydrolysate |
| CN103184244B (en) * | 2013-03-20 | 2014-08-13 | 中国科学院微生物研究所 | Method for synchronous enzymolysis and fermentation production of D-lactic acid by using cottonseed protein |
| CN103173501A (en) * | 2013-03-20 | 2013-06-26 | 中国科学院微生物研究所 | Method for producing D-lactic acid by using sodium hydroxide as neutralizer |
| CN104419657A (en) * | 2013-09-11 | 2015-03-18 | 中国石油化工股份有限公司 | D-lactic acid producing strain with high growth rate and acid producing velocity and application thereof |
| CN108925805A (en) * | 2018-08-01 | 2018-12-04 | 广西壮族自治区农业科学院经济作物研究所 | One preparation method for cultivating peanut dried orange peel fermented beverage |
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| CN101173240A (en) * | 2007-10-18 | 2008-05-07 | 中国科学院微生物研究所 | Method for producing D-lactic acid and brood-cell lactobacillus special for the same |
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| CN101173240A (en) * | 2007-10-18 | 2008-05-07 | 中国科学院微生物研究所 | Method for producing D-lactic acid and brood-cell lactobacillus special for the same |
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