CN101886095A - 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 atoms is arranged in the lactic acid molecules, so lactic acid has opticity.L-lactic acid is levorotation, and D-lactic 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 the excellent specific property of plastic material of raw material except having with the petroleum chemicals, also having maximum characteristics is biodegradabilities, but promptly at the occurring in nature natural degradation, do not cause environmental pollution, solved the white pollution problems that is perplexed for world's environmental protection.But many unstable properties of pure poly (l-lactic acid) (PLLA), and the poly-D-lactic acid of adding suitable proportion carries out polymerization, can improve performance, has widened the range of application of poly(lactic acid).D-lactic acid is the precursor of synthetic multiple chiral material as a chiral centre, has important use in the chiral material in fields such as low-toxin farm chemicals, weedicide and makeup is synthetic.
(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-lactic acid from glucose fermentation, fermentation can be produced the D-lactic acid of 40.7 grams per liters, inversion rate of glucose 67.8% in 72 hours.Chinese patent CN200610097453.6 discloses a kind of technology 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-lactic acid that the D-concentration of lactic acid that this inventive method obtains reaches as high as 162 grams per liters.Chinese patent CN200810098908.5 has reported the technology of lactobacillus Sporolactobacillus sp.Y2-8 bacterial strain and fermentation production of D-lactic acid thereof, and the D-lactic acid content reaches 9.1%～16.5% in the fermented liquid, and D-lactic acid optical purity reaches 99.1%.Chinese patent CN200810116194.6 has reported plant lactobacillus production D-lactic acid, and total lactic acid concn is 18% in the fermented liquid, D-lactic acid optical purity 84～86%.The fermentative Production L-lactic acid concn of report can reach 190 grams per liters at present, and optical purity can reach (Chinese patent CN200710176060.9) more than 99%.Compare with L-lactic acid, fermentative Production D-concentration of lactic acid and optical purity also need further raising.
Usually adopt yeast extract as nitrogenous source because lactic fermentation is produced, improved the production cost of lactic acid to a certain extent.At 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.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 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 acid proof equipment, also need carry out under comparatively high temps, consumes big energy.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 gentle more, helps preserving 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 the equipment input and the complicated operation degree of whole fermentation production process.If the proteolysis process can be integrated mutually with fermenting process, proteolysis and lactic fermentation are carried out in fermentor tank simultaneously, just can save independent proteolysis operation, thereby save space and facility investment, help reducing D-lactic acid-producing 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 in the report that the D-lactic fermentation is produced.
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, being small-molecular peptides and amino acid, and peanut meal enzymolysis process and fermentation production of D-lactic acid carried out synchronously the proteolysis in the peanut meal.
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 a water; The pH of described substratum is 5.5～6.5.
Also can be added with the pH regulator agent in the described special culture media, be preferably lime 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-lactic 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 reduced 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 intermittently and stirs, and 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 that synanthrin lactobacillus (Sporolactobacillus inulinus) CASDCGMCC No.2185 is inserted in the slant medium, cultivates 48～72 hours down at 37～47 ℃; Described slant culture based formulas is: glucose 10 grams per liters, and yeast powder 5 grams per liters, lime 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 insert in the seed culture medium through synanthrin lactobacillus (Sporolactobacillusinulinus) CASD of slant culture CGMCC No.2185 again, cultivates 30～36 hours down at 37～47 ℃; Described seed culture based formulas is: described seed culture based formulas is: glucose 40 grams per liters, and yeast powder 5 grams per liters, lime carbonate 3 grams per liters, surplus is a water; Described seed culture medium pH is 6～7.
The high-concentration D-lactic acid and the application of peanut meal in the high-concentration D-lactic acid of enzymatic hydrolysis and fermentation production synchronously that obtain with aforesaid method also belong to protection scope of the present invention.
The present invention has realized peanut meal is produced D-lactic 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 effectively saved the place and have taken and the equipment input.
3. employing the inventive method, can significantly improve the output of D-lactic acid, compare with present report (Chinese patent CN200810116194.6 report Lactic Acid from Fermentation Broth concentration is 180 grams per liters), the D-lactic acid production 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 fermentative preparation D-lactic 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, has purity height, production efficiency height, to advantage such as the plant and instrument requirement is low and with low cost, suits large area to popularize and uses.
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 the sterilization back), flavor protease is available from letter Bioisystech Co., Ltd of Beijing Novi (20,000 unit of activity/grams, add the sterilization back), compound protease is available from letter Bioisystech Co., Ltd of Beijing Novi (20,000 unit of activity/grams, add the sterilization back).
The basic step of utilizing the synchronous enzymatic hydrolysis and fermentation of peanut meal to produce D-lactic acid of the present invention is as follows:
(1) slant culture: CASDCGMCC No.2185 inserts slant medium with synanthrin lactobacillus (Sporolactobacillus inulinus), 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:, change cultured seed liquid over to fermention medium and carry out fermentation culture with the inoculum size of 5～10% volume ratios.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 reduced 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 centrifugal 5 minutes, get supernatant liquor dilution suitable multiple, detect glucose content, D-lactic acid content and L-lactic acid content with 6,000 rev/mins speed.Glucose concn is measured with bio-sensing analyser SBA-40C (Shandong Province academy sciences Biology Research Institute).L-lactic acid and D-lactic 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.D-lactic acid standard substance are Sigma-Aldrich company product, and article No. is L0625, and L-lactic acid standard substance are Sigma-Aldrich company product, and article No. is L1750.Under this chromatographic condition, the retention time of D-lactic acid standard substance is 9.724 minutes.
D-lactic acid optical purity method of calculation: D-lactic acid optical purity (%)=(D-lactic acid concn (grams per liter) ÷ [D-lactic acid concn (grams per liter)+L-lactic acid concn (grams per liter)]) * 100%[Bioresource Technology 101 (2010) 6494～6498]
Glucose acid invert ratio method of calculation: transformation efficiency (%)=(D-lactic acid concn (grams per liter) ÷ glucose consumption amount (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 being given an example does not limit protection scope of the present invention.
Embodiment 1 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-lactic 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, lime 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, lime 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, lime carbonate 70 grams per liters, surplus is a 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: CASDCGMCC No 2185 is inoculated on the slant medium with synanthrin lactobacillus (Sporolactobacillus inulinus), cultivates 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, inoculum size with 10% volume ratio is inoculated in the new seed culture medium then, 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), is placed on 37 ℃ of static cultivations, stir once, cultivated 42 hours, finish fermentation every vibration in 12 hours.
After the fermentation ends,, detect D-lactic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.3 repetitions are established in this experiment altogether.D-lactic acid content 112 ± 3 grams per liters, glucose content 20 ± 1 grams per liters, glucose acid invert ratio 97.4%, D-lactic acid optical purity reaches 99.2%.
Embodiment 2 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-lactic 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), is placed on 42 ℃ and leaves standstill cultivation, stir once, cultivated 42 hours, finish fermentation every vibration in 12 hours.
After the fermentation ends,, detect D-lactic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.3 repetitions are established in this experiment altogether.D-lactic acid content 129 ± 2 grams per liters, glucose content 0.3 ± 0.2 grams per liter, glucose acid invert ratio 96.0%, D-lactic acid optical purity reaches 99.1%.
Embodiment 3 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-lactic 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), is placed on 47 ℃ and leaves standstill cultivation, stir once, cultivated 42 hours, finish fermentation every vibration in 12 hours.
After the fermentation ends,, detect D-lactic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.3 repetitions are established in this experiment altogether.D-lactic acid content 128 ± 2 grams per liters, glucose content 3.7 ± 0.6 grams per liters, glucose acid invert ratio 97.5%, D-lactic acid optical purity reaches 99.3%.
Embodiment 4 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-lactic 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 cerelose 135 grams per liters simultaneously, neutral protease 0.5 grams per liter, and lime carbonate 70 grams per liters, surplus is a 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,, detect D-lactic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.3 repetitions are established in this experiment altogether, and the result shows, D-lactic acid content 102.0 ± 4 grams per liters, and glucose content 30 ± 2 grams per liters, glucose acid invert ratio 97.0%, D-lactic acid optical purity reaches 99.4%.
Embodiment 5 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-lactic 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 cerelose 135 grams per liters simultaneously, neutral protease 0.5 grams per liter, and lime carbonate 70 grams per liters, surplus is a 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,, detect D-lactic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.3 repetitions are established in this experiment altogether, and the result shows, D-lactic acid content 128 ± 5 grams per liters, and glucose content 3 ± 0.5 grams per liters, glucose acid invert ratio 96.5%, D-lactic acid optical purity reaches 99.2%.
Embodiment 6 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-lactic 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 cerelose 135 grams per liters simultaneously, neutral protease 0.5 grams per liter, and lime carbonate 70 grams per liters, surplus is a 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,, detect D-lactic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.3 repetitions are established in this experiment altogether, and the result shows, D-lactic 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 neutral protease 0.5 grams per liter respectively, 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, lime carbonate 70 grams per liters, surplus is a 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,, detect D-lactic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.3 repetitions are established in this experiment altogether, and the result shows, adds flavor protease, compound protease, the substratum of neutral protease produce D-lactic acid 108 ± 1 grams per liters, 112 ± 1 grams per liters respectively, 129 ± 3 grams per liters, glucose acid invert ratio is up to 98.0%, and D-lactic acid optical purity is up to 99.2%.
Embodiment 8 utilizes synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCCNo.2185 to produce D-lactic 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, lime carbonate 70 grams per liters, surplus is a 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,, detect D-lactic acid content, glucose content in the fermentation ends secondary fermentation liquid, calculate transformation efficiency, D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.3 repetitions are established in this experiment altogether, and the result shows, D-lactic 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-lactic 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, lime carbonate 70 grams per liters, surplus is a water, the pH of described fermention medium is 6.5,115 ℃ of sterilizations 20 minutes.Prepare sugared mother liquor and calcium carbonate powders again, 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, the 30mL seed culture fluid is inoculated in the new seed culture medium of 300mL then, 42 ℃ leave standstill cultivation 36 hours, make seed culture fluid;
(3) fermentation culture: the seed culture fluid that 300mL step (2) is made inserts 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, adds glucose 280 grams, add lime 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,, detect D-lactic acid content, glucose content in the fermented liquid, calculate transformation efficiency and D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.The result shows: D-lactic 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-lactic 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, the 30mL seed culture fluid is inoculated in the new seed culture medium of 300mL then, 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 inserts 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 lime carbonate, makes that glucose concn reaches 120～130 grams per liters in the fermented liquid.Finished fermentation in 70 hours.
After the fermentation ends,, detect D-lactic acid content, glucose content in the fermented liquid, calculate transformation efficiency and D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.The result shows: D-lactic 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-lactic acid.
Claims (10)
1. utilizing the synchronous enzymatic hydrolysis and fermentation of peanut meal to produce the special culture media of high-concentration D-lactic acid, is with the fermention medium of peanut meal as organic nitrogen source.
2. special culture media according to claim 1 is characterized in that: also be added with proteolytic enzyme in the described substratum.
3. special culture media according to claim 2 is characterized in that: described substratum 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 a water; The pH of described substratum is 5.5～6.5.
4. according to claim 1 or 2 or 3 described special culture medias, it is characterized in that: also be added with the pH regulator agent in the described substratum, be preferably lime carbonate, its content in fermention medium is 70 ± 2 grams per liters.
5. method of utilizing the synchronous enzymatic hydrolysis and fermentation of peanut meal to produce high-concentration D-lactic acid, be synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC No.2185 to be inoculated in each described special culture media of right 1-4 ferment, obtain high-concentration D-lactic acid.
6. method according to claim 5 is characterized in that: described inoculum size is 5～10% volume ratios; Described fermentation condition is: cultivated 42～70 hours for 37～47 ℃; Being preferably 42 ℃ cultivated 48 hours.
7. method according to claim 5 is characterized in that: proceed to 30～36 hours in fermentation culture, glucose concn is reduced to about 50 grams per liters, also need add glucose, makes that glucose concn reaches 120～130 grams per liters in the fermented liquid.
8. method according to claim 5 is characterized in that: described fermenting process adopts intermittently and stirs, and 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.
9. according to each described method of claim 5-8, it is characterized in that: 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 that synanthrin lactobacillus (Sporolactobacillusinulinus) CASD CGMCC No.2185 is inserted in the slant medium, cultivates 48～72 hours down at 37～47 ℃; Described slant culture based formulas is: glucose 10 grams per liters, and yeast powder 5 grams per liters, lime 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 that synanthrin lactobacillus (Sporolactobacillus inulinus) the CASDCGMCC No.2185 through slant culture is inserted in the seed culture medium again, cultivates 30～36 hours down at 37～47 ℃; Described seed culture based formulas is: described seed culture based formulas is: glucose 40 grams per liters, and yeast powder 5 grams per liters, lime carbonate 3 grams per liters, surplus is a water; Described seed culture medium pH is 6～7.
10. the application of peanut meal in the high-concentration D-lactic acid of enzymatic hydrolysis and fermentation production synchronously.
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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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|---|---|---|---|---|
| WO2011156945A1 (en) * | 2010-06-13 | 2011-12-22 | 天津工业生物技术研究所 | Method for fermentation production of high concentration of d-lactic acid with synchronous enzymolysis by using peanut meal and dedicated medium thereof |
| CN102643875A (en) * | 2012-04-24 | 2012-08-22 | 中国科学院微生物研究所 | Method for producing D-lactic acid by utilizing jerusalem artichoke hydrolysate |
| 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 |
| CN103184244A (en) * | 2013-03-20 | 2013-07-03 | 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 |
| 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 |
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| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952164A (en) * | 2006-11-10 | 2007-04-25 | 南京工业大学 | Combined fermentation process for producing D-lactic acid |
| CN101173241A (en) * | 2007-10-18 | 2008-05-07 | 中国科学院微生物研究所 | Method for producing L-lactic acid and isoduicitol lactobacillus special for the same |
| 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 |
| CN101302488A (en) * | 2008-07-04 | 2008-11-12 | 中国科学院微生物研究所 | Method for producing lactic acid and special plant bacterium lacticum |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2010
- 2010-06-13 CN CN2010102081486A patent/CN101886095B/en active Active
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952164A (en) * | 2006-11-10 | 2007-04-25 | 南京工业大学 | Combined fermentation process for producing D-lactic acid |
| CN101173241A (en) * | 2007-10-18 | 2008-05-07 | 中国科学院微生物研究所 | Method for producing L-lactic acid and isoduicitol lactobacillus special for the same |
| 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 |
| CN101302488A (en) * | 2008-07-04 | 2008-11-12 | 中国科学院微生物研究所 | Method for producing lactic acid and special plant bacterium lacticum |
Non-Patent Citations (3)
| Title |
|---|
| 《中国生物工程学会2006年学术年会暨全国生物反应器学术研讨会论文摘要集》 20060801 李政 等 用添加玉米浆的水解小麦胚芽蛋白生产L-乳酸 第73页第1-3行 1-10 , * |
| 《生物加工过程》 20040831 丁子建 等 芽胞乳杆菌发酵葡萄糖制备D（-）-乳酸的研究 30-36 1-10 第2卷, 第3期 * |
| 《过程工程学报》 20060228 丁绍峰 等 豆粕水解液为氮源细菌厌氧流加发酵生产L-乳酸 77-81 1-10 第6卷, 第1期 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011156945A1 (en) * | 2010-06-13 | 2011-12-22 | 天津工业生物技术研究所 | Method for fermentation production of high concentration of d-lactic acid with synchronous enzymolysis by using peanut meal and dedicated medium thereof |
| CN102643875A (en) * | 2012-04-24 | 2012-08-22 | 中国科学院微生物研究所 | Method for producing D-lactic acid by utilizing jerusalem artichoke hydrolysate |
| 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 |
| CN103184244A (en) * | 2013-03-20 | 2013-07-03 | 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 |
| 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 |
| 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 |
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