CN104561162A - Method for preparing lysine by fermentation - Google Patents
Method for preparing lysine by fermentation Download PDFInfo
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- CN104561162A CN104561162A CN201310503252.1A CN201310503252A CN104561162A CN 104561162 A CN104561162 A CN 104561162A CN 201310503252 A CN201310503252 A CN 201310503252A CN 104561162 A CN104561162 A CN 104561162A
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Abstract
The invention discloses a method for preparing lysine by fermentation. The method comprises the following steps: grafting a lysine fermentation strain into a lysine fermentation culture medium, and performing fermentation under the condition of feeding a carbon source and a nitrogen source to generate lysine. The fermentation process comprises a strain growth stage and a strain acid-production stage, wherein the fermentation temperature in the strain growth stage is 30-38 DEG C; the fermentation temperature in the strain acid-production stage is 32-42 DEG C; and the fermentation temperature in the strain growth stage is lower than that in the strain acid-production stage. By virtue of adopting the technical scheme for producing lysine, the lysine concentration and the fermentation conversion rate are effectively increased, the fermentation cycle is shortened, and the energy consumption is lowered.
Description
Technical field
The present invention relates to a kind of method of preparing lysine through fermentation, particularly, relate to a kind of method of preparing lysine through fermentation of the temperature of grading-controlling fermentation during the fermentation.
Background technology
Methionin is also known as 2,6-diaminocaproic acid, and molecular formula is C
6h
14o
2n
2, be white or the free-pouring crystalline powder of near-white, soluble in water, almost odorless.Usual stable in properties, easily lumps under high temperature, and under alkaline condition, heating is easily decomposed.One of Methionin or eight kinds of primary amino acids of needed by human, can promote human development, strengthen immunologic function, and be improved the effect of central nervous tissue function.Methionin is divided into L-type (left-handed) and D type (dextrorotation), only has L-type energy bioavailable.At present, Methionin has become world's second largest amino acids industry, and mainly as nutrition-fortifying agent, tasty agents, sweeting agent, amino acid derivative, amino acid salts etc. are for feedstuff industry, food service industry and pharmaceutical industries.Wherein, the 1B of 90% for fodder industry, 10% for food and medicine industry.
Before nineteen fifty, because Bio-synthetic pathway of lysine is not fully illustrated, the production of Methionin adopts extraction process, chemical synthesis and enzyme process mostly.Nineteen sixty Japan wood under wish that the scholars such as youth obtain a strain auxotropic variant with uviolizing Corynebacterium glutamicum, from then on industrially bring into use fermentative Production Methionin.Subsequently, other produce method of Methionin be fermented gradually method substitute.At present, the whole world more than 90% enterprise adopts fermentative Production Methionin.
CN102533891A discloses a kind of production method of Methionin, the method is by fermenting lysine bacterial classification access fermenting lysine substratum, under stream adds carbon source and stream adds the condition of nitrogenous source, carry out fermentation culture, in fermentation culture after 15 hours, in fermented liquid, stream adds the nutritive salt such as Repone K, magnesium sulfate and manganous sulfate.CN102533890A discloses a kind of production method of Methionin, the method is by fermenting lysine bacterial classification access fermenting lysine substratum, fermentation culture is carried out under stream adds carbon source and stream adds the condition of nitrogenous source, fermentation culture after 30 hours in fermentation culture 48 hours, in fermented liquid, add fermenting lysine bacterial classification.CN1928104 discloses a kind of Secondary Fermentation technology of lysine fermentation liquor, this technology is by being adjusted to suitable pH by the lysine fermentation liquor of fermentation ends, then cultured yeast is added wherein, carry out Secondary Fermentation, result reduces residual sugar, improve fermentation broth viscosity, improve Methionin grain size number and color and luster simultaneously.CN101104863 discloses a kind of fermenting lysine method, the method comprises the steps: bacterial classification after large seed bottle cultivates 9-11 hour, to move on to secondary seed tank from slant tube, amount of planting is for 0.2-0.3%, and culture temperature is 38-40 DEG C, and incubation time is 15-17 hour; Then move into three grades of seeding tanks, kind amount 5-6%, cultivate to move in fermentor tank after 9-11 hour and ferment.
But the method for above-mentioned preparing lysine through fermentation, produce lysine concentration and fermentation conversion rate all lower, and energy consumption is higher.
Summary of the invention
The object of the invention is to overcome adopt prior art to produce lysine concentration and fermentation conversion rate all lower, and the shortcoming that energy consumption is high, provides a kind of method of preparing lysine through fermentation, produces lysine concentration and fermentation conversion rate, and reduce energy consumption to improve.
The present inventor finds, adopt prior art preparing lysine through fermentation, produce lysine concentration and fermentation conversion rate all lower, and the high reason of energy consumption is, prior art is all carry out at that same temperature in whole fermenting process, or is controlled in certain scope by leavening temperature, or controls in the level higher than the fermentation later stage in the temperature of earlier fermentation, such as, classical fermenting lysine technique.
The present inventor is but surprised to find that, the temperature of the growth phase in earlier stage of fermented bacterium in lysine fermentation process is first controlled in lower level, and then at the product acid phase in later stage, temperature is controlled in higher level, although fermented bacterium can not be made like this to carry out raised growth at earlier fermentation, but the discovery that the present inventor is surprised, the fermented bacterium under this state produces sour after date its fermentation level and but obtains significant raising entering.This is mainly: on the one hand, the quick growth of bacterial classification obtains suppression to a certain extent, and can grow on normal level, make it enter the sour after date of product to be more prone to produce acid, thus its nutritive substance for own growth is reduced, nutritive substance for producing acid is improved, therefore, it is possible to improve glucose acid invert ratio; On the other hand, after entering large volume production acid phase, suitably improve leavening temperature, under the prerequisite ensureing dissolved oxygen amount, the enzyme system be more conducive to for producing acid plays a role, thus makes to produce the increase of acid amount.
Based on above discovery, the invention provides a kind of method of preparing lysine through fermentation, the method comprises, by in fermenting lysine bacterial classification access fermenting lysine substratum, under stream adds carbon source and stream adds the condition of nitrogenous source, carry out fermenting to generate Methionin, described fermenting process comprises the growth stage and bacterial classification produces acid phase, be 30-38 DEG C at the leavening temperature in growth stage, producing the leavening temperature of acid phase at bacterial classification is 32-42 DEG C, and at the leavening temperature in growth stage lower than the leavening temperature producing acid phase at bacterial classification.
Preferably, at the leavening temperature in growth stage 0.5-5 DEG C lower than the leavening temperature producing acid phase at bacterial classification.
Preferably, the method also comprises, and adds the step of alkaline matter in the process of described fermentation in fermention medium.
Adopt technique scheme to produce Methionin, effectively improve and produce lysine concentration and fermentation conversion rate, and reduce energy consumption.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
The invention provides a kind of method of preparing lysine through fermentation, the method comprises, a kind of method of preparing lysine through fermentation, the method comprises, by in fermenting lysine bacterial classification access fermenting lysine substratum, under stream adds carbon source and stream adds the condition of nitrogenous source, carry out fermenting to generate Methionin, described fermenting process comprises the growth stage and bacterial classification produces acid phase, be 30-38 DEG C at the leavening temperature in growth stage, the leavening temperature producing acid phase at bacterial classification is 32-42 DEG C, and produce the leavening temperature of acid phase at the leavening temperature in growth stage lower than at bacterial classification.
In the present invention, term " growth stage " refers to the fermentation time section that will start in fermenting lysine strain inoculation to fermention medium when reaching 5g/L/h to the generating rate of Methionin, and " bacterial classification product acid phase " refers to that the generating rate of Methionin is greater than the fermentation time section of 5g/L/h when fermentation ends.According to the practical experience of the present inventor, when constantly little by starting to fermentation 12 in fermenting lysine strain inoculation to fermention medium, the generating rate of Methionin can reach 5g/L/h, therefore, in the operating process of reality, using this time point as the growth stage and the dividing point of acid phase can also be produced, the sublevel segmentation that the temperature in fermenting process is carried out as above is controlled.
According to the present invention, the temperature in growth stage is first controlled at 30-38 DEG C, again temperature is controlled at 32-42 DEG C when fermented bacterium enters to produce after acid phase, ensure that the leavening temperature in the growth stage produces the leavening temperature of acid phase lower than bacterial classification simultaneously.Like this can the quick growth of stopping fermentation bacterial classification to a certain extent in the growth stage, make it be more prone to the development of product acid phase, thus fermented bacterium is reduced for the nutritive substance of own growth, glucose acid invert ratio is improved.When fermented bacterium enters product acid phase, because bacterial classification has entered the stationary phase of growth, start to synthesize tunning in a large number, fermented bacterium is also difficult to again to the future development of its raised growth, the various enzymes that now raising leavening temperature is conducive in lysine synthetic pathway play a role, thus, can fermentation level be improved.
Preferably, the leavening temperature when the growth stage is 32-36 DEG C, when the leavening temperature of bacterial classification product acid phase is 35-39 DEG C, is more conducive to the raising of fermentation level, a step-down less energy-consumption of going forward side by side.
The discovery that the present inventor is surprised, as the leavening temperature 0.5-5 DEG C lower than the leavening temperature of bacterial classification product acid phase in growth stage, during preferred 2-3.5 DEG C, during the fermentation when leavening temperature changes, fermented bacterium is less by the impact of leavening temperature change, thus can adapt to the environment that bacterial classification produces acid phase faster.
According to the present invention, any means well known in the art can be adopted to the method that the temperature in fermenting process controls, such as, can be controlled by heated by electrodes and/or cooling water circulation method.
In the present invention, with after inoculating and stream adds carbon source and stream adds nutrient solution in the prefermentor of nitrogenous source for benchmark, the inoculum size of fermenting lysine bacterial classification is 5-20 volume %, is preferably 10-15 volume %.
Wherein, the amount that stream adds carbon source makes the concentration of reducing sugar in nutrient solution control at 2-12 grams per liter, preferred 5-10 grams per liter, and the amount that stream adds nitrogenous source makes the concentration of nitrogen in nutrient solution control at 0.35-1.2 grams per liter, preferred 0.5-1 grams per liter." amount that stream adds carbon source makes the concentration of reducing sugar in nutrient solution control at 2-12 grams per liter; preferably 5-10 grams per liter; the amount that stream adds nitrogenous source makes the concentration of nitrogen in nutrient solution control at 0.35-1.2 grams per liter; preferably 0.5-1 grams per liter " refers to that adding by control flow check the speed that carbon source and stream adds nitrogenous source makes the concentration of reducing sugar in nutrient solution in whole fermentation culture process maintain 2-12 grams per liter herein, preferred 5-10 grams per liter, the concentration of nitrogen in nutrient solution is made to maintain 0.35-1.2 grams per liter, preferred 0.5-1 grams per liter.
According to the present invention, the carbon source that stream adds during the fermentation can for the carbon source added for fermenting lysine stream conventional in this area, and such as, described carbon source can be the saccharified liquid of starch materials and/or the saccharified liquid of cellulose family raw material.Wherein, described starch materials can be selected from one or more in corn, wheat, Chinese sorghum, paddy rice; Described cellulose family raw material can be selected from maize straw, wheat stalk, rice straw, sugarcane stalk, jowar stalk, cotton stalk, beanstalk and Herba Eichhorniae one or more.
In the present invention, described carbon source is preferably starchy material saccharification clear liquid.Starchy material saccharification clear liquid both can adopt dry method sugar refining technology to prepare, and wet method sugar refining technology also can be adopted to prepare.Simple from technique, facility investment is few, the lower aspect of production cost is considered, prepares preferably by dry method sugar refining technology.Dry method sugar refining technology refer to without immersion directly carry out fragmentation and enzymolysis.
Be prepared as example with the saccharification clear liquid of starchy material, described dry method sugar refining technology can comprise: by raw material pulverizing, by pulverize after product size mixing, and add amylase to starch carry out first time be hydrolyzed; Solid-liquid separation is carried out to first time hydrolysate, and add in the liquid phase component obtained saccharifying enzyme carry out second time hydrolysis, obtain starchy material saccharification clear liquid.Preferably, pulverize the percent of pass making starchy material cross 30 mesh sieves and be greater than 75%, the percent of pass more preferably crossing 30 mesh sieves is 100%.The method of sizing mixing is well known to those skilled in the art, but preferably, and the method for sizing mixing can comprise the product after being pulverized by starchy material and be added to the water and mix, and the add-on of water makes the degree Beaume of the slurries obtained can be 9-17B é °.Term " degree Beaume " is a kind of method representing strength of solution, is the number of degrees obtained by Beaumé scale detection solution.
According to the present invention, in first time hydrolysis, the dry weight basis of the product after pulverizing with every gram, diastatic consumption can be 10-30 enzyme activity unit, the temperature of enzymolysis can be 88-110 DEG C, and the time of enzymolysis can be 90-120 minute, and the pH value of enzymolysis can be 5.5-6.5.There is no particular limitation for the condition of solid-liquid separation, and preferably, the condition of solid-liquid separation makes the solid content in the liquid phase component obtained be 19-22 % by weight, is more preferably 20-21 % by weight.
According to the present invention, in second time hydrolysis, in every gram of liquid phase component, the consumption of saccharifying enzyme can be 50-100 enzyme activity unit, and the temperature of enzymolysis can be 55-65 DEG C, and the time of enzymolysis can be 50-80 hour, and the pH value of enzymolysis can be 4-5.
Enzyme activity unit of the present invention is defined as: pH value be 6.0, under temperature is the condition of 70 DEG C, the enzyme amount of 1 milligram of Starch Conversion needed for reducing sugar is an enzyme activity unit by 1 minute.
Amylase refers to can the general name of class of enzymes of starch-splitting glycosidic link, and described amylase generally comprises α-amylase, beta-amylase.
α-amylase is also known as starch Isosorbide-5-Nitrae-dextrinase, and it can cut the α-Isosorbide-5-Nitrae-glycosidic link of starch chain inside at random, brokenly, is maltose, the oligosaccharides containing 6 glucose units and the oligosaccharides with side chain by Starch Hydrolysis.The microorganism producing this enzyme mainly contains Bacillus subtilus, aspergillus niger, aspergillus oryzae and head mold.
Beta-amylase, also known as starch Isosorbide-5-Nitrae-maltoside enzyme, can cut Isosorbide-5-Nitrae-glycosidic link from starch molecule non reducing end, generate maltose.The product that this enzyme acts on starch is maltose and limit dextrin.This enzyme produces primarily of aspergillus, head mold and endomyces.
According to the present invention, preferably use α-amylase.
According to the present invention, saccharifying enzyme is preferably α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme.
According to the present invention, starchy material can be the various raw material containing starch that may be used for enzymolysis, preparing lysine through fermentation well known in the art, such as, can be selected from one or more in corn, potato class (as cassava) and wheat.
In the present invention, the kind of nitrogenous source is conventionally known to one of skill in the art, such as, can be one or more in urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, liquefied ammonia and ammoniacal liquor.Wherein, when nitrogenous source is ammonium salt, in nutrient solution, the concentration of nitrogen represents with the concentration of ammonium radical ion, and the concentration of nitrogen controls at 0.35-1.2 grams per liter, then the concentration of ammonium radical ion controls at 0.5-1.5 grams per liter.
The present inventor finds in an experiment, and it is good that the Continuous Flow of described Carbon and nitrogen sources adds the ferment effect added than intermittent flow, and therefore, the stream in the present invention adds preferably Continuous Flow and adds.
It will be understood by those skilled in the art that the fermentation culture of Methionin should be carried out in atmosphere.In order to effectively utilize the production capacity of fermentor tank, after inoculation and the volume that stream adds the nutrient solution that carbon source and stream add in the prefermentor of nitrogenous source is preferably the 40-60% of fermenter volume, carbon source is added and stream adds nitrogenous source along with stream, the volume of the nutrient solution in fermentor tank increases gradually, in order to ensure the air capacity in fermentor tank, be preferably stream to add carbon source and stream and add nitrogenous source to blowing during the 70-80% of fermenter volume, in order to ensure the bacterial classification quantity in blowing secondary fermentation tank, do not affect the fermentation culture in blowing secondary fermentation tank, blowing volume is preferably the 4-10% of nutrient solution volume in blowing prefermentor.
Method of the present invention is also included in the step adding alkaline matter in the process of described fermentation in fermention medium, and preferably, adding of described alkaline matter makes the pH value of fermention medium be 6.5-7.5.
Wherein, the kind of described alkaline matter has no particular limits, as long as the pH value of fermention medium can be regulated and do not affect the physiological activity of fermented bacterium, preferably, described alkaline matter is one or more in liquefied ammonia, ammoniacal liquor, ammonium sulfate, sodium hydroxide, potassium hydroxide, lithium hydroxide, hydrated barta, aluminium hydroxide, manganous hydroxide, zinc hydroxide, sodium carbonate, salt of wormwood, Quilonum Retard and ammoniacal liquor.
Preferably, in order to control the pH value of fermention medium preferably, described alkaline matter adds as a solution, wherein, the concentration of the solution containing described alkaline matter is not particularly limited, as long as the pH value of substratum effectively can be regulated, can not to damage fermented bacterium again and can not diluted medium largely simultaneously.Preferably, the concentration of described solution neutral and alkali material is 0.1-30 % by weight.
Wherein, also have no particular limits the feed postition of described alkaline matter, preferably, for the ease of controlling the pH value of fermention medium, the mode that described alkaline matter adds with Continuous Flow adds.
In the present invention, to the condition of fermentation culture without particular requirement, the condition that this area is conventional can be adopted, be preferably: air flow is 0.5-1.5 volume: (volume minute), and pressure is 0.05-0.1MPa.
In the present invention, to the kind of fermenting lysine bacterial classification without particular requirement, the bacterial classification that this area is conventional can be adopted, be preferably selected from intestinal bacteria, Corynebacterium glutamicum, Brevibacterium lactofermentus and brevibacterium flavum one or more; Preferred, described fermentation strain be selected from intestinal bacteria, Corynebacterium glutamicum, Brevibacterium lactofermentus and brevibacterium flavum two or more.
In the present invention, after preferred fermentation culture 45-50 hour, put tank.Tank of putting in the present invention refers to and is all released from fermentor tank by the nutrient solution in fermentor tank, namely stops fermentation.
It will be understood by those skilled in the art that to obtain the higher lysine product of purity, the inventive method also comprise from blowing or put tank solution extract Methionin.For extracting the method for Methionin without particular requirement, the various methods that this area is conventional can be adopted, such as, continuous ionic is adopted to exchange separating and extracting method, to blowing or put tank solution in add a large amount of vitriol oils and adjust pH to 2.0-3.0 to carry out acidifying, through metallic membrane or ceramic membrane filter removing thalline after acidifying, obtain Methionin membrane filtration liquid and Methionin clear liquid, or the lysine fermentation liquor after acidifying is obtained Methionin clear liquid through flocculation filtration except after thalline, exchange except the Methionin clear liquid after thalline adopts strongly acidic cation-exchange to carry out absorption, the saturated rear weak ammonia of resin absorption carries out wash-out, the Methionin eluted is through concentrated, salt acid for adjusting pH value, crystallization, solid-liquid separation, dry, obtain lysine hydrochloride finished product, or blowing or put tank solution in add acid, make lysine fermentation liquor acidifying, after filtration or the method removing thalline such as centrifugal.Calcium hydroxide adjust ph is added to 8.0-11.5 in Methionin clear liquid after removing thalline, the impurity such as the salt in Methionin clear liquid, colloid are made to generate insolubles, lysine solution is obtained after solid-liquid separation, content lysine solution being concentrated into Methionin in every milliliter of lysine solution is 0.6-0.8g, highly purified lysine solution can be obtained again through filtration, then through concentrated, salt acid for adjusting pH value, crystallization, solid-liquid separation, oven dry, lysine hydrochloride finished product is obtained.
In the present invention, the composition of nutrient solution is without particular requirement, can adopt the fermenting lysine nutrient solution that this area is conventional, such as, nutrient solution can contain starchy material saccharification clear liquid, molasses, corn steep liquor, ammonium sulfate, dipotassium hydrogen phosphate, magnesium sulfate, Threonine, methionine(Met) and L-glutamic acid.According to the present invention, in described nutrient solution, the content of each component can in very large range change, under preferable case, in often liter of nutrient solution, the content of starchy material saccharification clear liquid is 40-60 gram, the content of molasses is 15-30 gram, the content of trimethyl-glycine is 2-5 gram, the content of corn steep liquor is 20-70 gram, and the content of ammonium sulfate can be 0-40 gram, and the content of dipotassium hydrogen phosphate can be 0-1.5 gram, the content of magnesium sulfate is 2-5 gram, the content of Threonine is 0.1-0.3 gram, and the content of methionine(Met) is 0.1-0.3 gram, and the content of L-glutamic acid is 0-0.4 gram.
It will be understood by those skilled in the art that fermenting lysine bacterial classification is before being seeded in fermentation culture, adopt ordinary method by fermenting lysine bacterial classification through seed tank culture, and then bacterial classification is accessed in fermentation culture.Can by sampling sediments microscope inspection, OD(optical density in the cultivation degree of seed tank culture) pH-value determination pH observes product Methionin microbial growth, when being observed by aforesaid method, thalli morphology is normal, mensuration OD value stops cultivating when reaching more than 0.75, and then by the seed liquor of seeding tank access fermentation culture.Seed tank culture can adopt first class seed pot cultivation that secondary seed tank also can be adopted to cultivate, and first class seed pot is cultivated in a seeding tank, cultivate required cultivation degree by fermenting lysine bacterial classification always; Secondary seed tank proceeds to another seeding tank again after cultivating and namely first fermenting lysine bacterial classification being cultivated for some time in a seeding tank continues to cultivate, and cultivates required cultivation degree.Secondary seed tank is cultivated and is not particularly limited at the incubation time of each seeding tank, as long as finally can cultivate required cultivation degree.In order to easy to operate, seed tank culture of the present invention preferably adopts first class seed pot to cultivate.
Because fermenting lysine bacterial classification individuality is small, quantity is not easily added up, and the OD value of seed liquor is the optical density(OD) that tested seed liquor absorbs, the quantity of Methionin microorganism in seed liquor can be reflected, therefore, in this area, the general OD value that all adopts represents the quantity of Methionin microorganism in seed liquor, and the present invention also continues to use the employing OD value of this area to represent the custom of the quantity of Methionin microorganism in seed liquor.And in the present invention, OD value 722N visible spectrophotometer measures.
In seed tank culture base, the content of each component can in very large range change, under preferable case, in often liter of substratum, the content of starchy material saccharification clear liquid is 30-50 gram, the content of trimethyl-glycine can be 2-8 gram, the content of corn steep liquor (dry weight is 20-50 % by weight) is 30-80 gram, the content of potassium primary phosphate is 0.5-5 gram, the content of para-amino benzoic acid is 2-6 gram, the content of magnesium sulfate is 0.4-5 gram, and the content of ammonium sulfate is 10-18 gram, and the content of Threonine is 0.1-1 gram, the content of methionine(Met) is 0-0.3 gram, and the content of L-glutamic acid is 0-0.4 gram.
Embodiment
The present invention is further illustrated for following embodiment, but therefore do not limit the present invention.
In following embodiment and comparative example:
OD pH-value determination pH: the fermented liquid of sampling is carried out 26 times of dilutions, adopts 722N visible spectrophotometer, under wavelength 562 nanometer visible ray, measures light absorption value.
The concentration of reducing sugar in fermented liquid is measured according to the method for GB/T5009.7-2008.
The concentration of ammonia nitrogen in fermented liquid is measured according to the method for GB/T3595-2000.
The lysine concentration (in lysine hydrochloride) in nutrient solution is detected according to the method for GB10794-2009.
Single tank is for acid amount=(put the lysine concentration of tank × put tank volume+middle blowing lysine concentration × middle blowing volume).
Transformation efficiency (%)=mono-tank is for weight × 100% of acid amount/total reducing sugar, and wherein the weight of total reducing sugar comprises seeding tank sugar weight and fermentor tank sugar weight.
Preparation example 1
This preparation example is for illustration of the preparation of Methionin seed and fermention medium.
(1) pulverized by corn particle by mechanical workout by 100 parts by weight Corn of results, the percent of pass making Semen Maydis powder cross 30 mesh sieves is 80%.
(2) product after pulverizing is added water and sizes mixing to 12B é °, relative to the dry weight of every gram of crushed products, add the amylase (Novozymes Company, α-amylase) of 20 enzyme activity units, 100 ± 2.0 DEG C, pH be the condition of 6.0 ± 0.1 under enzymolysis 100 minutes, obtain enzymolysis product.Wherein, by enzymolysis product by carrying out press filtration with fluid pressure type plate-and-frame filter press, isolate enzymolysis clear liquid (solid content is 20 % by weight); Add the saccharifying enzyme (α-Isosorbide-5-Nitrae-D-Glucose lytic enzyme, Novozymes Company) of 80 enzyme activity units afterwards, 60 ± 1.0 DEG C, pH be the condition of 4.5 ± 0.1 under enzymolysis 72 hours, obtain starchy material saccharification clear liquid.
(3) the starchy material saccharification clear liquid preparation seed tank culture base using step (2) to obtain, specifically consist of: the substratum relative to often liter, the content of starchy material saccharification clear liquid is 40 grams, the content of corn steep liquor (dry weight is 35 % by weight) is 50 grams, and the content of ammonium sulfate is 14g, and the content of potassium primary phosphate is 3.0 grams, the content of magnesium sulfate is 2 grams, the content of Threonine is 0.8 gram, and the content of para-amino benzoic acid is 4mL, and the content of trimethyl-glycine is 5mL.Substratum is heated to 121 DEG C of sterilizings, maintains and be cooled to 37 DEG C after 20 minutes and keep constant.
(4) the starchy material saccharification clear liquid preparation fermentation culture using step (2) to obtain, specifically consist of: the nutrient solution relative to often liter, the content of starchy material saccharification clear liquid is 40 grams, the content of corn steep liquor (dry weight is 35 % by weight) is 50 grams, the content of Threonine is 0.2 gram, and the content of methionine(Met) is 0.2 gram, and the content of trimethyl-glycine is 3.6 grams, the content of molasses (Xinjiang, the place of production) is 20 grams, and the content of magnesium sulfate is 3 grams.Nutrient solution is heated to 121 DEG C of sterilizings and is cooled to 37 DEG C after 30 minutes and keeps constant, regulates pH to 6.9, obtain fermention medium with ammoniacal liquor.
Embodiment 1
The present embodiment is for illustration of the method for preparing lysine through fermentation provided by the invention.
Open the stirring in seed culture tank, adjustment tank pressure is 0.1MPa, passes into sterile air according to ventilation and substratum 1:1 volume ratio, regulates pH to 6.8 and keep constant with ammoniacal liquor.Cultivate after strain Escherichia coli (bacterial strain ZL01 original seed takes from Chinese industrial Microbiological Culture Collection administrative center) activation and propagation in access seeding tank, in culturing process, it is 3-10g/L that stream adds starchy material saccharification clear liquid control concentration of reduced sugar, the pH value that Feeding ammonia water controls solution is 6.8, and sampled microscopies every 120 minutes and measure OD value, stop cultivating when microscopy thalli morphology is normal and OD value reaches 0.8, obtain ripe seed liquor A.
Seed liquor A is joined in the fermention medium in preparation example 1 and start fermentation, wherein, in every milliliter of Preliminary fermentation substratum, the inoculum size of fermented bacterium is after 10 volume %(inoculate and stream adds the volume that carbon source and stream adds the substratum in the prefermentor of nitrogenous source is 50% of fermenter volume), fermentation condition comprises: pass into sterile air according to ventilation and culture volume 1:1, tank pressure is 0.07MPa, it is 8.0-10.0g/l that stream adds concentration of reduced sugar in starchy material saccharification clear liquid control fermented soln, it is 0.8-1.0g/l that stream adds ammonia nitrogen concentration in ammoniumsulphate soln control fermented soln, it is 6.8 ± 0.1 that stream hydro-oxidation sodium solution controls pH value of solution.Be mainly the growth stage to fermentation during 12 hours after inoculation, it is 35 DEG C that leavening temperature controls; Ferment 12 little up to fermentation ends for mainly to produce acid phase, leavening temperature control be 37 DEG C.
When liquid level starts discharge higher than during fermentor tank 75%, about 5% of each discharge fermentating liquid volume, loads in triangular flask and refrigerates in 4 DEG C after sealing.Fermentation culture put tank after 48 hours, measured the lysine concentration of fermentation termination lysine concentration and middle discharge, and calculating product acid concentration and glucose acid invert ratio are in table 1.
Embodiment 2
The present embodiment is for illustration of the method for preparing lysine through fermentation provided by the invention.
Open the stirring in seed culture tank, adjustment tank pressure is 0.1MPa, passes into sterile air according to ventilation and substratum 1:1 volume ratio, regulates pH to 6.8 and keep constant with ammoniacal liquor.Cultivate after species Corynebacterium glutamicum (bacterial strain BTC01 original seed takes from Chinese industrial Microbiological Culture Collection administrative center) activation and propagation in access seeding tank, culturing process, it is 3-10g/L that stream adds starchy material saccharification clear liquid control concentration of reduced sugar, the pH value that Feeding ammonia water controls solution is 6.8, and sampled microscopies every 120 minutes and measure OD value, stop cultivating when microscopy thalli morphology is normal and OD value reaches 0.8, obtain ripe seed liquor B.
Seed liquor B is joined in the fermention medium in preparation example 1 and start fermentation, wherein, in every milliliter of Preliminary fermentation substratum, the inoculum size of fermented bacterium is after 12 volume %(inoculate and stream adds the volume that carbon source and stream adds the substratum in the prefermentor of nitrogenous source is 60% of fermenter volume), fermentation condition comprises: pass into sterile air according to ventilation and culture volume 0.5:1, tank pressure is 0.1MPa, it is 7.0-9.0g/l that stream adds concentration of reduced sugar in starchy material saccharification clear liquid control fermented soln, it is 0.6-0.8g/l that stream adds ammonia nitrogen concentration in ammonium chloride solution and ammoniacal liquor control fermented soln, pH is 6.5 ± 0.1.Be mainly the growth stage to fermentation during 12 hours after inoculation, it is 32 DEG C that leavening temperature controls; Ferment 12 little up to fermentation ends for mainly to produce acid phase, leavening temperature control be 35.5 DEG C.
When liquid level starts discharge higher than during fermentor tank 80%, about 10% of each discharge fermentating liquid volume, loads in triangular flask and refrigerates in 4 DEG C after sealing.Fermentation culture put tank after 48 hours, measured the lysine concentration of fermentation termination lysine concentration and middle discharge, and calculating product acid concentration and glucose acid invert ratio are in table 1.
Embodiment 3
The present embodiment is for illustration of the method for preparing lysine through fermentation provided by the invention.
Open the stirring in seed culture tank, adjustment tank pressure is 0.1MPa, passes into sterile air according to ventilation and substratum 1:1 volume ratio, regulates pH to 6.8 and keep constant with ammoniacal liquor.Cultivate after brevibacterium flavum bacterial classification (bacterial strain BTC02 original seed takes from Chinese industrial Microbiological Culture Collection administrative center) activation and propagation in access seeding tank, culturing process, it is 3-10g/L that stream adds starchy material saccharification clear liquid control concentration of reduced sugar, the pH value that Feeding ammonia water controls solution is 6.8, and sampled microscopies every 120 minutes and measure OD value, stop cultivating when microscopy thalli morphology is normal and OD value reaches 0.8, obtain ripe seed liquor C.
Seed liquor C is joined in the fermention medium in preparation example 1 and start fermentation, wherein, in every milliliter of fermention medium, the inoculum size of fermented bacterium is after 15 volume %(inoculate and stream adds the volume that carbon source and stream adds the substratum in the prefermentor of nitrogenous source is 40% of fermenter volume), fermentation condition comprises: pass into sterile air according to ventilation and culture volume 1.5:1, tank pressure is 0.05MPa, it is 5.0-7.0g/l that stream adds concentration of reduced sugar in starchy material saccharification clear liquid control fermented soln, it is 0.5-0.7g/l that stream adds ammonia nitrogen concentration in ammonium phosphate solution control fermented soln, it is 7.5 ± 0.1 that stream adds sodium carbonate solution control pH value of solution.Be mainly the growth stage to fermentation during 12 hours after inoculation, it is 36 DEG C that leavening temperature controls; Ferment 12 little up to fermentation ends for mainly to produce acid phase, leavening temperature control be 39 DEG C.
When liquid level starts discharge higher than during fermentor tank 70%, about 5% of each discharge fermentating liquid volume, loads in triangular flask and refrigerates in 4 DEG C after sealing.Fermentation culture put tank after 48 hours, measured the lysine concentration of fermentation termination lysine concentration and middle discharge, and calculating product acid concentration and glucose acid invert ratio are in table 1.
Embodiment 4
The present embodiment is for illustration of the method for preparing lysine through fermentation provided by the invention.
Adopt the method for embodiment 3 to carry out the fermentation of Methionin, the leavening temperature unlike, growth stage is the leavening temperature that 31 DEG C of bacterial classifications produce acid phases is 34 DEG C.Produce acid concentration and fermentation turnover ratio as shown in table 1.
Embodiment 5
The present embodiment is for illustration of the method for preparing lysine through fermentation provided by the invention.
The method of embodiment 3 is adopted to carry out the fermentation of Methionin, unlike, the leavening temperature in growth stage is 38 DEG C, and the leavening temperature producing acid phase is 42 DEG C.Produce acid concentration and fermentation turnover ratio as shown in table 1.
Embodiment 6
The present embodiment is for illustration of the method for preparing lysine through fermentation provided by the invention.
The method of embodiment 3 is adopted to carry out the fermentation of Methionin, unlike, the leavening temperature in growth stage is 30 DEG C, and the leavening temperature producing acid phase is 35 DEG C.Produce acid concentration and fermentation turnover ratio as shown in table 1.
Embodiment 7
The present embodiment is for illustration of the method for preparing lysine through fermentation provided by the invention.
The method of embodiment 3 is adopted to carry out the fermentation of Methionin, unlike, after inoculation, extremely 18 hours periods of fermentation are mainly the growth stage, and it is 35 DEG C that leavening temperature controls; Ferment 18 little up to fermentation ends for mainly to produce acid phase, leavening temperature control be 37 DEG C.
Embodiment 8
The present embodiment is for illustration of the method for preparing lysine through fermentation provided by the invention.
The method of embodiment 3 is adopted to carry out the fermentation of Methionin, unlike, fermented bacterium is Brevibacterium lactofermentus bacterial classification (bacterial strain BTC03 original seed takes from Chinese industrial Microbiological Culture Collection administrative center).
Comparative example 1
Method prepared by the fermentation Methionin that this comparative example provides for illustration of prior art.
The method of embodiment 3 is adopted to carry out the fermentation of Methionin, unlike, do not adopt sectional type to control leavening temperature, but whole fermentation processes temperature is 37 DEG C.Produce acid concentration, fermentation period and fermentation turnover ratio as shown in table 1.
Table 1
| Embodiment/comparative example numbering | Produce acid concentration (g/100mL) | Transformation efficiency (%) |
| Embodiment 1 | 18.6 | 63.1 |
| Embodiment 2 | 18.2 | 62.7 |
| Embodiment 3 | 18.8 | 63.5 |
| Embodiment 4 | 16.5 | 62.1 |
| Embodiment 5 | 17.5 | 62.0 |
| Embodiment 6 | 17.8 | 61.9 |
| Embodiment 7 | 16.8 | 62.2 |
| Embodiment 8 | 18.3 | 62.5 |
| Comparative example 1 | 16.0 | 60.5 |
As can be seen from the data of upper table 1, compared with comparative example 1, embodiment 1-8 adopts the preparation method of citric acid provided by the invention, and sectional type controls leavening temperature during the fermentation, effectively improves the product acid concentration of citric acid, fermentation turnover ratio; Growth stage and the temperature and the temperature difference of producing acid phase, compared with embodiment 4-6, control, in the preferred scope of the present invention, to improve the fermentation level of citric acid further by embodiment 1.Embodiment 1 and embodiment 7 are compared and can be found out, be seeded to by fermented bacterium to the fermentation time period of 12 hours as distinguishing growth stage and the reference mark of producing acid phase in fermention medium, fermentation level can be further enhanced.
In addition, embodiment 1-8 adopts sectional type to control the fermentation process of leavening temperature, effectively can shorten fermentation period, improves equipment efficiency of usage, reduces the electric power in fermenting process and the consumption of power equal energy source; In addition, the fermentation and acid stage adopts higher leavening temperature, can save the recirculated cooling water needed for high-temperature material cooling, also play the effect reducing electric power and power consumption.
Therefore, the method adopting sectional type of the present invention control leavening temperature to prepare citric acid effectively can improve the fermentation level of citric acid, and reduces energy consumption.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (12)
1. the method for a preparing lysine through fermentation, the method comprises: by fermenting lysine bacterial classification access fermenting lysine substratum, under stream adds carbon source and stream adds the condition of nitrogenous source, carry out fermenting to generate Methionin, described fermenting process comprises the growth stage and bacterial classification produces acid phase, be 30-38 DEG C at the leavening temperature in growth stage, the leavening temperature producing acid phase at bacterial classification is 32-42 DEG C, and at the leavening temperature in growth stage lower than the leavening temperature producing acid phase at bacterial classification.
2. method according to claim 1 wherein, is 32-36 DEG C at the leavening temperature in growth stage, and the leavening temperature producing acid phase at bacterial classification is 35-39 DEG C.
3. method according to claim 1 and 2, wherein, at the leavening temperature in growth stage 0.5-5 DEG C lower than the leavening temperature producing acid phase at bacterial classification.
4. method according to claim 3, wherein, at the leavening temperature in growth stage 2-3.5 DEG C lower than the leavening temperature producing acid phase at bacterial classification.
5. method according to claim 1, wherein, the described growth stage refers to the fermentation time section that will start in fermenting lysine strain inoculation to fermention medium when reaching 5g/L/h to the generating rate of Methionin, and described bacterial classification produces acid phase and refers to that the generating rate of Methionin is greater than the fermentation time section of 5g/L/h when fermentation ends.
6. method according to claim 1, wherein, described stream adds as Continuous Flow and adds;
The carbon source that stream adds is the saccharification clear liquid of starch materials and/or the saccharification clear liquid of cellulose family raw material; The amount that stream adds carbon source makes the concentration of reducing sugar in nutrient solution control at 2-12 grams per liter;
The nitrogenous source that stream adds be selected from urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, liquefied ammonia and ammoniacal liquor one or more; The amount that stream adds nitrogenous source makes the concentration of nitrogen in nutrient solution control at 0.35-1.2 grams per liter.
7. according to the method in claim 1-6 described in any one, wherein, with after inoculating and stream adds culture medium before carbon source and stream add nitrogenous source for benchmark, the inoculum size of described fermenting lysine bacterial classification is 5-20 volume %.
8. method according to claim 1, wherein, after inoculation and stream adds the volume that carbon source and stream adds the substratum in the prefermentor of nitrogenous source is the 40-60% of fermenter volume, stream adds carbon source and stream and adds nitrogenous source to blowing during the 70-80% of fermenter volume, and blowing volume is the 4-10% of culture volume in blowing prefermentor.
9. according to the method in claim 1-8 described in any one, wherein, the method also comprises, and adds the step of alkaline matter in the process of described fermentation in fermention medium, and adding of described alkaline matter makes the pH value of fermention medium be 6.5-7.5.
10. method according to claim 6, wherein, described alkaline matter is selected from one or more in liquefied ammonia, ammoniacal liquor, ammonium sulfate, sodium hydroxide, potassium hydroxide, lithium hydroxide, hydrated barta, aluminium hydroxide, manganous hydroxide, zinc hydroxide, sodium carbonate, salt of wormwood, Quilonum Retard and ammoniacal liquor.
11. according to the method in claim 1-10 described in any one, and wherein, the condition of described fermentation culture is: air flow is 0.5-1.5 volume: (volume minute), and pressure is 0.05-0.1MPa, and the time is 45-50 hour.
12. methods according to claim 1, wherein, described fermented bacterium be selected from intestinal bacteria, Corynebacterium glutamicum, Brevibacterium lactofermentus and brevibacterium flavum one or more.
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