CN102533891B - Production method of lysine - Google Patents
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- CN102533891B CN102533891B CN201210009450.8A CN201210009450A CN102533891B CN 102533891 B CN102533891 B CN 102533891B CN 201210009450 A CN201210009450 A CN 201210009450A CN 102533891 B CN102533891 B CN 102533891B
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Abstract
The invention discloses a production method of lysine. The method comprises the step of inoculating lysine fermentation strains into lysine fermentation media for fermentation culture under the condition of feeding a carbon source and a nitrogen source. The method is characterized by feeding nutrient salts into fermentation liquor after fermentation culture for 15 hours, wherein the nutrient salts comprise potassium chloride, magnesium sulfate and manganese sulfate. The production method has the following advantages: the content of final lysine, the acid supply quantity per tank and the conversion rate can be improved, namely improving the yield of lysine, thus improving the economic benefits.
Description
Technical field
The present invention relates to a kind of production method of Methionin.
Background technology
At present China's fermenting lysine all adopts the method for batch fermentation, and fermentation adopts every tank drop into fermention medium and access certain fermenting lysine bacterial classification according to certain ratio, at stream, adds carbon source and stream adds under the condition of nitrogenous source, carries out fermentation culture.Carrying out along with fermentation, Methionin acidity improves gradually, and along with the stream of Carbon and nitrogen sources add and fermenting process in blowing, the concentration of other nutritive substances in fermentor tank beyond carbon, nitrogen reduces gradually, Methionin bacterial classification is old and feeble, self-dissolving too early, metabolic capacity declines, fermentation and acid speed is on a declining curve, finally because of fermentation and acid speed, slowly puts tank, causes the production capacity that has limited Methionin, cost is higher, and economic benefit is lower.
Summary of the invention
The object of the invention is in order to improve the production capacity of Methionin, thereby increase economic efficiency, a kind of production method of new Methionin is provided.
The present inventor surprisingly finds under study for action, in fermentation culture, after 15 hours, to stream in fermented liquid, add the nutritive salt that comprises Repone K, sal epsom and manganous sulfate, can greatly improve terminal lysine content, single tank for acid amount and transformation efficiency, improve Methionin production capacity, thereby increase economic efficiency.
Therefore, to achieve these goals, the invention provides a kind of production method of Methionin, described method comprises in fermenting lysine bacterial classification access fermenting lysine substratum, at stream, adds carbon source and stream adds under the condition of nitrogenous source, carries out fermentation culture, it is characterized in that, in fermentation culture, after 15 hours, in fermented liquid, flow Ensure Liquid salt, described nutritive salt comprises Repone K, sal epsom and manganous sulfate.
Preferably, before fermentation culture finished to fermentation culture after 15 hours in 6 hours, to stream in fermented liquid, add described nutritive salt.
The production method of Methionin provided by the invention, can improve terminal lysine content, single tank for acid amount and transformation efficiency, improves Methionin production capacity, thereby increases economic efficiency.
Other features and advantages of the present invention partly in detail are described the embodiment subsequently.
Embodiment
Below the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.
The invention provides a kind of production method of Methionin, the method comprises in fermenting lysine bacterial classification access fermenting lysine substratum, at stream, add carbon source and stream adds under the condition of nitrogenous source, carry out fermentation culture, in fermentation culture after 15 hours, in fermented liquid, flow Ensure Liquid salt, nutritive salt comprises Repone K, sal epsom and manganous sulfate.
In the present invention, the concept that fermention medium is known to the skilled person, refer to the nutriment with the artificial preparation maintaining for microorganism growth that microorganism fermentation is required, generally all contain carbohydrate, nitrogenous substances, inorganic salt (comprising trace element) and VITAMIN and water etc.The concept that fermented liquid is also known to the skilled person, refers to an access the liquid nutrient medium (this liquid nutrient medium is also alleged fermention medium in the present invention) of microorganism strains, products therefrom after cultivation after a while.
According to the present invention, although in fermentation culture after 15 hours, in fermented liquid, flow Ensure Liquid salt, nutritive salt comprises Repone K, sal epsom and manganous sulfate, can realize object of the present invention, improve terminal lysine content, single tank for acid amount and transformation efficiency, but in order further to reduce costs, and do not affect the raising of Methionin production capacity, under preferable case, before fermentation culture finished to fermentation culture after 15 hours in 6 hours, to stream in fermented liquid, add the nutritive salt that comprises Repone K, sal epsom and manganous sulfate.
In the present invention, take inoculate after and stream to add the fermention medium that carbon source and stream adds before nitrogenous source be benchmark, the inoculum size of fermenting lysine bacterial classification is 12-18 volume %.What those skilled in the art should understand that is, fermenting lysine bacterial classification in being seeded to fermention medium before, adopt ordinary method by fermenting lysine bacterial classification process seed tank culture, cultivation degree in seed tank culture can be observed producing Methionin microbial growth by sampling sediments microscope inspection, OD (optical density) pH-value determination pH, when by aforesaid method, observe thalli morphology normal, measure OD value and reach 0.75 and stop cultivation when above, the seed liquor in seeding tank is now called to mature seed liquid.And then by mature seed liquid access fermention medium.Therefore, in the present invention, the inoculum size of fermenting lysine bacterial classification is 12-18 volume %, and the volume that refers to the mature seed liquid in access fermention medium accounts for the long-pending 12-18% of access mature seed liquid post-fermentation and culture matrix.
Seed tank culture can adopt first class seed pot to cultivate also can adopt the cultivation of secondary seed tank, and required cultivation degree is cultivated in first class seed pot cultivation soon fermenting lysine bacterial classification in a seeding tank always; Secondary seed tank proceeds to another seeding tank again after cultivating and first fermenting lysine bacterial classification being cultivated to for some time in a seeding tank continues to cultivate, and cultivates required cultivation degree.Secondary seed tank is cultivated the not concrete restriction of incubation time at each seeding tank, as long as finally can cultivate required cultivation degree.For easy to operate, seed tank culture of the present invention preferably adopts first class seed pot to cultivate.
In the present invention, for the composition of seed tank culture base without particular requirement, the conventional seed tank culture base in this area can be adopted, for example, the preparation seed tank culture bases such as starchy material saccharification clear liquid, corn steep liquor, dipotassium hydrogen phosphate, sal epsom, ammonium sulfate, Threonine and methionine(Met) can be used.According to the present invention, in every liter of seed tank culture base, the consumption of each raw material can in very large range change, under preferable case, in every liter of seed tank culture base, the consumption of starchy material saccharification clear liquid can be 30-40 gram, the consumption of corn steep liquor (dry weight is 20-50 % by weight) can be 70-90 gram, the consumption of dipotassium hydrogen phosphate can be 0.5-1.5 gram, the consumption of sal epsom can be 0.4-1.1 gram, the consumption of ammonium sulfate can be 5-15 gram, the consumption of Threonine can be 0.1-0.6 gram, and the consumption of methionine(Met) can be 0.1-0.3 gram.
In order further to improve the production capacity of Methionin, the amount that stream adds Repone K preferably makes the concentration of Repone K in fermented liquid be controlled at 2-4 grams per liter, the amount that stream adds sal epsom preferably makes the concentration of sal epsom in fermented liquid be controlled at 1-3 grams per liter, and the amount that stream adds manganous sulfate preferably makes the concentration of manganous sulfate in fermented liquid be controlled at 0.02-0.06 grams per liter.The concentration of the nutritive salt adding for stream and flow velocity are without particular requirement, as long as the concentration of each nutritive salt in fermented liquid is controlled in above-mentioned scope.
In the present invention, the amount that stream adds carbon source makes the concentration of reducing sugar in fermented liquid be controlled at 5-10 grams per liter, and the amount that stream adds nitrogenous source makes the concentration of nitrogen in fermented liquid be controlled at 0.35-0.8 grams per liter." amount that stream adds carbon source makes the concentration of reducing sugar in fermented liquid be controlled at 5-10 grams per liter; the amount that stream adds nitrogenous source makes the concentration of nitrogen in fermented liquid be controlled at 0.35-0.8 grams per liter " refers to by controlling stream and adds speed that carbon source and stream adds nitrogenous source and make in whole fermentation culture process the concentration of reducing sugar in fermented liquid maintain 5-10 grams per liter herein, makes the concentration of nitrogen in fermented liquid maintain 0.35-0.8 grams per liter.
The present inventor finds in experiment, add, and the stream of nutritive salt adds for the stream of Carbon and nitrogen sources, and it is good that Continuous Flow adds the ferment effect adding than intermittent flow, and therefore, the stream in the present invention adds and is preferably Continuous Flow and adds.
In the present invention, fermentation culture is carried out in fermentor tank, in order effectively to utilize the production capacity of fermentor tank, after inoculation and stream add the 40-60% that volume that carbon source and stream adds the substratum in fermentor tank before nitrogenous source is preferably fermentor tank volume, along with stream adds carbon source and stream adds nitrogenous source, and fermentation culture is after 15 hours, in fermented liquid, flow Ensure Liquid salt, the volume of the substratum in fermentor tank increases gradually, in order to guarantee the air capacity in fermentor tank, blowing when being preferably stream and adding carbon source and stream and add nitrogenous source and stream Ensure Liquid salt to the 70-80% of fermentor tank volume, in order to guarantee 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 5-10% of culture volume in blowing prefermentor.
In prior art, by in fermenting lysine bacterial classification access fermenting lysine substratum, at stream, add carbon source and stream adds under the condition of nitrogenous source, carry out fermentation culture, carrying out along with fermentation, Methionin acidity improves gradually, nutritive substance reduces gradually, fermentation and acid speed is on a declining curve, after nutritive substance is reduced to a certain degree, fermentation and acid rate reduction is judged as fermentation termination to a certain extent, reaches fermentation termination and puts tank, put tank and refer to the substratum in fermentor tank is all emitted from fermentor tank, stop fermentation.In prior art, generally after fermentation culture 40-50 hour, put tank.The present invention is owing to after 15 hours, flowing Ensure Liquid salt in fermentation culture in fermented liquid, time that therefore can proper extension fermentation culture, preferably after fermentation culture 42-54 hour, puts tank.
It will be understood by those skilled in the art that in order to obtain the lysine product that purity is higher, the inventive method also comprises from blowing or puts the solution of tank extracts Methionin.For the method for extracting Methionin without particular requirement, can adopt the conventional the whole bag of tricks in this area, for example, adopt continuous ionic exchange separating and extracting method, to blowing or put in the solution of tank and add a large amount of vitriol oils to adjust pH to 2.0-3.0 to carry out acidifying, after acidifying, through metallic membrane or ceramic membrane filter, remove thalline, obtaining Methionin membrane filtration liquid is Methionin clear liquid, or the lysine fermentation liquor after acidifying is obtained to Methionin clear liquid through flocculation filtration except after thalline, except adopting strongly acidic cation-exchange, the Methionin clear liquid after thalline adsorbs exchange, resin absorption is saturated carries out wash-out afterwards with weak ammonia, the Methionin eluting is through concentrated, salt acid for adjusting pH value, crystallization, solid-liquid separation, dry, obtain lysine hydrochloride finished product, or at blowing or in putting the solution of tank, add acid, make lysine fermentation liquor acidifying, after filtration or the method such as centrifugal remove thalline.In Methionin clear liquid after removing thalline, add calcium hydroxide to regulate pH value to 8.0-11.5, make the impurity such as salt in Methionin clear liquid, colloid generate insolubles, after solid-liquid separation, obtain lysine solution, the content that lysine solution is concentrated into Methionin in every milliliter of lysine solution is 0.6-0.8g, through filtering, can obtain highly purified lysine solution again, then through concentrated, salt acid for adjusting pH value, crystallization, solid-liquid separation, oven dry, obtain lysine hydrochloride finished product.
In the present invention, to the condition of fermentation culture without particular requirement, can adopt the conventional condition in this area, for example, temperature is 35-38 ℃, and pressure is 0.05-0.1MPa, and pH value is 6.7-7.0, air flow be 0.5-1.2 cubic metres of air/cubic meter substratum/minute, air flow be preferably the substratum of 0.7-0.9 cubic metres of air/cubic meter/minute.
In the present invention, to the kind of fermenting lysine bacterial classification, without particular requirement, can adopt the conventional bacterial classification in this area, be preferably at least one in Corynebacterium glutamicum, intestinal bacteria and brevibacterium flavum.
In the present invention, carbon source is preferably starchy material saccharification clear liquid.
In the present invention, starchy material saccharification clear liquid both can adopt the preparation of dry method sugar refining technology, also can adopt the preparation of wet method sugar refining technology.Simple from technique, facility investment is few, the lower aspect of production cost is considered, preferably by dry method sugar refining technology, is prepared.Dry method sugar refining technology refers to that starchy material directly carries out fragmentation and enzymolysis without soaking.
Dry method sugar refining technology can comprise: starchy material is pulverized, and the product after starchy material is pulverized is sized mixing, and adds amylase to be hydrolyzed for the first time starch; Hydrolysate is for the first time carried out to solid-liquid separation, and add saccharifying enzyme to be hydrolyzed for the second time in the liquid phase component obtaining, obtain starchy material saccharification clear liquid.Preferably, pulverize the percent of pass that makes starchy material cross 30 mesh sieves and be greater than 75%, the percent of pass of 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 that the product after starchy material is pulverized is added to the water and mix, and it can be 9-17B é ° that the add-on of water makes the degree Beaume of the slurries that obtain.Term " degree Beaume " means a kind of method of strength of solution, is to detect by Beaumé scale the number of degrees that solution obtains.
According to the present invention, in hydrolysis for the first time, with the dry weight basis of the product after every gram of pulverizing, diastatic consumption can be 10-30 enzyme activity unit, the temperature of enzymolysis can be 88-92 ℃, and the time of enzymolysis can be 90-120 minute, and the pH value of enzymolysis can be 5.5-6.0.There is no particular limitation for the condition of solid-liquid separation, and preferably, the solid content in the liquid phase component that the condition of solid-liquid separation makes to obtain is 19-22 % by weight, more preferably 20-21 % by weight.
According to the present invention, in hydrolysis for the second time, in every gram of liquid phase component, the consumption of saccharifying enzyme can be 110-130 enzyme activity unit, and the temperature of enzymolysis can be 55-65 ℃, and the time of enzymolysis can be 420-600 minute, and the pH value of enzymolysis can be 4.0-4.5.
Enzyme activity unit of the present invention is defined as: in pH value, be 6.0, temperature is that under the condition of 70 ℃, 1 minute is converted into the required enzyme amount of reducing sugar by 1 milligram of starch is an enzyme activity unit.
Amylase refers to the general name of class of enzymes that can starch-splitting glycosidic link, and described amylase generally comprises α-amylase, beta-amylase.
α-amylase claims again starch Isosorbide-5-Nitrae-dextrinase, and it can cut the α-Isosorbide-5-Nitrae-glycosidic link of starch chain inside at random, brokenly, by Starch Hydrolysis be maltose, the oligosaccharides that contains 6 glucose units and with the oligosaccharides of side chain.The microorganism that produces this enzyme mainly has Bacillus subtilus, aspergillus niger, aspergillus oryzae and head mold.
Beta-amylase claims again starch Isosorbide-5-Nitrae-maltoside enzyme, can cut Isosorbide-5-Nitrae-glycosidic link from starch molecule non reducing end, generates maltose.The product that this enzyme acts on starch is maltose and limit dextrin.This enzyme is mainly produced by 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 variously can, for the raw material that contains starch of enzymolysis, preparing lysine through fermentation, for example, can be selected from one or more in corn, potato class (as cassava) and wheat for well known in the art.
In the present invention, the kind of nitrogenous source is conventionally known to one of skill in the art, for example, can be ammonium salt.When nitrogenous source is ammonium salt, in substratum, the concentration of nitrogen represents with the concentration of ammonium radical ion, and the concentration of nitrogen is controlled at 0.35-0.8 grams per liter, and the concentration of ammonium radical ion is controlled at 0.5-1.0 grams per liter.
In the present invention, the composition of fermention medium is without particular requirement, can adopt the conventional fermenting lysine substratum in this area, for example, can use the preparation fermention mediums such as starchy material saccharification clear liquid, molasses, corn steep liquor, ammonium sulfate, dipotassium hydrogen phosphate, sal epsom, Threonine, methionine(Met) and L-glutamic acid.According to the present invention, in every liter of fermention medium, the consumption of each raw material can in very large range change, under preferable case, in every liter of fermention medium, the consumption of starchy material saccharification clear liquid can be 40-60 gram, the consumption of molasses can be 30-50 gram, the consumption of corn steep liquor (dry weight is 20-50 % by weight) can be 20-40 gram, the consumption of ammonium sulfate can be 20-40 gram, the consumption of dipotassium hydrogen phosphate can be 0.5-1.5 gram, the consumption of sal epsom can be 0.4-0.6 gram, the consumption of Threonine can be 0.1-0.3 gram, the consumption of methionine(Met) can be 0.1-0.3 gram, the consumption of L-glutamic acid can be 0.2-0.4 gram.
In addition, it will be understood by those skilled in the art that the bacterial classification that access seeding tank is cultivated is the bacterial classification after the laggard row multiplication culture of overactivation.The common practise that activation and multiplication culture are this area, does not repeat them here.
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 characterictic described in above-mentioned embodiment, in reconcilable situation, can combine by any suitable mode, for fear of unnecessary repetition, the present invention is to the explanation no longer separately of various possible array modes.
In addition, between various embodiment of the present invention, also can carry out arbitrary combination, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
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 to 26 times of dilutions, adopt 722N visible spectrophotometer, under wavelength 562 nanometer visible rays, measure light absorption value, be OD value, by the cumulative volume of the OD value * extension rate ÷ fermented liquid obtaining, in the numerical value calculating reflection unit volume fermented liquid, produce the quantity of Methionin microorganism.
According to the method for GB/T5009.7-2008, measure the concentration of reducing sugar in fermented liquid.
According to the method for GB3595-83, measure the concentration of ammonium radical ion in fermented liquid.
According to the method for GB3595-1996, measure the concentration of Repone K in fermented liquid.
According to the method for GB/T671-1998, measure the concentration of sal epsom in fermented liquid.
According to the method for GB/T15899-1995, measure the concentration of manganous sulfate in fermented liquid.
According to the lysine concentration (in lysine hydrochloride) in GB10794-89 standard detection substratum.
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 sugar weight and fermentor tank sugar weight for seeding tank.
Embodiment 1
The present embodiment is for illustrating the production method of Methionin provided by the invention.
(1) 100 weight part corns of results are pulverized corn particle by mechanical workout, the percent of pass that makes Semen Maydis powder cross 30 mesh sieves is 80%.
(2) product after pulverizing being added to water sizes mixing to 12B é °, with respect to the dry weight of every gram of crushed products, add the amylase (Novozymes Company, α-amylase) of 20 enzyme activity units, under the condition that is 5.5 at 90 ℃, pH, enzymolysis is 100 minutes, obtains enzymolysis product.Wherein, enzymolysis product, by carrying out press filtration with fluid pressure type sheet frame pressure filter, is isolated to enzymolysis clear liquid (solid content is 20 % by weight); The saccharifying enzyme (α-Isosorbide-5-Nitrae-glucose hydrolysis Mei， Novozymes Company) that adds afterwards 115 enzyme activity units, under the condition that is 4.5 at 60 ℃, pH, enzymolysis is 420 minutes, obtains starchy material saccharification clear liquid.
(3) the starchy material saccharification clear liquid preparation seed tank culture base that uses step (2) to obtain, specifically consist of: with respect to the substratum of every liter, the consumption of starchy material saccharification clear liquid is 35 grams, the consumption of corn steep liquor (dry weight is 35 % by weight) is 80 grams, the consumption of dipotassium hydrogen phosphate is 1.0 grams, and the consumption of sal epsom is 0.5 gram, and the consumption of ammonium sulfate is 10 grams, the consumption of Threonine is 0.2 gram, and the consumption of methionine(Met) is 0.2 gram.Substratum is heated to 121 ℃ of sterilizations, is cooled to 37 ℃ and keep constant after maintaining 20 minutes.Open and stir, adjusting tank pressure is 0.1MPa, according to ventilation and 1: 0.5 volume ratio of substratum, passes into sterile air, with ammoniacal liquor, regulates pH to 6.8 and keeps constant.Then will after brevibacterium flavum bacterial classification (bacterial strain original seed FB42 is purchased from Southern Yangtze University) activation and propagation, in access seeding tank, cultivate, in culturing process, every 120 minutes, sample microscopies and measure OD value, when microscopy thalli morphology is normal and OD value reaches 0.8, stop cultivating, obtain mature seed liquid.
(4) the starchy material saccharification clear liquid preparation fermention medium that uses step (2) to obtain, specifically consist of: with respect to every liter of fermention medium, the consumption of starchy material saccharification clear liquid is 50 grams, the consumption of molasses (Xinjiang, the place of production) is 40 grams, the consumption of corn steep liquor (dry weight is 35 % by weight) is 30 grams, the consumption of ammonium sulfate is 30 grams, the consumption of dipotassium hydrogen phosphate is 1.0 grams, the consumption of sal epsom is 0.5 gram, the consumption of Threonine is 0.2 gram, the consumption of methionine(Met) is 0.2 gram, and the consumption of L-glutamic acid is 0.3 gram.Substratum is heated to 121 ℃ of sterilizations and is cooled to 37 ℃ and keep constant after 30 minutes, with ammoniacal liquor, regulates pH to 6.9.
(5) in fermentor tank, pack the fermention medium that step (4) prepares into, fermention medium volume is 50% of fermentor tank volume.Use the mature seed liquid of step (3) gained, in the substratum of access fermentor tank, carry out fermentation culture, take postvaccinal fermention medium as benchmark, the inoculum size of the mature seed liquid of step (3) gained is 15 volume %.After inoculation, Continuous Flow adds starchy material saccharification clear liquid and the ammonium sulfate that step (2) obtains, the amount that stream adds the starchy material saccharification clear liquid that step (2) obtains makes the concentration of reducing sugar in fermented liquid be controlled at 6-8 grams per liter, the amount that stream adds ammonium sulfate makes the concentration of ammonium radical ion in fermented liquid be controlled at 0.6-0.8 grams per liter, tank pressure is controlled as 0.1MPa, it is 37 ℃ that leavening temperature is controlled, air flow be 0.7 cubic metres of air/cubic meter substratum/minute, and with liquefied ammonia, regulate pH to maintain 6.9 to carry out fermentation culture, before fermentation culture finished to fermentation culture after 15 hours in 6 hours, to stream in fermented liquid, add Repone K, sal epsom and manganous sulfate, the amount that stream adds Repone K makes the concentration of Repone K in fermented liquid be controlled at 2-4 grams per liter, the amount that stream adds sal epsom makes the concentration of sal epsom in fermented liquid be controlled at 1-3 grams per liter, the amount that stream adds manganous sulfate makes the concentration of manganous sulfate in fermented liquid be controlled at 0.02-0.06 grams per liter.
Stream adds starchy material saccharification clear liquid and the ammonium sulfate that step (2) obtains, and stream add Repone K, sal epsom and manganous sulfate to fermentor tank volume 75% time blowing, blowing volume be in blowing prefermentor culture volume 8%.Fermentation culture was put tank after 48 hours, measured and put the lysine concentration (being terminal lysine content, lower same) of tank and the lysine concentration of middle blowing, calculated single tank and supplied acid amount and transformation efficiency in Table 1.
Embodiment 2
The present embodiment is for illustrating the production method of Methionin provided by the invention.
The preparation method of starchy material saccharification clear liquid, seed tank culture based formulas, seeding tank mature seed liquid cultural method, fermentor cultivation based formulas are all identical with embodiment 1.
Culture volume in fermentor tank is 40% of fermentor tank volume.By carrying out fermentation culture in the substratum of mature seed liquid access fermentor tank, take postvaccinal fermention medium as benchmark, the inoculum size of seed liquor is 12 volume %.After inoculation, Continuous Flow adds starchy material saccharification clear liquid and the ammonium sulfate making, the amount that stream adds the starchy material saccharification clear liquid making makes the concentration of reducing sugar in fermented liquid be controlled at 5-7 grams per liter, the amount that stream adds ammonium sulfate makes the concentration of ammonium radical ion in fermented liquid be controlled at 0.5-0.7 grams per liter, tank pressure is controlled as 0.08MPa, it is 35 ℃ that leavening temperature is controlled, air flow be 0.8 cubic metres of air/cubic meter substratum/minute, and with liquefied ammonia, regulate pH to maintain 6.7 to carry out fermentation culture, before fermentation culture finished to fermentation culture after 15 hours in 6 hours, to stream in fermented liquid, add Repone K, sal epsom and manganous sulfate, the amount that stream adds Repone K makes the concentration of Repone K in fermented liquid be controlled at 2-4 grams per liter, the amount that stream adds sal epsom makes the concentration of sal epsom in fermented liquid be controlled at 1-3 grams per liter, the amount that stream adds manganous sulfate makes the concentration of manganous sulfate in fermented liquid be controlled at 0.02-0.06 grams per liter.
Stream adds starchy material saccharification clear liquid and the ammonium sulfate making, and stream add Repone K, sal epsom and manganous sulfate to fermentor tank volume 70% time blowing, blowing volume be in blowing prefermentor culture volume 5%.Fermentation culture was put tank after 42 hours, measured and put the lysine concentration of tank and the lysine concentration of middle blowing, calculated single tank and supplied acid amount and transformation efficiency in Table 1.
Embodiment 3
The present embodiment is for illustrating the production method of Methionin provided by the invention.
The preparation method of starchy material saccharification clear liquid, seed tank culture based formulas, seeding tank mature seed liquid cultural method, fermentor cultivation based formulas are all identical with embodiment 1.
Culture volume in fermentor tank is 60% of fermentor tank volume.By carrying out fermentation culture in the substratum of mature seed liquid access fermentor tank, take postvaccinal fermention medium as benchmark, the inoculum size of seed liquor is 18 volume %.After inoculation, Continuous Flow adds starchy material saccharification clear liquid and the ammonium sulfate making, the amount that stream adds the starchy material saccharification clear liquid making makes the concentration of reducing sugar in fermented liquid be controlled at 8-10 grams per liter, the amount that stream adds ammonium sulfate makes the concentration of ammonium radical ion in fermented liquid be controlled at 0.8-1.0 grams per liter, tank pressure is controlled as 0.05MPa, it is 38 ℃ that leavening temperature is controlled, air flow be 0.9 cubic metres of air/cubic meter substratum/minute, and with liquefied ammonia, regulate pH to maintain 7.0 to carry out fermentation culture, before fermentation culture finished to fermentation culture after 15 hours in 6 hours, to stream in fermented liquid, add Repone K, sal epsom and manganous sulfate, the amount that stream adds Repone K makes the concentration of Repone K in fermented liquid be controlled at 2-4 grams per liter, the amount that stream adds sal epsom makes the concentration of sal epsom in fermented liquid be controlled at 1-3 grams per liter, the amount that stream adds manganous sulfate makes the concentration of manganous sulfate in fermented liquid be controlled at 0.02-0.06 grams per liter.
Stream adds starchy material saccharification clear liquid and the ammonium sulfate making, and stream add Repone K, sal epsom and manganous sulfate to fermentor tank volume 80% time blowing, blowing volume be in blowing prefermentor culture volume 10%.Fermentation culture was put tank after 54 hours, measured and put the lysine concentration of tank and the lysine concentration of middle blowing, calculated single tank and supplied acid amount and transformation efficiency in Table 1.
Comparative example 1
According to the method for embodiment 1, produce Methionin, different, in fermentation culture process, to stream in fermented liquid, do not add Repone K, sal epsom and manganous sulfate.Fermentation culture was put tank after 48 hours, measured and put the lysine concentration of tank and the lysine concentration of middle blowing, calculated single tank and supplied acid amount and transformation efficiency in Table 1.
Table 1
| Terminal lysine content (g/100mL) | Single tank is for acid amount (t) | Transformation efficiency (%) | |
| Embodiment 1 | 18.35 | 52.62 | 63.25 |
| Embodiment 2 | 18.20 | 50.04 | 60.09 |
| Embodiment 3 | 18.12 | 53.51 | 62.35 |
| Comparative example 1 | 17.34 | 48.03 | 59.95 |
As can be seen from Table 1, adopt the inventive method fermentative production Methionin, can obviously improve terminal lysine content, single tank for acid amount and transformation efficiency.
The production method of Methionin provided by the invention, by in fermentation culture after 15 hours, to stream in fermented liquid, add the nutritive salt that comprises Repone K, sal epsom and manganous sulfate, terminal lysine content, single tank have been improved for acid amount and transformation efficiency, improve Methionin production capacity, thereby improved economic benefit.
Claims (12)
1. the production method of a Methionin, described method comprises in fermenting lysine bacterial classification access fermenting lysine substratum, at stream, add carbon source and stream adds under the condition of nitrogenous source, carry out fermentation culture, it is characterized in that, in fermentation culture after 15 hours, in fermented liquid, flow Ensure Liquid salt, described nutritive salt comprises Repone K, sal epsom and manganous sulfate, the amount that stream adds Repone K makes the concentration of Repone K in fermented liquid be controlled at 2-4 grams per liter, the amount that stream adds sal epsom makes the concentration of sal epsom in fermented liquid be controlled at 1-3 grams per liter, the amount that stream adds manganous sulfate makes the concentration of manganous sulfate in fermented liquid be controlled at 0.02-0.06 grams per liter.
2. method according to claim 1, wherein, before fermentation culture finished to fermentation culture after 15 hours in 6 hours, adds described nutritive salt to stream in fermented liquid.
3. method according to claim 1 and 2, wherein, take inoculate after and stream to add the fermention medium that carbon source and stream adds before nitrogenous source be benchmark, the inoculum size of described fermenting lysine bacterial classification is 12-18 volume %.
4. method according to claim 3, wherein, the amount that described stream adds carbon source makes the concentration of reducing sugar in fermented liquid be controlled at 5-10 grams per liter, and the amount that described stream adds nitrogenous source makes the concentration of nitrogen in fermented liquid be controlled at 0.35-0.8 grams per liter.
5. method according to claim 1 and 2, wherein, described stream adds as Continuous Flow and adds.
6. method according to claim 1 and 2, wherein, described fermentation culture is carried out in fermentor tank, after inoculation and stream to add the volume that carbon source and stream adds the substratum in fermentor tank before nitrogenous source be the 40-60% of fermentor tank volume, blowing when stream adds Carbon and nitrogen sources and stream Ensure Liquid salt to the 70-80% of fermentor tank volume, blowing volume is the 5-10% of culture volume in blowing prefermentor.
7. method according to claim 6, wherein, puts tank after fermentation culture 42-54 hour.
8. method according to claim 7, wherein, described method also comprise from described blowing or described in put the solution of tank and extract Methionin.
9. method according to claim 1 and 2, wherein, the condition of described fermentation culture is: temperature is 35-38 ℃, pressure is 0.05-0.1MPa, pH value is 6.7-7.0, air flow be 0.5-1.2 cubic metres of air/cubic meter substratum/minute.
10. method according to claim 1 and 2, wherein, described fermenting lysine bacterial classification is at least one in Corynebacterium glutamicum, intestinal bacteria and brevibacterium flavum.
11. methods according to claim 1 and 2, wherein, described carbon source is starchy material saccharification clear liquid.
12. methods according to claim 1 and 2, wherein, described nitrogenous source is ammonium salt.
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| CN106755156A (en) * | 2016-12-28 | 2017-05-31 | 安徽丰原发酵技术工程研究有限公司 | A kind of fermentation process of L lysines |
| CN110872606A (en) * | 2019-09-02 | 2020-03-10 | 黑龙江成福食品集团有限公司 | Preparation method of L-lysine sulfate and by-product thereof |
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