CN102533889B - Method for continuously fermenting lysine - Google Patents
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- CN102533889B CN102533889B CN201210009366.6A CN201210009366A CN102533889B CN 102533889 B CN102533889 B CN 102533889B CN 201210009366 A CN201210009366 A CN 201210009366A CN 102533889 B CN102533889 B CN 102533889B
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Abstract
The invention discloses a method for continuously fermenting lysine, which comprises the following steps: inoculating a lysine fermentation strain into a lysine fermentation medium; under the conditions of a flow-feed carbon source and a flow-feed nitrogen source, and carrying out fermentation and culture. The invention is characterized in that nutrient substances required by lysine fermentation and culture are added into the fermentation liquid 20 hours after the fermentation and culture are started. The method disclosed by the invention greatly prolongs the fermentation and culture time, enhances the metabolic capability of the lysine strain, can carry out fermentation all along under the conditions that the equipment does not goes wrong and the fermentation medium is not contaminated by bacteria, increases the acid supply amount per unit time, and enhances the equipment utilization rate, thereby lowering the lysine fermentation cost.
Description
Technical field
The present invention relates to a kind of method of fermenting lysine, especially relate to a kind of method that Methionin continuously ferments.
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.Along with the carrying out of fermentation, Methionin acidity improves gradually, and cumulative volume increases, and nutritive substance concentration reduces gradually, and 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, enters extraction process after fermentation ends.
Carrying out along with fermentation, Nutrious fermented material is reduced to a certain degree, the metabolic capacity of Methionin bacterial classification declines, cause fermentation and acid speed slowly to put tank, and will carry out cleaning, inspection, the sterilizing of tank after putting tank, and will again load substratum, access Methionin bacterial classification restarts fermentation culture, the time of the fermentation culture of every batch of tank is shorter, has virtually increased the quantity in auxiliary cycle, causes the service efficiency of fermentation equipment to reduce.
Summary of the invention
The object of the invention is, in order to overcome the lower defect of the service efficiency of fermentation equipment in prior art, provides a kind of method of new fermenting lysine.
The present inventor is surprised to find, by in fermentation culture after 20 hours, to stream in fermented liquid, add fermenting lysine and cultivate required nutritive substance, can make the metabolic capacity of Methionin bacterial classification substantially not decline, thereby can make fermentation and acid speed substantially remain unchanged, thereby without putting tank, fermenting process is carried out continuously.
Therefore, to achieve these goals, the invention provides a kind of method that Methionin continuously ferments, 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 20 hours, to stream in fermented liquid, add fermenting lysine and cultivate required nutritive substance.
Preferably, described method was also included in fermentation culture after 20 hours, to stream in fermented liquid, added fermenting lysine bacterial classification.
The method that Methionin provided by the invention continuously ferments, greatly extended the time of fermentation culture, and do not affect transformation efficiency, improved the metabolic capacity of Methionin bacterial classification, in the situation that equipment does not break down with not microbiological contamination of fermention medium, fermentation can go on always, has improved the unit time for acid amount, and improved plant factor, and then reduced fermenting lysine cost.
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 method that Methionin continuously ferments, described method comprises in fermenting lysine bacterial classification access fermenting lysine substratum, in stream sugaring and stream, add under the condition of organic nitrogen source, carry out fermentation culture, in fermentation culture after 20 hours, to stream in fermented liquid, add fermenting lysine and cultivate required nutritive substance.
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.
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 by sampling sediments microscope inspection, OD (optical density) pH-value determination pH is observed producing Methionin microbial growth, normal when observing thalli morphology by aforesaid method, measuring OD value reaches 0.75 and stops cultivation when above, 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, magnesium sulfate, 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 magnesium sulfate 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 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.
In the present invention, in fermented liquid, the concentration of reducing sugar adds carbon source by stream and controls, in fermented liquid, the concentration of nitrogen adds nitrogenous source by stream and controls, carrying out along with fermentation, in fermented liquid, the content of other nutritive ingredients also decreases, fermentation in fermentation culture, after 20 hours, by stream in fermented liquid, adds fermenting lysine and cultivates the concentration that required nutritive substance carrys out other nutritive ingredients in controlled fermentation liquid, so that can continue to go on.As long as the amount adding for composition and the stream of nutritive substance can make fermentation continue to go on, under preferable case, nutritive substance is the fermention medium that concentration doubles, with respect to after every liter of inoculation and stream add carbon source and stream and add the fermention medium before nitrogenous source, the flow acceleration of nutritive substance is 0.007-0.012 l/h.
In the present invention, fermenting lysine bacterial classification can constantly be bred in fermention medium, produce new fermenting lysine bacterial classification, in fermentation culture after 20 hours, to stream in fermented liquid, add fermenting lysine and cultivate required nutritive substance, in theory, in the situation that equipment does not break down with not microbiological contamination of substratum, fermentation can go on always.In order to make fermentation continue better to carry out, the inventive method was preferably also included in fermentation culture after 20 hours, to stream in fermented liquid, added fermenting lysine bacterial classification.It is 0.95-1.35 that the amount that fermenting lysine bacterial classification stream adds preferably makes the OD value of fermented liquid.What it will be understood by those skilled in the art that fermenting lysine bacterial classification that this place stream adds refers to is also the mature seed liquid after seed tank culture.
The present inventor finds in experiment, add, and the stream of nutritive substance and fermenting lysine bacterial classification 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 20 hours, to stream in fermented liquid, add fermenting lysine and cultivate required nutritive substance, 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 material 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 fermentating liquid 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 in fermentation culture after 20 hours, to stream in fermented liquid, add fermenting lysine and cultivate required nutritive substance, therefore, in theory, in the situation that equipment does not break down with not microbiological contamination of substratum, fermentation can go on always, but in actual production, can put at any time tank according to actual needs, stop fermentation, but the present invention will be longer than prior art far away in the time that puts the cultivation of tank primary fermentation, put the time that tank primary fermentation is cultivated.
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, magnesium sulfate, 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 magnesium sulfate 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 the present invention, nutritive substance is the fermention medium that concentration doubles, and refers to that nutritive substance is the substratum that the consumption of each raw material in every liter of fermention medium doubles to obtain.
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 lysine concentration (in lysine hydrochloride) in GB10794-89 standard detection fermented liquid.
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.
Unit time measures/(fermentation period+auxiliary cycle) for acid for acid=mono-tank
Fermentation period is the time putting the cultivation of tank primary fermentation; The auxiliary cycle is between adjacent tank is criticized, to carry out cleaning, inspection, the sterilizing of tank, and again loads the time of substratum etc., and adjacent tank does not carry out the interval time of fermentation culture between criticizing.
Plant factor=fermentation period/(fermentation period+auxiliary cycle)
Embodiment 1
The present embodiment is for illustrating the preparation 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 enzyme, 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 every liter of seed tank culture base, 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 magnesium sulfate 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 magnesium sulfate 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, in fermentation culture after 20 hours, to stream in fermented liquid, add fermention medium and the mature seed liquid that concentration doubles, with respect to after every liter of inoculation and stream add carbon source and stream and add the fermention medium before nitrogenous source, the flow acceleration of the fermention medium that concentration doubles is 0.01 l/h, it is 0.95-1.35 that the amount that stream adds ripe seed liquor makes the OD value of fermented liquid.
Stream adds starchy material saccharification clear liquid and the ammonium sulfate that step (2) obtains, and stream add fermention medium that concentration doubles and mature seed liquid to fermentor tank volume 75% time blowing, blowing volume be in blowing prefermentor culture volume 8%.Fermentation culture was put tank after 58 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 and plant factor in Table 1 for sour amount, transformation efficiency, unit time.
Embodiment 2
The present embodiment is for illustrating the preparation 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, in fermentation culture after 20 hours, to stream in fermented liquid, add fermention medium and the mature seed liquid that concentration doubles, with respect to after every liter of inoculation and stream add carbon source and stream and add the fermention medium before nitrogenous source, the flow acceleration of the fermention medium that concentration doubles is 0.007 l/h, it is 0.95-1.35 that the amount that stream adds ripe seed liquor makes the OD value of fermented liquid.
Stream adds starchy material saccharification clear liquid and the ammonium sulfate making, and stream add fermention medium that concentration doubles and mature seed liquid to fermentor tank volume 70% time blowing, blowing volume be in blowing prefermentor culture volume 5%.Fermentation culture was put tank after 68 hours, measured and put the lysine concentration of tank and the lysine concentration of middle blowing, calculated single tank and supplied acid and plant factor in Table 1 for sour amount, transformation efficiency, unit time.
Embodiment 3
The present embodiment is for illustrating the preparation 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, in fermentation culture after 20 hours, to stream in fermented liquid, add fermention medium and the mature seed liquid that concentration doubles, with respect to after every liter of inoculation and stream add carbon source and stream and add the fermention medium before nitrogenous source, the flow acceleration of the fermention medium that concentration doubles is 0.012 l/h, it is 0.95-1.35 that the amount that stream adds ripe seed liquor makes the OD value of fermented liquid.
Stream adds starchy material saccharification clear liquid and the ammonium sulfate making, and stream add fermention medium that concentration doubles and mature seed liquid to fermentor tank volume 80% time blowing, blowing volume be in blowing prefermentor culture volume 10%.Fermentation culture was put tank after 96 hours, measured and put the lysine concentration of tank and the lysine concentration of middle blowing, calculated single tank and supplied acid and plant factor in Table 1 for sour amount, transformation efficiency, unit time.
Comparative example 1
Method according to embodiment 1 is prepared Methionin, different is, in fermentation culture process, to stream in fermented liquid, do not add fermenting lysine and cultivate required nutritive substance and mature seed liquid, fermentation culture was put tank after 48 hours, mensuration is put the lysine concentration of tank and the lysine concentration of middle blowing, calculates single tank and supplies acid and plant factor in Table 1 for sour amount, transformation efficiency, unit time.
Table 1
As can be seen from Table 1, the inventive method can extend fermentation period, and transformation efficiency is not had to impact substantially; Adopt the inventive method preparing lysine through fermentation, can obviously improve the unit time for acid and plant factor.
The method that Methionin provided by the invention continuously ferments, greatly extended the time of fermentation culture, and do not affect transformation efficiency, improved the metabolic capacity of Methionin bacterial classification, in the situation that equipment does not break down with not microbiological contamination of substratum, fermentation can go on always, has improved the unit time for acid amount, improve plant factor, and then reduced fermenting lysine cost.
Claims (11)
1. the method that Methionin continuously ferments, 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 20 hours, to stream in fermented liquid, add fermenting lysine and cultivate required nutritive substance and fermenting lysine bacterial classification, wherein, described fermenting lysine bacterial classification is brevibacterium flavum.
2. method according to claim 1, 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 %.
3. method according to claim 2, 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.
4. according to the method described in any one in claim 1-3, wherein, described nutritive substance is the fermention medium that concentration doubles, with respect to after every liter of inoculation and stream add carbon source and stream and add the fermention medium before nitrogenous source, the flow acceleration of described nutritive substance is 0.007-0.012 l/h.
5. according to the method described in any one in claim 1-3, wherein, it is 0.95-1.35 that the amount that fermenting lysine bacterial classification stream adds makes the OD value of fermented liquid.
6. according to the method described in any one in claim 1-3, wherein, described stream adds as Continuous Flow and adds.
7. according to the method described in any one in claim 1-3, 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 material to the 70-80% of fermentor tank volume, blowing volume is the 5-10% of fermentating liquid volume in blowing prefermentor.
8. method according to claim 7, wherein, described method also comprises from the solution of described blowing extracts Methionin.
9. according to the method described in any one in claim 1-3, wherein, the condition of described fermentation culture is: 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.
10. according to the method described in any one in claim 1-3, wherein, described carbon source is starchy material saccharification clear liquid.
11. according to the method described in any one in claim 1-3, and wherein, described nitrogenous source is ammonium salt.
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| CN104561162A (en) * | 2013-10-23 | 2015-04-29 | 中粮营养健康研究院有限公司 | Method for preparing lysine by fermentation |
| CN104726511B (en) * | 2013-12-24 | 2018-08-07 | 中粮生物化学（安徽）股份有限公司 | A kind of method of preparing lysine through fermentation |
| CN103820509B (en) * | 2014-01-24 | 2016-10-19 | 安徽丰原发酵技术工程研究有限公司 | The 1B fermentation process that short productivity of a kind of cycle is high |
| CN103992964B (en) * | 2014-03-21 | 2016-10-12 | 山东寿光巨能金玉米开发有限公司 | A kind of resistant to high pH value strain and fermentative Production lysine method |
| CN106148444A (en) * | 2016-08-31 | 2016-11-23 | 南京工业大学 | Method for producing L-lysine by multistage continuous fermentation |
| CN106434291B (en) * | 2016-08-31 | 2019-03-05 | 寿光金远东变性淀粉有限公司 | A kind of device and method of uninterrupted culture production lysine |
| CN110129386A (en) * | 2019-05-31 | 2019-08-16 | 绥化象屿金谷生化科技有限公司 | A method of optimization amino acid fermentation |
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