CN1276091C - Feedback material-replenishing process of producing nisin - Google Patents
Feedback material-replenishing process of producing nisin Download PDFInfo
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Abstract
The present invention relates to a feedback material-replenishing process for producing nisin, which comprises: preparing a strain liquid via a conventional method; then, carrying out anaerobic fermentation. The present invention is characterized in that during anaerobic fermentation, the proportional relation between substrate consumption amount and acid production amount is used, a mixed liquid of the substrate and alkali is added to control the pH value of the system and to maintain the concentration of the substrate in the system within a proper range, so that no substrate inhibition and no limitation are produced. The process utilizes pH value feedback to control material replenishment, and the production process is simplified; under the condition of not increasing additional equipment, the fermentation yield (titer) of the nisin is greatly increased, and the process is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of production nisin method of (claiming nisin again), relate in particular to a kind of method of adding organic carbon source (substrate) production nisin by the pH feedback control.
Technical background
Nisin is a bacterioid element that is produced by Lactococcus lactis, and molecular weight is about 3500Da.Because it can suppress most gram positive bacterium, especially genus bacillus, carboxylic bacterium and the staphylococcus etc. that cause food spoilage there is had strong inhibitory effects, is now used as nontoxic, safe food preservatives by more than 50 countries and regions approvals.Nisin has very big potentiality in the milk-product Application for Field.In addition, on pasteur sterile milk goods, tinned pre-, meat product and medicine and veterinary drug, also all have broad application prospects.
The manufacturer of nisin mainly contains the Aplin ﹠amp of Britain; Barrett company and Chinese Yinxiang Biological Engineering Co., Ltd., Zhejiang Prov, what all adopt is the batch fermentation pattern.In this production model, it is extremely important for the fermentation of nisin to keep certain substrate (as sucrose) concentration.If initial sucrose concentration is not high enough, then substrate (sucrose) is exhausted very soon, and to detectability, thalline stops growing, and tiring of nisin is not high; Otherwise, if when disposable feeding intake, add more sucrose, can produce substrate again and suppress, influence thalli growth.
In order to address the above problem, generally adopt the way of feed supplement during the fermentation, the sucrose that promptly when beginning, only adds an amount of (more than the limiting concentration), continue to add sucrose then in some way again, to keep sucrose concentration in suitable scope, make it neither to produce substrate and suppress, also be not construed as limiting (shortage).Fed-batch fermentation is widely used in the industrial production.It is several to be divided into fed-batch, continuous feeding and repetition feed supplement by the feed supplement mode.Continuous feeding can be divided into constant speed again, the exponential sum variable flow adds.Though the method for these feed supplements can be eliminated the inhibition or the shortage of restricted substrate, the specific growth rate of control thalline improves the output of purpose product and the high-density culture that realizes cell.But these existing feed supplement methods all are to operate according to the program of prior setting, because the polytropy of fermentative production, variant between each batch, and sugar can not onlinely detect, the actual change of substrate in the fermenting process so be difficult to know for sure, its feed supplement has blindness, influences production control.
Summary of the invention
The objective of the invention is to overcome existing method of producing nisin and when feed supplement, have blindness, influence production control, make the defective that fermentation yield is low, providing a kind of changes by the pH that detects in the fermented liquid, utilize the feed supplement of pH value feedback control, thereby the simplification production technique, the method that nisin is produced in the feedback feed supplement that the fermentation yield (tiring) of nisin is increased substantially.
The objective of the invention is to realize by the following technical solutions:
The method of nisin is produced in feedback feed supplement provided by the invention, comprise and use ordinary method preparation kind of liquid, carry out anaerobically fermenting then, it is characterized in that: when anaerobically fermenting, utilize the proportionlity between the generation of base consumption amount and acid, come the pH of the hierarchy of control by the mixed flow liquid feeding that adds substrate and alkali, with the concentration of substrate in the maintenance system in a suitable scope, make it neither to produce substrate and suppress, also be not construed as limiting.
The concrete steps of this method are as follows:
1) plants the liquid preparation: preparation seed culture medium, sterilization according to a conventional method; Bacterial classification is inserted seed culture medium, and anaerobism is cultured to logarithmic phase under suitable temperature;
Described bacterial classification is Lactococcus lactis (Lactococcus lactis), comprises Lactococcus lactis ATCC11454 (USS strain library), Lactococcus lactis NIZO22186 (Dutch dairy products institute), Lactococcus lactis JCM7638 (Japanese microbial preservation center).
2) anaerobically fermenting: the kind liquid that step 1) is made inserts the fermention medium through conventional method sterilization, the temperature that control is suitable, and appropriateness stirs, stuffiness or feed an amount of nitrogen to keep anaerobic environment; In the fermenting process, along with substrate conversion is a lactic acid, thereby cause the pH of fermented liquid to reduce, when the pH of fermented liquid reduces to setting point when following, the mixed flow liquid feeding that adds substrate and alkali gos up to setting point until pH; This fermenting process stops when tiring of nisin reaches the highest.
Wherein, initial substrate concentrations is 1～20 grams per liter in the fermention medium, preferred 10 grams per liters.
Inoculum size is generally 1～10%.
The culture temperature of anaerobically fermenting is good with 25～35 ℃.
The pH setting point of fermented liquid is between 6.0～8.0.
The concentration of substrate is 50～600 grams per liters in the described stream liquid feeding, and the ratio of alkali and substrate is 3～14 milligramequivalents alkali/gram substrate.
Described substrate is sucrose, glucose, wood sugar, fructose, maltose.
Described alkali is sodium hydroxide, potassium hydroxide, yellow soda ash, sodium bicarbonate or ammonium hydroxide, preferred sodium hydroxide.
The feedback control of pH value can be finished by commercially available pH electrode (transmitter) and instrument, also can be by manually finishing.
For nisin produced bacterium, owing to belong to homotype lactic fermentation type, lactic acid is synthetic to exist the better linearity relation with sugar consumption, and this just provides precondition for the enforcement of pH feedback control.Simultaneously, in nisin produced bacterium, sugar consumption gene and nisin synthetic gene were positioned on the same operon, and sucrose synthesizes nisin and has regulating and controlling effect.Therefore, by pH feedback control sucrose concentration, can reach preferable nisin output.
The method of nisin is produced in feedback feed supplement provided by the invention, be based on such design: because nisin produces the bacterium Lactococcus lactis is that homology lactic acid produces bacterium, part substrate (as sucrose) is converted into lactic acid, thereby cause the pH of fermentation system to reduce, need to add alkali during the fermentation and keep the stable of pH.The present invention fits over substrate and alkali in the same solution by this relation, thereby regulates the pH value with the lactic acid that produces with fermentation in this mixed solution according to the relation of base consumption speed and lactic acid generation speed.Adding to fermentation system in the process of this mixing solutions with adjusting pH, by pH being controlled in the very narrow scope, make the alkali number (by equivalent) that adds equate with product acid amount, so when adding this mixed solution control pH, just in time replenished the amount of substrate that consumes in company with the amount of substrate that adds fermentation system, thereby made concentration of substrate maintain certain limit.Thereby, as long as pH is controlled in the very narrow scope, just can guarantee that the concentration of substrate in the fermentation system only fluctuates in very narrow scope.Such method can only add alkali and substrate in the feed supplement jar under the situation that does not change production unit, just can realize the control of concentration of substrate in the fermented liquid in control pH, promptly controls adding of substrate indirectly by the feedback of pH signal.And the control of pH is proven technique very, can use commercially available pH electrode (transmitter), control instruments and topworks to finish very easily.Set to intend the scope of the pH value of control with instrument, control automatically by instrument.
Compared with prior art, the advantage that the method for nisin is produced in feedback feed supplement provided by the invention is: by utilizing adding of pH signal feedback control substrate, thereby controlled the concentration of substrate in the fermented liquid indirectly, reach the purpose of controlling concentration of substrate more accurately, and can improve the output of purpose product nisin.Do not need to increase extras during the fermentation, and the mixed solution of substrate of adding and alkali do not need sterilization, simple to operate, and can shorten the production cycle, obtain higher nisin output, be suitable for suitability for industrialized production.
Embodiment
Further specify below in conjunction with embodiment:
Embodiment 1,
The microbial strains of using is Lactococcus lactis (Lactococcus lactis subsp.lactis) ATCC 11454.Carry out pH feedback control fed-batch fermentation with above-mentioned bacterial strains, used substratum has two kinds:
1) seed culture medium: sucrose 10 grams per liters, yeast powder 10 grams per liters, potassium primary phosphate 10 grams per liters, sodium-chlor 2 grams per liters, sal epsom 0.2 grams per liter.
2) fermention medium: sucrose 2 grams per liters, yeast powder 10 grams per liters, soy peptone 10 grams per liters, potassium primary phosphate 10 grams per liters, sodium-chlor 2 grams per liters, sal epsom 0.2 grams per liter.
Is 6.80 by prescription mixing back with NaOH and hydrochloric acid adjust pH with above-mentioned 2 kinds of medium components and distilled water, in 121 ℃ of sterilizations.
Bacterial classification inserts seed culture medium, 30 ℃ leave standstill cultivation 12 hours, inoculum size by 1% inserts in the 1 liter of automatic control fermentor tank that contains fermention medium, leavening temperature is controlled at 29-31 ℃, pH is controlled at 6.75-6.85, the nitrogen that feeds filtration sterilization carries out anaerobically fermenting, 100 rev/mins of mixing speed.Control pH and sugared concentration by stream with sucrose and sodium hydroxide mixed solution in the fermenting process, sucrose concentration is 300 grams per liters in the stream liquid feeding, and naoh concentration is 135 grams per liters.When pH reduced to 6.75, the mixed solution of beginning auto-feeding sucrose and sodium hydroxide rose to 6.85 to pH.Fermentation to the 10 hours, nisin are tired and are reached 4961IU/ml.Remaining sugar concentration maintains the 2-4 grams per liter during the fermentation.Compare with the highest the tiring (2300IU/ml, the 9th hour, total sucrose 45 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 1.1 times.
Embodiment 2,
Other fermentation conditions are with embodiment 1.Sucrose concentration 10 grams per liters of fermention medium are controlled pH and sugared concentration by stream with sucrose and sodium hydroxide mixed solution in the fermenting process, and sucrose concentration is 300 grams per liters in the stream liquid feeding, and naoh concentration is 135 grams per liters.When pH reduced to 6.75, the mixed solution of beginning auto-feeding sucrose and sodium hydroxide rose to 6.85 to pH.Fermentation to the 9 hours, nisin reaches 5011IU/ml.Remaining sugar concentration maintains the 9.5-10.2 grams per liter during the fermentation.Compare with the highest the tiring (2300IU/ml, the 9th hour, total sucrose 45 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 1.2 times.
Embodiment 3,
Other fermentation conditions are with embodiment 1.Sucrose concentration 5 grams per liters of fermention medium are controlled pH and sugared concentration by stream with sucrose and sodium hydroxide mixed solution in the fermenting process, and sucrose concentration is 300 grams per liters in the stream liquid feeding, and naoh concentration is 135 grams per liters.When pH reduced to 6.75, the mixed solution of beginning auto-feeding sucrose and sodium hydroxide rose to 6.85 to pH.Fermentation to the 10 hours, nisin are tired and are reached 4016IU/ml.Residual sugar maintains the 4.9-5.9 grams per liter during the fermentation.Compare with the highest the tiring (2300IU/ml, the 9th hour, total sucrose 45 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 0.8 times.
Embodiment 4,
Other fermentation conditions are with embodiment 1.Sucrose concentration 20 grams per liters of fermention medium are controlled pH and sugared concentration by stream with sucrose and sodium hydroxide mixed solution in the fermenting process, and sucrose concentration is 300 grams per liters in the stream liquid feeding, and naoh concentration is 135 grams per liters.When pH reduced to 6.75, the mixed solution of beginning auto-feeding sucrose and sodium hydroxide rose to 6.85 to pH.Fermentation to the 10 hours, nisin are tired and are reached 3545IU/ml.Residual sugar maintains the 19.9-21.2 grams per liter during the fermentation.Compare with the highest the tiring (2300IU/ml, the 9th hour, total sucrose 45 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 0.6 times.
Embodiment 5,
Other fermentation conditions are with embodiment 1, change the sucrose in seed culture medium and the fermention medium into glucose, the fermention medium glucose concn is 2 grams per liters, add glucose and potassium hydroxide mixed solution control pH and sugared concentration by stream in the fermenting process, glucose concn is 400 grams per liters in the stream liquid feeding, and concentration of potassium hydroxide is 239 grams per liters.When pH reduced to 6.75, the mixed solution of beginning auto-feeding glucose and potassium hydroxide rose to 6.85 to pH.Fermentation to the 9 hours, nisin reaches 4980IU/ml.Remaining sugar concentration maintains the 2-3 grams per liter during the fermentation.Compare with the highest the tiring (2330IU/ml, the 9th hour, total glucose 48 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 1.12 times.
Embodiment 6,
Other fermentation conditions are with embodiment 1, change the sucrose in seed culture medium and the fermention medium into glucose, the fermention medium glucose concn is 10 grams per liters, the pH of fermention medium is adjusted to 7.0, leavening temperature is controlled at 28-30 ℃, and pH is controlled at 6.95-7.05, adds glucose and ammonium hydroxide mixed solution control pH and sugared concentration by stream in the fermenting process, glucose concn is 600 grams per liters in the stream liquid feeding, and ammonium hydroxide concentration is 224 grams per liters.When pH reduced to 6.95, the mixed solution of beginning auto-feeding glucose and ammonium hydroxide rose to 7.05 to pH.Fermentation to the 9 hours, nisin reaches 4842IU/ml.Remaining sugar concentration maintains the 9.5-10.3 grams per liter during the fermentation.Compare with the highest the tiring (2330IU/ml, the 9th hour, total glucose 48 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 1.08 times.
Embodiment 7,
Other fermentation conditions are with embodiment 1, change the sucrose in seed culture medium and the fermention medium into wood sugar, the fermention medium xylose concentration is 5 grams per liters, the pH of fermention medium is adjusted to 7.20, leavening temperature is controlled at 27-29 ℃, and pH is controlled at 7.15-7.25, adds wood sugar and potassium hydroxide mixed solution control pH and sugared concentration by stream in the fermenting process, xylose concentration is 300 grams per liters in the stream liquid feeding, and concentration of potassium hydroxide is 224 grams per liters.When pH reduced to 7.15, the mixed solution of beginning auto-feeding wood sugar and potassium hydroxide rose to 7.25 to pH.Fermentation to the 9 hours, nisin reaches 3340IU/ml.Remaining sugar concentration maintains the 4.5-5.3 grams per liter during the fermentation.Compare with the highest the tiring (1810IU/ml, the 10th hour, total wood sugar 48 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 0.84 times.
Embodiment 8,
Other fermentation conditions are with embodiment 1, change the sucrose in seed culture medium and the fermention medium into wood sugar, the fermention medium xylose concentration is 20 grams per liters, the pH of fermention medium is adjusted to 6.50, leavening temperature is controlled at 29-31 ℃, and pH is controlled at 6.45-6.55, adds wood sugar and sodium bicarbonate mixed solution control pH and sugared concentration by stream in the fermenting process, xylose concentration is 200 grams per liters in the stream liquid feeding, and sodium bicarbonate concentration is 224 grams per liters.When pH reduced to 6.45, the mixed solution of beginning auto-feeding wood sugar and sodium bicarbonate rose to 6.55 to pH.Fermentation to the 9 hours, nisin reaches 3142IU/ml.Remaining sugar concentration maintains the 19.5-20.6 grams per liter during the fermentation.Compare with the highest the tiring (1810IU/ml, the 10th hour, total wood sugar 48 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 0.7 times.
Embodiment 9,
Other fermentation conditions are with embodiment 1, change the sucrose in seed culture medium and the fermention medium into fructose, the fermention medium fructose concentration is 10 grams per liters, the pH of fermention medium is adjusted to 6.70, leavening temperature is controlled at 30-32 ℃, and pH is controlled at 6.65-6.75, adds fructose and sodium hydroxide mixed solution control pH and sugared concentration by stream in the fermenting process, fructose concentration is 400 grams per liters in the stream liquid feeding, and naoh concentration is 171 grams per liters.When pH reduced to 6.65, the mixed solution of beginning auto-feeding fructose and sodium hydroxide rose to 6.75 to pH.Fermentation to the 11 hours, nisin reaches 3240IU/ml.Remaining sugar concentration maintains the 9.6-10.3 grams per liter during the fermentation.Compare with the highest the tiring (1650IU/ml, the 11st hour, total fructose 48 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 0.96 times.
Embodiment 10,
Other fermentation conditions are with embodiment 1, change the sucrose in seed culture medium and the fermention medium into fructose, the fermention medium fructose concentration is 15 grams per liters, leavening temperature is controlled at 31-33 ℃, pH is controlled at 6.75-6.85, add fructose and yellow soda ash mixed solution control pH and sugared concentration by stream in the fermenting process, fructose concentration is 300 grams per liters in the stream liquid feeding, and concentration of sodium carbonate is 170 grams per liters.When pH reduced to 6.75, the mixed solution of beginning auto-feeding fructose and potassium hydroxide rose to 6.85 to pH.Fermentation to the 11 hours, nisin reaches 3652IU/ml.Remaining sugar concentration maintains the 14.4-15.3 grams per liter during the fermentation.Compare with the highest the tiring (1650IU/ml, the 11st hour, total fructose 48 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 1.2 times.
Embodiment 11,
Other fermentation conditions are with embodiment 1, change the sucrose in seed culture medium and the fermention medium into maltose, the fermention medium maltose concentration is 5 grams per liters, leavening temperature is controlled at 30-32 ℃, pH is controlled at 6.75-6.85, control pH and sugared concentration by stream with maltose and potassium hydroxide mixed solution in the fermenting process, maltose concentration is 300 grams per liters in the stream liquid feeding, and concentration of potassium hydroxide is 189 grams per liters.When pH reduced to 6.75, the mixed solution of beginning auto-feeding maltose and potassium hydroxide rose to 6.85 to pH.Fermentation to the 9 hours, nisin reaches 3642IU/ml.Remaining sugar concentration maintains the 4.4-5.3 grams per liter during the fermentation.Compare with the highest the tiring (1980IU/ml, the 10th hour, total maltose 48 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 0.8 times.
Embodiment 12,
Other fermentation conditions are with embodiment 1, change the sucrose in seed culture medium and the fermention medium into maltose, the fermention medium maltose concentration is 15 grams per liters, the pH of fermention medium is adjusted to 6.60, pH is controlled at 6.55-6.65, control pH and sugared concentration by stream with maltose and ammonium hydroxide mixed solution in the fermenting process, maltose concentration is 400 grams per liters in the stream liquid feeding, and ammonium hydroxide concentration is 158 grams per liters.When pH reduced to 6.55, the mixed solution of beginning auto-feeding maltose and ammonium hydroxide rose to 6.65 to pH.Fermentation to the 9 hours, nisin reaches 3940IU/ml.Remaining sugar concentration maintains the 14.3-15.3 grams per liter during the fermentation.Compare with the highest the tiring (1980IU/ml, the 10th hour, total maltose 48 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 1.0 times.
Embodiment 13,
The microbial strains of using is Lactococcus lactis NIZO22186.Carry out pH feedback control fed-batch fermentation with above-mentioned bacterial strains, used substratum has two kinds:
1) seed culture medium: sucrose 10 grams per liters, yeast powder 8 grams per liters, potassium primary phosphate 8 grams per liters, sodium-chlor 2 grams per liters, sal epsom 0.2 grams per liter.
2) fermention medium: sucrose 10 grams per liters, yeast powder 8 grams per liters, polyprotein peptone 5 grams per liters, potassium primary phosphate 8 grams per liters, sodium-chlor 2 grams per liters, sal epsom 0.2 grams per liter.
Is 6.80 by prescription mixing back with NaOH and hydrochloric acid adjust pH with above-mentioned 2 kinds of medium components and distilled water, in 121 ℃ of sterilizations.
Bacterial classification inserts seed culture medium, 30 ℃ leave standstill cultivation 12 hours, inoculum size by 1% inserts in the 1 liter of automatic control fermentor tank that contains fermention medium, leavening temperature is controlled at 29-31 ℃, pH is controlled at 6.75-6.85, the nitrogen that feeds filtration sterilization carries out anaerobically fermenting, 100 rev/mins of mixing speed.Control pH and sugared concentration by stream with sucrose and sodium hydroxide mixed solution in the fermenting process, sucrose concentration is 300 grams per liters in the stream liquid feeding, and naoh concentration is 112 grams per liters.When pH reduced to 6.75, the mixed solution of beginning auto-feeding sucrose and sodium hydroxide rose to 6.85 to pH.Fermentation to the 12 hours, nisin are tired and are reached 5055IU/ml.Remaining sugar concentration maintains the 9.5-10.4 grams per liter during the fermentation, and it is 50 grams per liters that stream adds up sucrose concentration.Compare with the highest the tiring (1743IU/ml, the 12nd hour, total sucrose 45 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 1.9 times.
Embodiment 14,
Other fermentation condition is with embodiment 13.Change the sucrose in seed culture medium and the fermention medium into maltose, the fermention medium maltose concentration is 15 grams per liters, the pH of fermention medium is adjusted to 6.60, pH is controlled at 6.55-6.65, control pH and sugared concentration by stream with maltose and potassium hydroxide mixed solution in the fermenting process, maltose concentration is 300 grams per liters in the stream liquid feeding, and concentration of potassium hydroxide is 157 grams per liters.When pH reduced to 6.55, the mixed solution of beginning auto-feeding maltose and potassium hydroxide rose to 6.65 to pH.Fermentation to 12 hour, nisin is tired and is reached 3980IU/ml, and remaining sugar concentration maintains the 14.6-15.3 grams per liter during the fermentation, and it is 50 grams per liters that stream adds up maltose concentration.Compare with the batch fermentation (1743IU/ml, the 12nd hour, total maltose 50 grams per liters) of no-feed supplement, tire and improved 1.28 times.
Embodiment 15,
Other fermentation condition is with embodiment 13.Change the sucrose in seed culture medium and the fermention medium into glucose, the fermention medium glucose concn is 5 grams per liters, the pH of fermention medium is adjusted to 7.0, pH is controlled at 6.95-7.05, add glucose and ammonium hydroxide mixed solution control pH and sugared concentration by stream in the fermenting process, glucose concn is 300 grams per liters in the stream liquid feeding, and ammonium hydroxide concentration is 93 grams per liters.When pH reduced to 6.95, the mixed solution of beginning auto-feeding glucose and potassium hydroxide rose to 7.05 to pH.Fermentation to 12 hour, nisin is tired and is reached 4350IU/ml, and remaining sugar concentration maintains the 4.5-5.4 grams per liter during the fermentation, and it is 50 grams per liters that stream adds up glucose concn.Compare with the batch fermentation (1743IU/ml, the 12nd hour, total glucose 50 grams per liters) of no-feed supplement, tire and improved 1.5 times.
Embodiment 16,
The microbial strains of using is Lactococcus lactis JCM7638.Carry out pH feedback control fed-batch fermentation with above-mentioned bacterial strains, used substratum has two kinds:
1) seed culture medium: sucrose 10 grams per liters, yeast powder 10 grams per liters, potassium primary phosphate 8 grams per liters, sodium-chlor 2 grams per liters, sal epsom 0.2 grams per liter.
2) fermention medium: sucrose 10 grams per liters, yeast powder 10 grams per liters, polyprotein peptone 8 grams per liters, potassium primary phosphate 8 grams per liters, sodium-chlor 2 grams per liters, sal epsom 0.2 grams per liter.
Is 6.80 by prescription mixing back with NaOH and hydrochloric acid adjust pH with above-mentioned 2 kinds of medium components and distilled water, in 121 ℃ of sterilizations.
Bacterial classification inserts seed culture medium, 30 ℃ leave standstill cultivation 12 hours, inoculum size by 1% inserts in the 1 liter of automatic control fermentor tank that contains fermention medium, leavening temperature is controlled at 29-31 ℃, pH is controlled at 6.75-6.85, the nitrogen that feeds filtration sterilization carries out anaerobically fermenting, 100 rev/mins of mixing speed.Control pH and sugared concentration by stream with sucrose and sodium hydroxide mixed solution in the fermenting process, sucrose concentration is 300 grams per liters in the stream liquid feeding, and naoh concentration is 93 grams per liters.When pH reduced to 6.75, the mixed solution of beginning auto-feeding sucrose and sodium hydroxide rose to 6.85 to pH.Fermentation to the 16 hours, nisin are tired and are reached 4573IU/ml.Remaining sugar concentration maintains the 9.6-10.5 grams per liter during the fermentation.Compare with the highest the tiring (1759IU/ml, the 16th hour, total sucrose 45 grams per liters) of the batch fermentation of no-feed supplement, tire and improved 1.6 times.
Embodiment 17,
Other fermentation condition is with embodiment 16.Change the sucrose in seed culture medium and the fermention medium into maltose, the fermention medium maltose concentration is 15 grams per liters, the pH of fermention medium is adjusted to 6.60, pH is controlled at 6.55-6.65, control pH and sugared concentration by stream with maltose and potassium hydroxide mixed solution in the fermenting process, maltose concentration is 300 grams per liters in the stream liquid feeding, and concentration of potassium hydroxide is 131 grams per liters.When pH reduced to 6.55, the mixed solution of beginning auto-feeding maltose and potassium hydroxide rose to 6.65 to pH.Fermentation to 16 hour, nisin is tired and is reached 3780IU/ml, and remaining sugar concentration maintains the 14.6-15.3 grams per liter during the fermentation, and it is 50 grams per liters that stream adds up maltose concentration.Compare with the batch fermentation (1759IU/ml, the 16th hour, total maltose 50 grams per liters) of no-feed supplement, tire and improved 1.15 times.
Embodiment 18,
Other fermentation condition is with embodiment 16.Change the sucrose in seed culture medium and the fermention medium into glucose, the fermention medium glucose concn is 5 grams per liters, the pH of fermention medium is adjusted to 7.0, pH is controlled at 6.95-7.05, add glucose and ammonium hydroxide mixed solution control pH and sugared concentration by stream in the fermenting process, glucose concn is 300 grams per liters in the stream liquid feeding, and ammonium hydroxide concentration is 78 grams per liters.When pH reduced to 6.95, the mixed solution of beginning auto-feeding glucose and potassium hydroxide rose to 7.05 to pH.Fermentation to 12 hour, nisin is tired and is reached 4170IU/ml, and remaining sugar concentration maintains the 4.5-5.4 grams per liter during the fermentation, and it is 50 grams per liters that stream adds up glucose concn.Compare with the batch fermentation (1759IU/ml, the 16th hour, total glucose 50 grams per liters) of no-feed supplement, tire and improved 1.37 times.
Claims (7)
1, a kind of method of feeding back feed supplement production nisin, comprise and use ordinary method preparation kind of liquid, carry out anaerobically fermenting then, it is characterized in that: when anaerobically fermenting, utilize the proportionlity between the generation of base consumption amount and acid, control the pH of fermented liquid, to keep concentration of substrate in the fermented liquid in a suitable scope by the mixed flow liquid feeding that adds substrate and alkali, make it neither to produce substrate and suppress, also be not construed as limiting;
The pH setting point of described fermented liquid is between 6.0～8.0;
The concentration of substrate is 50～600 grams per liters in the described stream liquid feeding, and the ratio of alkali and substrate is 3～14 milligramequivalents alkali/gram substrate;
Described substrate is sucrose, glucose, wood sugar, fructose or maltose.
2, the method for nisin is produced in feedback feed supplement as claimed in claim 1, and it is characterized in that: the bacterial classification of described preparation kind liquid is Lactococcus lactis Lactococcus lactis.
3, the method for nisin is produced in feedback feed supplement as claimed in claim 2, and it is characterized in that: described Lactococcus lactis is the Lactococcus lactis ATCC11454 of USS strain library preservation.
4, the method for nisin is produced in feedback feed supplement as claimed in claim 1, and it is characterized in that: in the described anaerobically fermenting, initial substrate concentrations is 1～20 grams per liter in the fermention medium.
5, the method for nisin is produced in feedback feed supplement as claimed in claim 1, and it is characterized in that: described alkali is sodium hydroxide, potassium hydroxide, yellow soda ash, sodium bicarbonate or ammonium hydroxide.
6, the method for nisin is produced in feedback feed supplement as claimed in claim 1, and it is characterized in that: in the described anaerobically fermenting, culture temperature is 25～35 ℃.
7, the method for nisin is produced in feedback feed supplement as claimed in claim 1, and it is characterized in that: the feedback control of pH value is finished by commercially available pH electrode and instrument, or by manually finishing.
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