CN1252244C - Ammonia-resistant L(+)-lactic acid-producing bacteria and production method of L(+)-lactic acid - Google Patents
Ammonia-resistant L(+)-lactic acid-producing bacteria and production method of L(+)-lactic acid Download PDFInfo
- Publication number
- CN1252244C CN1252244C CN 99119265 CN99119265A CN1252244C CN 1252244 C CN1252244 C CN 1252244C CN 99119265 CN99119265 CN 99119265 CN 99119265 A CN99119265 A CN 99119265A CN 1252244 C CN1252244 C CN 1252244C
- Authority
- CN
- China
- Prior art keywords
- lactic acid
- ammonia
- rhizopus
- bacterial strain
- production method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
This new L(+)-lactic acid productive bacterium [e.g. Rhizopus sp. MK98-1156 strain (FERM BP-6776), etc.], is obtained by mutating Rhizopus sp. MK96 with nitrosoguanidine having a resistance to ammonia and controllable pH of a culturing solution by using ammonia as neutralizing agents and producible L(+)-lactic acid in a high yield.
Description
The present invention relates to belong to L (+)-lactic acid-producing bacteria Rhizopus, that have ammonia-resistance and use this bacterial classification to produce the method for L (+)-lactic acid, relate to L (+)-lactic acid-producing bacteria of head mold MK96 (Rhizopus sp.MK96) variation as maternal plant being obtained with nitrosoguanidine in more detail with ammonia-resistance, that is to say, relate to head mold MK96-1156 itself and under the condition of aerobic, regulate the pH of nutrient solution as neutralizing agent, thereby prepare the method for L (+)-lactic acid with high yield with ammonia with them.
Lactic acid can be used as the preparation that foodstuff additive are used for drinks, refreshment drink, salted vegetables, soy sauce, bread, beer etc., in addition, in industry such as leather, fiber, plastics, also is utilized, and be a kind of useful compound.
At present, using fermentative Production lactic acid, generally is that the bacterium that the lactobacillus genus (Lactobacillus) that will belong to Schizophyta, lactococcus (Lactococcus) etc. are known as lactic acid bacteria class is cultivated under the condition of anaerobic, carries out lactic fermentation.The substratum that uses in the culturing process of milk-acid bacteria is as main raw material, and to the nutrition sources such as yeast extract that wherein add as auxiliary material with sugar, starch etc.It is simple that the lactic fermentation that utilizes these lactobacilluss (Lactobacillus), galactococcus bacterium such as (Lactococcus) to carry out has process, and the advantage that the lactic acid yield is high, but in general, because the price height of auxiliary materials such as yeast extract, so the price of L (+)-lactic acid that obtains is also just high.In addition because the sizes of these bacterium is below the 1 μ m, with substratum separate the comparison difficulty, it is poor to cultivate the operability of handling.
On the other hand, also there is the thread fungus of using the Rhizopus class in containing the nutrient solution of lime carbonate, to cultivate the method for producing lactic acid by aerobic.The yield that obtains lactic acid from sugar is at 70-80%, because monose such as glucose can be as carbon source, and the composition of substratum only requires a spot of inorganic salt removing sugar, so the impurity in the back substratum of fermenting is fewer.For example, (L.Kristofikova such as Kristofikova, M.Rosenberg, A.Valnove, J.Sajbidor and M.Certik Folia Microbiol, 36 (5), 451-455 (1991)) with glucose as carbon source, produce 79 grams per liter lactic acid by unrooted rhizopus CCM8109 (Rhizopusarrhizus CCM8109).
In addition, the method for using Rhizopus oryzae NRRL395 (Rhizopus oryzae NRRL395) thalline to produce lactic acid is also arranged.For example, and Yang etc. (C.W.Yang,, Zhongjing Lu and Geroge T.Tsaso; ApplidBiochemistry and Biotechology vol.51/52 p57-71,1995) method of describing is under the condition of aerobic, cultivates this thalline with the nutrient solution that has added lime carbonate, obtains yield and be 78% lactic acid.
In addition, the thalline of above-mentioned Rhizopus forms aggregate (granular precipitation) easily, owing to can simply separate with substratum, so separation and purification lactic acid and this granular precipitation from the substratum after the fermentation easily.Utilized the method for this granular sedimentary advantage, the Rhizopus oryzae NRRL395 (Rhizopus oryzaeNRRL395) of use has been arranged, in the nutrient solution of wood sugar, made it to form the granular precipitation of thalline, with the method for this granular precipitation by glucose production lactic acid as carbon source.For example, (C.R.Soccol, B.Marin, M..Raimbault such as Soccol; Appl Microbiol Biotechnol 41,286-290 (1991)) under the condition of aerobic, the granular precipitation of the thalline that fermentation culture obtains in the nutrient solution that contains neutralizing agent lime carbonate obtains yield and is about 75% lactic acid.Adopt this method, in containing the nutrient solution of lime carbonate, cultivate this granular precipitation,, can produce lactic acid by successive by run out of at every turn as in new nutrient solution, cultivating again behind the glucose of carbon source.
Like this, under the condition of aerobic, cultivate the bacterial strain of Rhizopus, when being raw material production L (+)-lactic acid, compare, have multiple advantage with the occasion of using milk-acid bacteria with the grape sugar and starch.
But, under the condition of aerobic, cultivate rhizopus, the yield that produces lactic acid is generally lower, and fermentation needs 7-10 days mostly, and the time of producing lactic acid is long.Therefore, compare, lack industrial applicibility with the fermentation of using milk-acid bacteria under oxygen free condition.
In addition, though above-mentioned Soccol etc. have also reported the cultured continuously that reuses rhizopus, when being to use this bacterial strain, the output of 3 later lactic acid reduces greatly, in order to prevent the generation of this thing, just must add urea as nitrogenous source in nutrient solution.
In addition, general employing lactic acid-producing bacteria is produced in the method for L (+) lactic acid, for L (+)-lactic acid that suppresses to produce hinders the generation of product, and in and the acidity of nutrient solution, must use lime carbonate and sodium hydroxide as in the neutralizing agent and L (+)-lactic acid.When obtaining lactic acid with high yield, it is at present known that to carry out the employed neutralizing agent of lactic fermentation with Rhizopus nearly all be lime carbonate, for separation and purification lactic acid from the calcium lactate that generates, except using the sedimentary method of interpolation sulfuric acid separating calcium sulphate, there is not other method, owing to generate a large amount of byproduct calcium sulfate, industrial also be a problem.Some the time when using ammonia to obtain DL-Lactic acid ammonium salt, because ammonia has hindered the activity of thalline, so be easy to occurring unfavorable situation aspect yield and the productivity as neutralizing agent.And above-mentioned Soccol etc. point out, do not use the neutralizing agent of the acid of conduct generations such as lime carbonate, but separate L (+)-lactic acid with poly-4-V-Pyrol RC (PVP) resin absorption, owing to cell is received the problem that damage exists output to reduce.
In addition, can produce lactic acid under the effect by the carbohydrate that contains polysaccharide various enzymes in thalline, if the nutritional requirement complexity of using of bacterial strain own, just must in nutrient solution, add various compositions, the lactic acid that separates the lactic acid-producing bacteria generation like this from nutrient solution is just relatively more difficult, is not easy to prepare the lactic acid that yield is good, purity is high.In addition, because culture condition generated by product, easy and simple to handle for the lactic acid purification phase just must select by product to generate few culture condition.On the other hand,, preferably reuse lactic acid-producing bacteria for high efficiency use thalline, if but generally reuse bacterial strain then be difficult to satisfy fully its nutritional requirement, it is many to generate the low occasion of lactic acid ability.Therefore, must select the strong bacterial strain of reusing property.
The present invention provides a kind of use nitrosoguanidine to make head mold MK96 (Rhizopussp.MK96) variation obtain having L (+)-lactic acid-producing bacteria of ammonia-resistance in view of above-mentioned problem.
In addition, also provide a kind of head mold MK96-1156 the bacterial strain of (Rhizopus sp.MK96-1156).
In addition, also provide a kind of method that above-mentioned bacterial strains is produced L (+)-lactic acid of under aerobic conditions, cultivating.
In addition, also provide a kind of head mold MK96 (Rhizopus sp.MK96).
Among the present invention,, just can use so long as belong to Rhizopus, have L (+)-lactic acid-producing bacteria of ammonia-resistance.Belong to microorganism Rhizopus, that have lactic acid fermentation ability, for example can enumerate unrooted rhizopus (Rhizopus arrhizus), moral row rhizopus equinus (Rhizopus delemar), Java head mold (Rhizopusjavanic), the head mold that turns black (Rhizopus nigricans), Rhizopus oryzae (Rhizopus oryzae), Rhizopus stolonifer (Rhizopus stolonifer) etc., wherein preferred Rhizopus oryzae (Rhizopus oryzae) or unrooted rhizopus (Rhizopus arrhizus), the ability height that L (+)-lactic acid generates.This be because these belong to Rhizopus, mushroom with lactic acid fermentation ability can be particulate state, the block cultivation.
L (+)-lactic acid-producing bacteria that uses among the present invention also must have ammonia-resistance.As such bacterial strain, there are present inventors to adopt following method to make the variant head mold MK96-1156 (Rhizopus sp.MK96-1156) that new isolated maternal plant head mold MK96 (Rhizopussp.MK96) sudden change obtains from soil.That is to say, the present inventor is in order to address the above problem, with L (+)-lactic acid-producing bacteria of obtaining having ammonia-resistance is purpose, newly isolate the thalline of the Rhizopus that can generate L (+)-lactic acid from nature, handle to make it having ammonia-resistance through variation, thereby obtain having higher L (+)-bacterial strain of lactic acid generative capacity.Therefore, even when using ammonia, also can produce L (+)-lactic acid, and can not cause yield to reduce as neutralizing agent.
(1) the 1st of maternal plant the screening
Sample thief 1g from paddy field soil makes it outstanding turbid with the 5ml sterile purified water, is modulated into sample stoste.With its suitably the final diluent that obtains of dilution (コ Application ラ-ジ) is excellent to be got 0.1ml respectively and is coated in the separation of the potato agar substratum that contains L (+)-lactic acid as shown in table 1 with on the nutrient agar with Kang Ladishi.Cultivate in 24 ℃ thermostatic bath the inoculation back, since the 3rd day, takes out the bacterium colony that grows up with platinum filament, is transplanted to have on the slant agar substratum of forming shown in the table 1.Pedotheque is got 1 time 50 strains approximately, and pedotheque is got to add up to for 100 times and isolated 3500 strains approximately and infer it is the bacterial strain of fungi.In these fungies, from the slant agar substratum, select 800 strains to think that form passes through bacterial strain near the bacterium of Rhizopus as 1 screening.In addition, the composition of the various nutrient agars shown in the table 1 is to represent with the g number in 1 liter of distilled water.
Table 1
| Separate and use agar matrix | Slant culture agar matrix | |
| Potato sucrose L (+)-lactic acid agar pH (before the sterilization) | 200 20 1 20 7(NaOH) | 200 20 1 20 7(NaOH) |
| With autoclave (1kg/cm 2) G sterilization 15 minutes | With autoclave (1kg/cm 2) G sterilization 15 minutes |
(2) adopt the test tube shaking culture to carry out the 2nd screening
Spore on the aforementioned slant agar substratum is got 1 platinum filament, be inoculated on the preceding substratum shown in the 10ml table 2, in test tube shaking culture device, cultivated 2 days.With 1ml this before culture medium inoculated in the test tube that the production substratum shown in the 10ml table 2 is housed, adopt and the same method shaking culture of preceding cultivation 1 day.Then, add lime carbonate 1g, further shaking culture is 2 days.With culture supernatant バ-カ-サ マ-ソ Application method colorimetric assay L (+)-lactic acid.For all bacterial strain 800 strains of passing through for 1 time, investigate its L (+)-lactic acid accumulation, the bacterial strain that the soil sample that discovery is gathered near the paddy field Shizuoka county Teng Zhi city obtains has the highest L (+)-lactic acid accumulation therein, with this bacterial strain called after head mold MK96 (RhizopussD.MK96).
Table 2
| Preceding substratum | Generate substratum | |
| Glucose (NH 4) 2SO 4 KH 2PO 4 ZnSO 4·7H 2O MgSO 4·7H 2O pH (before the sterilization) | 100 1.35 0.3 0.04 0.15 7(NaOH) | 120 3.02 0.25 0.04 0.25 7(NaOH) |
| With autoclave (1kg/cm 2) G sterilization 15 minutes | With autoclave (1kg/cm 2) G sterilization 15 minutes |
(3) the mycology feature of head mold MK96
Narrate the mycology property of this bacterium below.
(a) morphological specificity; With the potato agar substratum 24 ℃ of morphological features of cultivating down the head mold MK96 bacterial strain that obtains have following some:
1. during spore (sporangium) has every;
2. only form sporocyst, spherical in shape;
3. sporangiophore (sporangiophore) is pale asphyxia;
Rhizoid (Rhizoid) and stolon (stolon) are 4. arranged;
5. sporangiophore begins growth by the base portion of rhizoid;
6. sporocyst and subiculum are all very flourishing.
(b) physiologic character; Use Cai Shi (Czapek) substratum shown in the table 3, the influence of culture temperature and initial pH when this bacterium is cultivated in research.The result is shown in table 4-7.
Table 3
| The composition of Cai Shi (Czapek) substratum (all being 1 liter of g number in the distilled water) | |
| NaNO 3 KH 2PO 4 MgSO 4·7H 2O KCl FeSO 4·7H 2O sucrose | 2 1 0.5 0.5 0.01 30 |
| PH 6.0, usefulness autoclave (1kg/cm 2) the G sterilization | |
Table 4:(b-1) propagation temperature
| Temperature (℃) | Propagation | Temperature (℃) | Propagation |
| 20 25 30 35 | + + ++ ++ | 40 45 50 55 | + - - - |
| Can judge this bacterium breeding critical temperature at 40-45 ℃ according to the result | |||
Table 5:(b-2) propagation pH (30 ℃ of propagation temperature)
| pH | Propagation | pH | Propagation |
| 2 2.5 3 3.5 4.5 | - - + + + | 5.5 6 6.5 7 8 | ++ ++ + + ± |
Table 6:(b-3) various carbohydrate with whether breed
| Conversion sucrose is cultivated down for 24 ℃ on the potato agar substratum that has added the carbohydrate shown in following, observes its breeding state | |||
| Arabinose D-Fructose D-Glucose lactose D-mannital Alpha-Methyl-(+) glucoside D-gossypose sucrose | ++ ++ ++ ++ + ± + ++ | Dextrin D-galactolipin inulin maltose D-MANNOSE L-rhamnose D-wood sugar | ++ ++ + ++ ++ + + |
Table 7:(b-4) various carbohydrate with whether breed
| A situation arises for gas add various hydro carbons in Cai Shi (Czapek) substratum after | |||
| Arabinose D-Fructose D-Glucose lactose D-mannital Alpha-Methyl-(+) glucoside D-gossypose sucrose | - + + - - - ± + | Dextrin D-galactolipin inulin maltose D-MANNOSE L-rhamnose D-wood sugar | ± + - ± + - - |
(4) identify
From above morphology and physiological feature, carry out the mycology qualification test of this bacterium (head mold MK96).Criteria for classification (Zycha.H. according to Zycha etc., Siepmann and G.limmemann:Mucorales, lehre (1969)), because the sporocyst form of this bacterium is sphere or spheroid, form, have by many spores in every, this bacterium obviously should be the zygomycetes of Mucor (Mukoraceae) (Mucor) section.In addition, retrieve according to Mucor (Mukoraceae) purpose that Hesseltine (Hesseltine) and Ellis (Ellis) etc. propose, because the sporocyst of this bacterium possesses apophysis, sporangiophore has mycelia, upright with the rhizoid formation part of rhizoid and mycelia, so this bacterium should belong to Rhizopus (Rhizopus) (Rhizopus).
Rhizopus report now has 15 kinds, but according to (Inui T. such as Inui, Takeda Y.andIizuka H., Taxonomical studies on geus Rhizopus, The Journal of Generaland Applied Microbiology, 11,1-121 (1965)) key that proposes, contrast with the Rhizopus oryzae Went and Prinsen-Geerlings that is considered to the nearly edge of this bacterium, carry out the detailed evaluation of this bacterium simultaneously.
From morphological feature and judgements such as physiologic character czapek'S medium, this bacterial strain is the bacterial strain with the very near edge of Rhizopus oryzae Went and Prinsen-Geerlings.But as shown in table 8, with the type culture comparison of Rhizopus oryzae (Rhizopus oryzae), the growing amount of L (+)-lactic acid exceeds nearly 4 times.From this result as can be seen this bacterial strain be novel strain, called after head mold MK96 (Rhizopus sp.MK96) bacterial strain.Head mold MK96 (Rhizopus sp.MK96) is useful as the maternal plant of following useful dissociant head mold MK96-1156 (Rhizopus sp.MK96-1156).In addition, this bacterial strain is stored in Industrial Technology Institute microbiological industry technical institute of Ministry of International Trade and Industry of Japan on July 7th, 1998, preserving number is FERM P-16876, and this preservation is converted to preservation based on budapest treaty, be kept at same place, preserving number is FERM BP-6776.
Table 8
| Bacterial strain (R) | The growing amount of L (+)-lactic acid (g/ liter) |
| Rhizopus oryzae IFO4707 IFO4798 IFO5378 IFO5379 IFO5380 IFO5384 IFO31005 Rhizopus sp.MK96 | 8.5 11.5 11.2 13.2 11.2 10.6 9.78 40.6 |
(5) handle by sudden change, thereby induce L (+)-lactic acid-producing bacteria with ammonia-resistance to head mold MK96
Nitrosoguanidine (NTG) variation; Above-mentioned head mold MK96 bacterial strain is had on the slant agar substratum of composition as shown in table 1, cultivating 10 days for 24 ℃ times.On the inclined-plane, add the 5ml aqua sterilisa, well take off spore after, make the spore suspension liquid by cotton layer with the gauze parcel, separate removing the centrifugal that spore compiles, only collect spore.These spores are placed in the Tris malate suspension liquid outstanding turbid, after adding NTG and reaching 1mg/ml, at 24 ℃ of vibration spore suspension liquids 2.5 hours, 150 minutes down.Then, in the spore that centrifugation obtains, add the 5ml aqua sterilisa, carry out centrifugation once more.Carry out 2 this operations, with behind the spore wash clean, the concentration gradient that is coated in according to L (+)-DL-Lactic acid ammonium salt is that the nutrient agar (culture dish) that 0～100g/l makes is gone up cultivation 3 days (concentration gradient plating method, Science under 24 ℃ of conditions fully, 116,46-48 (1952)).Then, take out, cultivated 10 days down at 24 ℃ growing the bacterium colony that gets up than higher part breeding in L (+)-DL-Lactic acid ammonium salt salt concn.
(6) selection of L (+)-lactic acid-producing bacteria
The dissociant that above-mentioned (5) of 1 platinum filament are obtained is inoculated into and contains in the test tube of producing substratum shown in the 10ml table 2, cultivates in test tube shaking culture device after 1 day under 35 ℃, adds the lime carbonate 1g of sterilization, cultivates 2 days again.Adopt L (+)-lactic acid in バ-カ-エ マ-ソ Application method colorimetric assay culture supernatant.In all bacterial strains, select flask to cultivate the growing amount of back L (+)-lactic acid than 10 strains of higher bacterial strain, relatively cultivate with ammonia neutral method in the jar fermenter shown in the aftermentioned embodiment 1, general's bacterial strain that wherein L (+)-lactic acid growing amount is the highest is as head mold MK96-1156 (Rhizopus sp.MK96-1156).This bacterial strain is stored in Industrial Technology Institute microbiological industry technical institute of Ministry of International Trade and Industry of Japan on July 7th, 1998, preserve number is FERM P-16877, this preservation is converted to preservation based on budapest treaty, is kept at same place, preserving number is FERM BP-6777.
(7) production method of L (+)-lactic acid
Make L (+)-lactic acid-producing bacteria with ammonia-resistance used in the present invention produce L by the aerobic cultivation
(+)-lactic acid.The present invention can use spore or the mycelia that separates the head mold MK96-1156 bacterial strain that obtains, and is inoculated on the substratum that contains nutrition source, makes its propagation under the condition of aerobic, thereby produces L (+)-lactic acid.
L (+)-lactic acid-producing bacteria with ammonia-resistance is cultivated in the present invention's separation, under the condition of aerobic, they in batches, semi-batch or successive cultivate in aeration-agitation type or bubble-column-type bio-reactor, further, by being accompanied by the batch culture repeatedly that thalline utilizes once more, can accessing L (+)-lactic acid of high yield and produce high efficiency.The preparation method of L (+)-lactic acid below is described.
As the nutrition source of L (+)-lactic acid-producing bacteria with ammonia-resistance, the common operable nutrition source that can assimilate as hydro carbons, nitrogenous source, inorganics etc.For example can enumerate W-Gum, hominy grits, starch, dextrin, maltose, glucose, glycerine, sucrose, molasses etc., can use separately or as mixture as carbon source.Can enumerate inorganic or organic nitrogen sources such as ammonium sulfate, SODIUMNITRATE, soyflour, corn steep liquor, gluten meal, meat extract, fat meat meal tankage, yeast extract, dry yeast, cottonseed meal, peptone, wheatgerm, fish meal, minced meat, defatted rice bran, defatted meat bone meal, malt extract, corn gluten meal as nitrogenous source, can use separately or as mixture.Can enumerate various inorganic salt such as lime carbonate, sodium-chlor, Repone K, sal epsom, Sodium Bromide, Sodium Tetraborate or potassium primary phosphate, zinc sulfate, sal epsom as inorganic salt, can use separately or as mixture.In addition, heavy metals such as the iron of the interpolation trace of corresponding necessity, manganese, zinc, cobalt, molybdic acid.In addition, get final product, can use any nutrition source, also can use any well-known fungus culture material so long as produce the material of L (+)-lactic acid.Also have,, also can add the defoamer of senior alcohols, various siloxanes etc. such as soybean oil, linseed oil wet goods vegetables oil, Stearyl alcohol in order to eliminate the foamed phenomenon in heat sterilization and the culturing process.The proportioning of aforesaid nutrition source has no particular limits, and can change in very wide scope, can determine optimum nutrition source and proportioning easily by simple bench-scale testing according to working conditions.
In addition, in order to make pH after nutritional medium is sterilized before cultivation about 5-7, preferably use sodium hydroxide, ammoniacal liquor or ammonia to regulate the pH value.
In addition, there is not specific restriction for the bacterium method of planting that belongs to Rhizopus among the present invention, has L (+)-lactic acid-producing bacteria spore of ammonia-resistance, usually can use above-mentioned spore outstanding turbid in following liquid, this suspension liquid direct inoculation to fermentation with the method for growing bacterium on the liquid nutrient medium.
As the outstanding purifying method of above-mentioned spore, for example can use on nutrient agar and cultivate, in the Rhizopus that forms this spore, have and mix stirring method after adding liquid in the thalline of L (+)-lactic acid-producing bacteria of ammonia-resistance.In addition; normally used as aforesaid liquid is sterilized water; also can use and add a small amount of (for example whole liquid being accounted for the degree of 0.01 weight %), for example the sterilized water of Triton tensio-active agent, acyl group anhydro sorbitols etc. such as Tweens tensio-active agent, Triton X series such as tween 80 at the operable material in biochemical field.According to the effective dispersal spore in sterilized water of this operation, thereby can access uniform suspension liquid.
With of the cultivation of this nutritional medium, can common employed liquid culture carry out when preparing L (+)-lactic acid by general fungi in principle this bacterial strain.The occasion of liquid culture can be used and leave standstill any of cultivation, stir culture, shaking culture or aerated culture etc.Preferred especially shaking culture, deep aeration-agitation are cultivated among the present invention.When cultivating, the preferred 0.05-3.0vvm of import volume of preferred aerobic cultivation, oxygen or pure oxygen, more preferably 0.2-1.0vvm.Culture apparatus for satisfying this condition does not have specific limited, but preferred bubbling air or pure oxygen or both mixed gass on one side, on one side with any cultivation the in stirring-type reactor or the bubble-column-type reactor.Particularly thereby the bubble-column-type reactor has mycelia and can not be wrapped in the advantage that can prevent on the agitating vane that mycelia itself is damaged.
And, cultivate importing successive that operation can be by air or oxygen, each in new nutrient solution batch-wise, or by supplying with cultivating of new nutrient solution semi-batch.And, can reuse thalline when carrying out in batches.
Do not limit for culture temperature is special,, can produce L (+)-lactic acid and get final product as long as do not hinder this bacterial strain propagation in fact, but generally at 20-40 ℃, the temperature in preferred 35-40 ℃ the scope.
In addition, incubation time is preferably according to the consumption of carbohydrate sources such as glucose and produce that the time of L (+)-lactic acid, the kind of reactor etc. are comprehensive to be judged.Head mold MK96-1156 particularly of the present invention owing to produce the active strong of L (+)-lactic acid, for example adopts batch-wise occasion preferred 20-80 hour, more preferably to obtain L (+)-lactic acid fully in 30-50 hour at 20-100 hour.On the other hand, in this L (+)-milk-acid bacteria because the bacterial strain that uses continuously the activity that causes producing L (+)-lactic acid to reduce is less, thereby also can batch-type, semi-batch reuse or successive uses.The continuous occasion of using, the activity of can this bacterial strain of investigation at any time producing L (+)-lactic acid is adjusted the suitable culture time.
In addition, the preferred 4-8 of the pH of substratum, more preferably 5-7.Be because the condition of production in this scope is good.In order to reach this purpose, preferably before giving medium sterilization, add lime carbonate or in culturing process, add aqueous sodium hydroxide solution or ammoniacal liquor or ammonia etc. with pH transmitter or pH controller, automatically keep suitable pH value.Particularly, preferably regulate the pH value among the present invention by importing ammoniacal liquor or ammonia.Reason be following some.
That is to say, the occasion of producing on a large scale, the general lime carbonate that uses in L (+)-lactic fermentation still, when separation and purification free L (+) from nutrient solution-lactic acid, can bring very large difficulty in the time of most of.When lime carbonate uses as neutralizing agent,, when producing free L (+)-lactic acid, just must add sulfuric acid, make it to generate the calcium sulfate of indissoluble, thereby separated because L (+)-lactic acid that generates is transformed into the form of L (+)-calcium lactate.Therefore, a large amount of calcium sulfate that generate when handling mass production just necessarily require a large amount of labours and funds.
On the other hand, when using ammoniacal liquor or ammonia as neutralizing agent, L (+)-lactic acid of generation becomes the form of ammonium salt.When this L (+)-lactic acid ammonium salt adopts two-way electrodialysis to generate free L (+)-lactic acid, can reclaim produced simultaneously ammonia, thereby in fermentation, reuse.In addition, add alcohols such as butanols, directly make L (+)-lactic acid ammonia esterification, also can when obtaining L (+)-lactate, reclaim ammonia, thereby in fermentation, reuse.And, also can add the sulfuric acid of calculated amount, make it to generate the useful ammonium sulfate of as fertilizer sources, adopt method separation L (+)-lactic acid such as extraction, the processing of ammonia is had diversity.
General ammonia has inhibition to the propagation of microorganism, if the concentration of ammonia reaches more than the certain limit, almost can hinder the carrying out of propagation.But the lactic acid-producing bacteria that belongs to Rhizopus that uses among the present invention is L (+)-lactic acid-producing bacteria with ammonia-resistance, even in the presence of ammonia, also can bring into play the ability of outstanding production L (+)-lactic acid.Therefore, production L (+)-lactic acid that this processing can high yield is easy to comprise the aftertreatment of handling the salt that generates, separation and purification L (+)-lactic acid etc. simultaneously.
According to the present invention, can provide a kind of L (+)-lactic acid-producing bacteria with ammonia-resistance.Use this bacterial strain, by under the condition of aerobic, cultivating, production L (+)-lactic acid that can high yield.In addition,, can remove the inhibition that neutralizing agent or product bring by in substratum, adding ammonia, and production L (+)-lactic acid that uses ammonia can not cause L (+)-lactic acid-producing ability to reduce, can high yield.Because preceding cultivation is carried out 1 time and got final product, the repeated batch fermentation of this bacterial strain can shorten the production time, and yield is higher.
Because the preparation method according to above-mentioned L (+)-lactic acid can obtain L (+)-lactic acid less expensively, therefore can be used as the preparation that foodstuff additive are used for drinks, refreshment drink, salted vegetables, soy sauce, bread, beer etc., in addition also can be as extensively utilizing in the preparation of leather, fiber, plastics etc. in industry.
Below, the present invention is described in conjunction with the embodiments, but the present invention is not limited thereto.In addition, " % " expression " weight % ".
The batch fermentation production of embodiment 1:L (+)-lactic acid (aeration-agitation type reactor)
On the slant agar substratum shown in the table 1, cultivate head mold MK96-1156, collect spore then.Be inoculated in the 500ml Erlenmeyer flask that preceding substratum 100ml as shown in table 9 is housed, making spore concentration is 10
6Individual/ml.Use rotation electromagnetic shaker (rotation number 170rpm, rotation radius 2cm) that it was cultivated 15 minutes down at 30 ℃.
Then, in that the aforementioned preceding nutrient solution of the middle interpolation of 2.5 liters of jar fermenters (ball water chestnut バ イ オ エ Application ジ (strain), Tokyo) that the based 1.5 liter is cultivated in the production shown in the table 9 is housed, make it to become 10% (v/v) that cultivates base unit weight.At rotating speed is that 300rpm, air flow are that 0.5vvm, culture temperature are under 35 ℃ the condition its aeration-agitation to be cultivated.After cultivating beginning, be 5.5, add 10% ammoniacal liquor by the pH controller in order to keep the pH value.Fermenting process is as shown in table 10.
Before inoculate behind the nutrient solution 24 hours, sugared consumption enlivened, and meanwhile begins to produce L (+)-lactic acid.L (+)-lactic acid of 76 grams per liters (amount of substratum is 1.64 liters during end) is produced in glucose completely dissolve after 72 hours.At this moment the turnout of L (+)-lactic acid is 25g/ liter/day, is benchmark with the glucose that adds, and the productive rate of L (+)-lactic acid surpasses 69.2%.For passing through in the ammonia and production L (+)-lactic acid, this is very high yield.
Table 9
| The composition (g) of preceding substratum | Produce the composition of substratum | |
| Starch glucose (NH 4) 2SO 4 KH 2PO 4 ZnSO 4 MgSO 4·7H 2O CaCO 3Tap water pH (before the sterilization) | 50 50 1.35 0.3 0.04 0.25 10.0 make total amount reach 1 liter 6.8 | -120 1.35 0.25 0.04 0.15-make total amount reach 1 liter 4.8 |
| With autoclave (1kg/cm 2) G sterilization 15 minutes | ||
Table 10
| Time (h) | Glucose (g/ liter) | L (+)-lactic acid (g/ liter) |
| 0 20 30 44 65 72 | 120 112 96 77 13 0 | 0 1 20 33 68 76 |
Comparative example 1
Use is as the head mold MK96 bacterial strain of head mold MK96-1156 starter strain, under the culture condition identical with embodiment 1, uses jar fermenter to cultivate.Its result is as shown in table 11.
As can be seen from Table 11, even head mold MK96 bacterial strain was cultivated beginning after 72 hours, the accumulation of L (+)-lactic acid is below 50% of head mold MK96-1156 just also, only accumulated 30g/ liter, final liquid measure and be L (+)-lactic acid of 1.54 liters.Glucose with adding is the productive rate that benchmark calculates, and is 25.7% after 72 hours.From this comparative example also as can be seen, head mold MK96-1156 is very outstanding as the generation bacterium of L (+)-lactic acid.
Table 11
| Time (h) | Glucose (g/ liter) | L (+)-lactic acid (g/ liter) |
| 0 10 20 30 44 65 72 | 120 118 115 95 81 70 62 | 0 0 0.2 12 19 25 30 |
Comparative example 2
Except that using Rhizopus oryzae NRRL395 to replace the head mold MK96-1156, under the condition identical, operate with embodiment 1.Final liquid measure after 147 hours is 1.50 liters, and yield is 44.2%.The result is as shown in table 12.
As can be seen from Table 12, if with former Rhizopus oryzae NRRL395 bacterial strain relatively, this head mold MK96-1156 bacterial strain is to produce L (+)-high lactic-acid-producing strain of lactic acid yield.
Table 12
| Time (h) | Glucose (g/ liter) | L (+)-lactic acid (g/ liter) |
| 0 9 24 45 53 103 147 | 120 112 89 62 54 20 0 | 0 3 16 24 26 44 53 |
Embodiment 2; The batch production of L (+)-lactic acid in the bubble-column-type reactor
On the slant agar substratum of forming shown in the table 1, cultivate head mold MK96-1156, collect spore then.Be inoculated in the 500ml Erlenmeyer flask that the preceding substratum 100ml shown in the table 9 is housed, making spore concentration is 10
6Individual/ml.Use rotation electromagnetic shaker (rotating speed 170rpm, rotation radius 2cm) that it was cultivated 15 minutes down at 30 ℃.
Then, in 2.5 liters of bubble-column-type reactors that the production cultivation based 1.5 liter shown in the table 9 is housed, add aforementioned preceding nutrient solution, make it to become 10% (v/v) that cultivates base unit weight.At air flow is that 1.0vvm, culture temperature are under 35 ℃ the condition, and only ventilation is cultivated.After cultivating beginning, be 5.5, add 10% ammoniacal liquor by the pH controller in order to keep the pH value.Culturing process is as shown in table 13.
As can be seen from Table 13, compare with the aeration-agitation type reactor shown in the embodiment 1, the production rate of L (+)-lactic acid is fast, has accumulated 81g/ liter (cultivating base unit weight during end is 1.72 liters) after 50 hours, and its productive rate of glucose that adds is 77.4% relatively.Take all factors into consideration preferred bubble-column-type reactor from the aspects such as easy degree that manufacturing cost, running cost and the scale of fermenter are magnified.
Table 13
| Time (h) | Glucose (g/ liter) | L (+)-lactic acid (g/ liter) |
| 0 8 23 28 31 35 47 50 | 120 118 101 78 65 41 4 0 | 0 0.1 12 26 38 53 78 81 |
The repeated batch fermentation production of embodiment 3:L (+)-lactic acid (bubble-column-type reactor)
Adopt the method identical, use 100 liters larger bubbles tower reactor to carry out batch culture repeatedly with embodiment 2.The amount of the production substratum that adds for the 1st time is 65 liters.In addition, the ammoniacal liquor of use 28% is adjusted the pH value.As shown in table 14, confirm to cultivate after the glucose completely dissolve as carbon source in the 2nd day of beginning back, stop ventilation, make the mycelia piece of generation be deposited in the bottom of reactor.Take out supernatant liquor then, obtain containing about 10 liters of the precipitation part of mycelia piece.Add the production substratum after just having sterilized, make total amount reach 65 liters, ventilating once more begins to cultivate.This batch culture repeatedly comprises once common 9 times repeatedly at first.
As can be seen from Table 14, necessary fate finishes to cultivate after shortening from the 2nd time to the 6th time 1.5 days with the increase of number of times in 1 batch culture, and the 7th time is later on to finish after 2 days to cultivate.After 15.5 days, finish, carried out batch culture repeatedly altogether 9 times.In common batch culture, because fermentation needs 2.5 days, the washing of reactor needs 0.5 day again, can only carry out 5 times batch culture in 15.5 days.In contrast to this, though head mold MK96-1156 of the present invention 9 times repeatedly batch culture still have good productivity, compare with this known method and have about 1.5 times lactic acid-producing, yield is very high.In addition, repeatedly in the batch culture,, compare, can reduce labour cost significantly with all must modulate preceding nutrient solution in the former batch culture at every turn because initial once get final product only carried out in preceding cultivation at this.
Table 4
| Number of times | Time (h) | Total fate (d) | Glucose (d/L) | L (+)-lactic acid (g/L) |
| 1 | 0 12 24 48 | 0 0.5 1.0 2.0 | 120 112 75 0 | 0 3 24 85 |
| 2 | 0 24 36 | 2.0 3.0 3.5 | 101 46 0 | 8 50 88 |
| 3 | 0 24 36 | 3.5 4.0 5.0 | 104 44 0 | 9 50 86 |
| 4 | 0 24 36 | 5.0 6.0 6.5 | 107 45 0 | 8 51 89 |
| 5 | 0 24 36 | 6.5 7.5 8.0 | 101 42 0 | 7 53 89 |
| 6 | 0 24 36 | 8.0 9.0 9.5 | 105 49 0 | 7 44 80 |
| 7 | 0 24 36 48 | 9.5 10.5 11.0 11.5 | 100 40 10 0 | 8 43 65 72 |
Table 4 (continuing)
| 8 | 0 24 48 | 11.5 12.5 13.5 | 99 36 0 | 6 44 72 |
| 9 | 0 24 48 | 13.5 14.5 15.5 | 95 34 0 | 9 45 68 |
Embodiment 4: use bubble-column-type reactor batch production L (+)-lactic acid
As producing substratum, except that in embodiment 2 employed compositions, having added hominy grits 1g/ liter, adopt identical operation, use the bubble-column-type reactor to ferment.Final liquid measure after 36 hours is 1.65 liters, and yield is 81.6%.The result is as shown in Table 15.Production rate has obtained significantly improving, and also can obtain higher productive rate simultaneously.
Table 15
| Time (h) | Glucose (g/ liter) | L (+)-lactic acid (g/ liter) |
| 0 5 20 24 27 30 36 | 119.0 121.5 77.2 64.4 50.0 37.6 0 | 0 0.5 35.0 45.5 57.5 68.0 89.0 |
Claims (6)
1. preserve number is L (+)-lactic acid-producing bacteria with ammonia-resistance of FERM BP-6777.
2.L the production method of (+)-lactic acid is characterized in that cultivating the described bacterial strain of claim 1 under aerobic conditions, adjust the pH of nutrient solution by adding ammonia.
3. the production method of L as claimed in claim 2 (+)-lactic acid, it is characterized in that above-mentioned aerobic cultivate by air or oxygen importing continuously, each in new nutrient solution in batches, or cultivate by supplying with new nutrient solution semi-batch ground.
4. the production method of L as claimed in claim 3 (+)-lactic acid when it is characterized in that above-mentioned aerobic cultivation is carried out with batch-type, is not carried out preceding cultivation, carries out batch-wise cultivation and reuse thalline.
5. the production method of L as claimed in claim 4 (+)-lactic acid is characterized in that above-mentioned aerobic is cultivated employing aeration-agitation type bio-reactor or the bubble-column-type bio-reactor carries out.
6. head mold MK96 (Rhizopus sp.MK96), it is preserved number is FERM BP-6776.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP209713/1998 | 1998-07-24 | ||
| JP209713/98 | 1998-07-24 | ||
| JP20971398A JP4132253B2 (en) | 1998-07-24 | 1998-07-24 | Ammonia-resistant L (+)-lactic acid-producing bacterium and L (+)-lactic acid production method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1254016A CN1254016A (en) | 2000-05-24 |
| CN1252244C true CN1252244C (en) | 2006-04-19 |
Family
ID=16577423
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99119265 Expired - Fee Related CN1252244C (en) | 1998-07-24 | 1999-07-24 | Ammonia-resistant L(+)-lactic acid-producing bacteria and production method of L(+)-lactic acid |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP4132253B2 (en) |
| CN (1) | CN1252244C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104245918A (en) * | 2012-04-19 | 2014-12-24 | 花王株式会社 | Method for producing lactic acid |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1332035C (en) * | 2003-07-03 | 2007-08-15 | 新疆威仕达生物工程股份有限公司 | Feed-batch fermentation process for preparing L-lactic acid |
| JP4469568B2 (en) | 2003-07-09 | 2010-05-26 | 三菱化学株式会社 | Method for producing organic acid |
| EP1672077B1 (en) | 2003-08-28 | 2015-01-21 | Mitsubishi Chemical Corporation | Process for producing succinic acid |
| EP1672067B1 (en) | 2003-09-17 | 2015-11-11 | Mitsubishi Chemical Corporation | Process for producing non-amino organic acid |
| BRPI0510919A (en) | 2004-05-20 | 2008-05-20 | Ajinomoto Kk | succinic acid producing bacteria and process for producing succinic acid |
| JP2006246846A (en) * | 2005-03-14 | 2006-09-21 | Musashino Chemical Laboratory Ltd | Method for producing lactic acid |
| BRPI0617491B1 (en) | 2005-10-18 | 2021-04-27 | Ajinomoto Co., Inc | PROCESS FOR THE PRODUCTION OF SUCCINIC ACID |
| US7993888B2 (en) | 2006-02-24 | 2011-08-09 | Mitsubishi Chemical Corporation | Bacterium having enhanced 2-oxoglutarate dehydrogenase activity |
| CN108474012B (en) * | 2015-12-24 | 2022-01-18 | 花王株式会社 | Process for producing organic acid |
-
1998
- 1998-07-24 JP JP20971398A patent/JP4132253B2/en not_active Expired - Fee Related
-
1999
- 1999-07-24 CN CN 99119265 patent/CN1252244C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104245918A (en) * | 2012-04-19 | 2014-12-24 | 花王株式会社 | Method for producing lactic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2000037196A (en) | 2000-02-08 |
| CN1254016A (en) | 2000-05-24 |
| JP4132253B2 (en) | 2008-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tay et al. | Production of L (+)‐lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor | |
| Taskin et al. | Efficient production of l-lactic acid from chicken feather protein hydrolysate and sugar beet molasses by the newly isolated Rhizopus oryzae TS-61 | |
| Liao et al. | Studying pellet formation of a filamentous fungus Rhizopus oryzae to enhance organic acid production | |
| CN101611767B (en) | Method for producing microbial fermentation bait for sea cucumbers | |
| Jin et al. | Rhizopus arrhizus–a producer for simultaneous saccharification and fermentation of starch waste materials to L (+)-lactic acid | |
| CN1252244C (en) | Ammonia-resistant L(+)-lactic acid-producing bacteria and production method of L(+)-lactic acid | |
| CN100334200C (en) | Glutamic acid capable of having high-yield glutamine | |
| CN1181423A (en) | Process for producing 2 -keto -L -gulonic acid or salt thereof | |
| Afifi | Enhancement of lactic acid production by utilizing liquid potato | |
| Lim et al. | Optimization of medium by orthogonal matrix method for submerged mycelial culture and exopolysaccharide production in Collybia maculata | |
| CN107201315A (en) | A kind of Paecilomyces thermaphila mutagenic strain and its method of mutagenesis and application | |
| US8343741B2 (en) | Pelletization process to control filamentous fungi morphology for enhanced reactor rheology bioproduct formation | |
| CN103773720B (en) | A kind of preparation method of microbial fertilizer | |
| Yao et al. | Utilization of protein extract from dairy manure as a nitrogen source by Rhizopus oryzae NRRL-395 for L-lactic acid production | |
| CN107058119A (en) | A kind of method for improving Cordyceps militaris liquid state fermentation production cordycepin and thermostable protein production of enzyme | |
| CN1840653A (en) | Mutant strain of lactobacillus casei, its preparation method and fermentation production of L-lactic acid | |
| CN113564215A (en) | Preparation method of biosurfactant taking carbon dioxide and/or lignocellulose as substrates | |
| Bermek et al. | The effect of fungal morphology on ligninolytic enzyme production by a recently isolated wood-degrading fungus Trichophyton rubrum LSK-27 | |
| KR19990022043A (en) | New Cellulose Producing Bacteria | |
| CN101223282A (en) | Method for fermentative production of N-acetyl-D-glucosamine by microorganism | |
| CN111019995A (en) | Method for producing vanillin by fermentation with eugenol as substrate | |
| CN115197977A (en) | Method for synthesizing lactic acid from lignocellulose by using fungus-bacterium mixed system with complementary functions | |
| CN1027084C (en) | Process for preparing L-sorbose | |
| CN1260004A (en) | Strain producing remarkable amount of epsilon-poly-L-Lysine and process for producing same | |
| KR101261002B1 (en) | Stenotrophomonas nitritireducens and Manufacturing method for hydroxy-fatty acid with high yield by using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20060419 Termination date: 20160724 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |