CN103305437B - L-ammonium lactate tolerant bacterium and application thereof - Google Patents
L-ammonium lactate tolerant bacterium and application thereof Download PDFInfo
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Abstract
The invention discloses an L-ammonium lactate tolerant bacterial strain and an application thereof in producing L-ammonium lactate through fermentation. According to the invention, low-energy nitrogen ions are injected in mutant original strains, and then the strains are screened by using a high-sugar tablet, a succinic acid tablet and an ammonium lactate tablet so as to obtain high-sugar-resistant and EMP approach reinforced strains which do not decompose and use a lactic acid. In a 3L fermentation tank, the strains are subjected to cultivation and acclimatization in a culture medium filled with critical-concentration ammonium lactate, and a bacteria liquid subjected to acclimatization of 72 hours is used as an original bacteria liquid of the next mutagenesis and acclimatization. The process is repeated until a target strain (Lactobacillus casei) LC-An226 is screened. The strain has the characteristics of good ammonium lactate tolerance and high saccharic acid conversion rate. In a 5L fermentation tank, the output of L-ammonium lactate in the fermentation liquor reaches 206 g/L, and increased by 58.46% compared with the production of L-ammonium lactate by using original strains through fermentation, therefore, the strain has great social significance and economic values.
Description
Technical field
The present invention relates to a strain Pfansteihl ammonium tolerance bacterial strain and the application in fermentation lactic acid producing ammonium thereof, belong to microbial fermentation technology field.
Background technology
DL-Lactic acid ammonium salt can be used for the dermopathic medicine of production for treating at pharmacy field, also can be used for the additive of animal-feed.DL-Lactic acid ammonium salt solution can make the transition and obtain pure lactic acid solution through Zeo-karb.With DL-Lactic acid ammonium salt and ethanol direct esterification, can generate " green solvent " ethyl lactate, ethyl lactate purposes most with prospects is to utilize the features such as it is nontoxic, solvability good, not volatile, replace the many environmentally harmful solvent using at present, as: halo class, ethers and the chloro-fluorocarbon solvent that damages the ozone layer etc.Ethyl lactate is used for essence, spices, food and liquor industry in traditional consumer field, is good foodstuff additive; In addition, ethyl lactate can be used as the lubricant of slugging in pharmaceutical industry, as intermediate of medicine pindolol etc.Visible, Pfansteihl ammonium has very large researching value and development prospect.
Lactic acid can obtain by chemical synthesis, microbe fermentation method (homotype lactic fermentation, heterolactic fermentation) and enzyme process several method are synthetic.Because microorganism fermentation can specialty obtain Pfansteihl and pollution-free, so fermentation method is widely used in the production of Pfansteihl.Mainly with Rhizopus oryzae, moral formula Bacterium lacticum etc., carry out Pfansteihl fermentative production both at home and abroad at present.Head mold belongs to heterolactic fermentation, and nutritional requirement is simple, but transformation efficiency is relatively low, and tunning optical purity is not high.Lactic-acid-bacterium belongs to homotype lactic fermentation, and transformation efficiency is high, and product optical purity is also higher, belongs to photoheterotrophy type microorganism, must provide multiple nutrients material and somatomedin by the external world, as amino acid, VITAMIN, nucleic acid base etc.
It is strong that occurring in nature produces Pfansteihl ability, and can be applicable to industrial bacterial classification and only have lactobacillus, bacillus, the streptococcus in the Sino-German Rhizopus of mould and bacterium.In order to improve the output of L-lactic acid fermentation, reduce manufacture cost, improve the quality of production, its production process has been carried out to large quantity research both at home and abroad, mainly comprise: produce induction mutation of bacterium improvement, the exploitation of low price raw material, the aspects such as optimization of zymotechnique.As can be seen here, improve the study hotspot that Pfansteihl tunning optical purity, raising fermentation glucose acid invert ratio, the sugared ability of the raising resistance to height of product etc. have become L-lactic acid fermentation.
The traditional technology of fermentation production of L-lactic acid is to adopt different calcium carbonate add-ons to eliminate in fermentation to suppress because lactic acid generates the acid causing, and regulates fermented liquid pH value to make bacterial classification obtain higher lactic acid production at applicable pH value bottom fermentation.Calcium carbonate is as neutralizing agent, and fermented liquid main component is crude lactic acid calcium.Crude lactic acid calcium need obtain crude lactic acid and byproduct calcium sulfate through vitriol oil acidolysis, and calcium sulfate is difficult to further be utilized, and a large amount of calcium sulfate accumulations can cause environmental pollution.The present invention be take ammoniacal liquor and is carried out controlled fermentation liquid pH value as neutralizing agent, and the product directly obtaining is DL-Lactic acid ammonium salt.But the accumulation of DL-Lactic acid ammonium salt can cause product to suppress, the output of DL-Lactic acid ammonium salt is caused to reducing to a certain extent.
Summary of the invention
The object of the invention is can cause product to suppress in order to solve the accumulation of DL-Lactic acid ammonium salt in fermenting process, the output of DL-Lactic acid ammonium salt is reduced, a strain Pfansteihl ammonium tolerance bacterial strain is provided, the output of its L-lactic acid fermentation ammonium is significantly improved.
In order to address the above problem, the technical solution used in the present invention is as follows:
A Pfansteihl ammonium tolerance bacterial strain, its Classification And Nomenclature is lactobacterium casei (Lactobacillus casei) LC-An226, on April 3rd, 2013, has been preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC M 2013123.
Bacterial strain---lactobacterium casei (Lactobacillus casei) LC-An226 screening method is in Pfansteihl ammonium of the present invention tolerance: starting strain lactobacterium casei LC-N235, after Low energy N+ ions mutagenesis, utilizes that high sugar is dull and stereotyped, succsinic acid is dull and stereotyped, DL-Lactic acid ammonium salt plate screening obtains resistance to high sugar, EMP Embden Meyerbof Parnas pathway strengthening and do not decompose the bacterial strain that utilizes lactic acid.Obtained strains, in 3L fermentor tank, is cultivated domestication in being added with the kind liquid of threshold concentration DL-Lactic acid ammonium salt, with ammoniacal liquor, is that neutralizing agent carrys out controlled fermentation liquid pH value.Bacterium liquid using domestication after 72 hours goes down to posterity for several times as the bacterium liquid that sets out of next round Inducing and taming.Repeat said process, until screen aimed strain lactobacterium casei (Lactobacillus casei) LC-An226 of resisting high-concentration Pfansteihl ammonium.
It is as follows that the morphology of bacterial strain of the present invention and Physiology and biochemistry are learned feature:
Colony colour: faint yellow
Bacterium colony size: 3mm ± 1mm
Colonial morphology: circular, bacterium colony smooth surface is moistening
Gramstaining: the positive
Microscopy form: thick, straight, shaft-like, chaining
Aerobic mode: amphimicrobian
Nutritional mode: chemoheterotrophy
Suitable growth temperature: 45～55 ℃
Suitable growth PH:6.0～7.0
Catalase reaction: feminine gender
Catalase reaction: feminine gender
Gelatine liquefication reaction: feminine gender
Glucose fermentation: aerogenesis not
Mutagenesis screening and the acclimation method of resistance to ammonium of Pfansteihl ammonium tolerance bacterium provided by the present invention, concrete steps are as follows:
(a) collecting cells: starting strain lactobacterium casei LC-N235 is made to bacteria suspension, and adjusting bacteria concentration is 10
6individual/milliliter.
(b) Low energy N+ ions mutagenesis: the bacteria suspension of getting in the step (a) of 0.1mL is evenly coated on aseptic plate, dries up with sterile wind, at Implantation Energy, is 20 KeV, and implantation dosage is 2 * 10
15ions/cm
2under that it is carried out to nitrogen is ion implantation.After ion implantation, take out plate, under gnotobasis, with 1mL sterilized water wash-out, be applied on high glucose medium, at 45～55 ℃, be inverted and cultivate 48 hours.
(c) the dull and stereotyped primary dcreening operation of succsinic acid: the well-grown that step (b) is screened and the large bacterial strain of transparent circle are chosen and be connected on succsinic acid plate culture medium, are inverted and cultivate 48 hours at 45～55 ℃.
(d) the dull and stereotyped multiple sieve of DL-Lactic acid ammonium salt: the ratio starting strain that step (c) is screened postpones to occur that single bacterium colony of bacterium colony is chosen is connected on DL-Lactic acid ammonium salt plate culture medium, cultivates bacterium at 45～55 ℃ 48 hours.
(e) ammoniacal liquor domestication: the lactobacterium casei access slant medium that step (d) is filtered out, at 45～55 ℃, cultivate 24 hours, get slant culture access domestication substratum, in 3L fermentor tank, spread cultivation, and sour with its product of ammonia neutralization of mass concentration 25%, control pH value in 6.2, tame 96 hours.The starting strain that the bacterium liquid of then take after domestication is next round mutagenesis, repeats above-mentioned steps until screen aimed strain.
In above-mentioned screening method: the component that the high glucose medium that step (b) adopts comprises following mass percent: carbon source 25%～35%, nitrogenous source 1.0%～3.0%, inorganic salt 0.2%～0.8%, neutralizing agent 2%～4%, agar 1.2%～1.5%, all the other are water, PH6.0～7.0; Wherein said carbon source is one or both the mixing in grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and phosphoric acid salt; Neutralizing agent is light calcium carbonate.The succsinic acid plate culture medium that step (c) adopts replaces the carbon source in high glucose medium, other components unchanged with 25%～35% succsinic acid.The DL-Lactic acid ammonium salt plate culture medium that step (d) adopts replaces the carbon source in high glucose medium, other components unchanged with 2%～3% DL-Lactic acid ammonium salt.
In above-mentioned screening method: the component that the slant medium that step (e) adopts comprises following mass percent: carbon source 0.3%～0.6%, nitrogenous source 1.0%～3.0%, inorganic salt 0.2%～0.8%, neutralizing agent 2%～4%, agar 1.5%～1.8%, all the other are water, PH6.0～7.0; Wherein said carbon source is one or both the mixing in grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and phosphoric acid salt; Neutralizing agent is light calcium carbonate.
In above-mentioned screening method: the component that the domestication substratum that step (e) adopts comprises following mass percent: carbon source 15%～25%, nitrogenous source 1.0%～3.0%, inorganic salt 0.2%～0.8%, DL-Lactic acid ammonium salt 8%～24%, neutralizing agent 12%～20%, all the other are water, PH6.0～7.0; Wherein said carbon source is one or both the mixing in grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and phosphoric acid salt; Neutralizing agent is the ammoniacal liquor of mass concentration 25%.
The application of lactobacterium casei (Lactobacillus casei) LC-An226 in L-lactic acid fermentation ammonium, comprises the following steps:
1) dull and stereotyped cultivation: lactobacterium casei LC-An226 is seeded on plate culture medium and is cultivated, and culture temperature is 45～55 ℃, and incubation time is 48 hours.
2) slant culture: the dull and stereotyped lactobacterium casei LC-An226 cultivating of step 1) is seeded to slant medium and cultivates, culture temperature is 45～55 ℃, and incubation time is 24 hours.
3) slant culture of the lactobacterium casei LC-An226 fermentation lactic acid producing ammonium: by step 2) is seeded in fermention medium to be cultivated, and culture temperature is 45～55 ℃, and mixing speed is 80 rpm, and pH is stabilized in 6 ~ 7, fermentation time 48～96 hours.
The component that the plate culture medium that step 1) adopts comprises following mass percent: carbon source 1%～3%, nitrogenous source 1.0%～3.0%, inorganic salt 0.2%～0.8%, neutralizing agent 2%～4%, agar 1.2%～1.5%, all the other are water, PH6.0～7.0; Wherein said carbon source is one or both the mixing in grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and phosphoric acid salt; Neutralizing agent is light calcium carbonate.
Step 2) component that the slant medium adopting comprises following mass percent: carbon source 0.3%～0.6%, nitrogenous source 1.0%～3.0%, inorganic salt 0.2%～0.8%, neutralizing agent 2%～4%, agar 1.5%～1.8%, all the other are water, PH6.0～7.0; Wherein said carbon source is one or both the mixing in grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and phosphoric acid salt; Neutralizing agent is light calcium carbonate.
The component that the fermention medium that step 3) adopts comprises following mass percent: carbon source 10%～25%, nitrogenous source 1.0%～3.0%, inorganic salt 0.2%～0.8%,, neutralizing agent 20%～50%, all the other are water, PH6.0～7.0; Wherein said carbon source is one or both the mixing in grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and phosphoric acid salt; Neutralizing agent is the ammoniacal liquor of mass concentration 25%.
Beneficial effect:
It is high that Pfansteihl ammonium provided by the invention tolerance bacterial strain has glucose acid invert ratio, and Pfansteihl ammonium that can enduring high-concentration, removes the product of general bacterial strain in L-lactic acid fermentation ammonium process and suppress, and gained DL-Lactic acid ammonium salt purity is high; In 5L fermentor tank, end product Pfansteihl ammonium content has reached 206g/L, than original starting strain L-lactic acid fermentation ammonium, has improved 128.9%, has great social effect and economic worth.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described concrete material proportion of embodiment, processing condition and result thereof be only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1
The method of Low energy N+ ions mutagenesis and the domestication of resistance to ammonium is carried out lactobacterium casei starting strain in the present embodiment explanation.
The concrete steps of carrying out the first step Low energy N+ ions mutagenesis screening and the domestication of resistance to ammonium are as follows:
(a) collecting cells: get 45～55 ℃ of constant temperature culture fresh inclined-plane of lactobacterium casei LC-N235 of 48 hours and add sterilized water 10mL, scrape and wash lower bacterium colony and favour in the 250mL test tube with little granulated glass sphere, on quick vortex mixer, mix, break up bacterium colony, three pull-up fat filtered through gauze, filtrate simple staining microscopy, adjusts bacterial concentration, until get 0.1ml bacteria suspension, evenly to coat 100cm culture dish acellular overlapping.
(b) Low energy N+ ions mutagenesis: get bacteria suspension in 0.1mL step (a) and evenly coat on aseptic plate, microscopy is acellular, and overlapping person carries out Low energy N+ ions.This experiment Low energy N+ ions machine is ion beam bioengineering center fixture.Under the energy of 20KeV, to lactobacterium casei, carry out ion implantation.Implantation dosage is 2 * 10
15ions/cm
2, target chamber vacuum tightness is 10
-3pa, injects with 20S pulsed, interval 15s, and in target chamber, the control sample of injecting is not accepted in placement.After ion implantation, take out plate, under gnotobasis, with 1mL sterilized water wash-out, be applied on high sugared plate culture medium, at 45～55 ℃, be inverted and cultivate 48 hours.
(c) screening of mutagenic strain:
The dull and stereotyped primary dcreening operation of succsinic acid: single bacterium colony that the well-grown that step (b) is screened and transparent circle are large is chosen and is connected on succsinic acid plate culture medium, cultivates 48 hours at 45～55 ℃, filters out the bacterial strain that postpones to occur bacterium colony than starting strain.
(d) the dull and stereotyped multiple sieve of DL-Lactic acid ammonium salt: the well-grown screening on succsinic acid flat board and the large single bacterium colony of transparent circle are chosen and be connected on DL-Lactic acid ammonium salt plate culture medium, be inverted and cultivate 48 hours at 45～55 ℃, the bacterial strain that screening is not grown on DL-Lactic acid ammonium salt flat board.
(e) determining of DL-Lactic acid ammonium salt threshold concentration: single colony inoculation that primary dcreening operation finishing screen is chosen is to slant medium, at 45～55 ℃, cultivate 24 hours, slant culture access has been added in the domestication substratum of 8% DL-Lactic acid ammonium salt, in 3L fermentor tank, in 50 ℃, under the constant agitation rotating speed of 90rpm, domestication is cultivated.With ammonia neutralization, it produces acid, makes nutrient solution pH value be stabilized in 6.5, tames 72 hours.By the concentration of DL-Lactic acid ammonium salt in bio-sensing analysis-e/or determining domestication substratum, the critical DL-Lactic acid ammonium salt concentration of domestication using the DL-Lactic acid ammonium salt concentration recording as next round mutagenesis.
(f) ammoniacal liquor gradient domestication: single colony inoculation that the above step finishing screen of process is chosen is to slant medium, at 45～55 ℃, cultivate 24 hours, in the domestication substratum that slant culture access is contained to threshold concentration DL-Lactic acid ammonium salt, in 3L fermentor tank, in 45～55 ℃, under the constant agitation rotating speed of 90rpm, domestication is cultivated.With ammonia neutralization, it produces acid, makes nutrient solution pH value be stabilized in 6.2.Tame 72 to 96 hours.The kind liquid of then usining after domestication, as the bacterium liquid that sets out of next round mutagenesis screening, repeats the step of above mutagenesis, primary dcreening operation, multiple sieve and domestication, until filter out aimed strain.
Wherein, the culture medium prescription using (% is mass percent)
Height sugar plate culture medium used in step (b) is: glucose 30%, Tryptones 1%, yeast extract paste 0.3%, bitter salt 0.05%, light calcium carbonate 3%, agar 1.5%, all the other be distilled water (glucose 0.07MPa wherein, sterilizing is 20 minutes separately, light calcium carbonate 0.1MPa, independent sterilizing 40min), PH6.5.
In step (c), succsinic acid plate culture medium used replaces in high glucose medium 30% glucose, other components unchanged with 25% succsinic acid.In step (d), DL-Lactic acid ammonium salt plate culture medium used replaces in high glucose medium 30% glucose, other components unchanged with 2% DL-Lactic acid ammonium salt.
In step (e) and step (f), slant medium used is: glucose 0.5%, and Tryptones 1%, yeast extract paste 0.3%, bitter salt 0.05%, light calcium carbonate 0.3%, agar 1.8%, all the other are distilled water, PH6.5.
In step (e) and step (f), domestication substratum used is: glucose 6%, DL-Lactic acid ammonium salt 8%, Tryptones 1%, yeast extract paste 0.3%, potassium primary phosphate 0.15%, all the other are water, and sour with its product of ammonia neutralization of mass concentration 25%, make its substratum PH be stable at 6.2,3L fermentor tank stirring velocity and be stabilized in 90 rpms.
embodiment 2
The genetic stability of the present embodiment explanation lactobacterium casei LC-An226.The fermentation test result that goes down to posterity is as shown in table 2
The genetic stability of table 2 lactobacterium casei (lactobacillus casei) LC-An226
| Passage number | Pfansteihl ammonium output (g/L) |
| 1 | 205 |
| 2 | 200 |
| 3 | 202 |
| 4 | 195 |
| 5 | 192 |
| 6 | 192 |
From genetic stability experimental result, through 6 continuous passages, mutant strain lactobacterium casei LC-An226 L-lactic acid fermentation ammonium output is more stable, has good mitotic stability, can be used as the production bacterial strain of further research and development.
embodiment 3
The present embodiment explanation mutant strain lactobacterium casei LC-An226 produces Pfansteihl ammonium in 5L fermentation cylinder for fermentation.
Culture medium prescription described in the present embodiment (% is mass percent):
Dull and stereotyped minimum medium: glucose 0.5%, Tryptones 1%, yeast extract paste 0.3%, bitter salt 0.05%, light calcium carbonate 3%, agar 1.8%, all the other are distilled water, PH6.5.
Slant medium is: glucose 0.5%, and Tryptones 1%, yeast extract paste 0.3%, bitter salt 0.05%, light calcium carbonate 0.3%, agar 1.8%, all the other are distilled water, PH6.5.
Fermention medium is: glucose 22%, and Tryptones 1%, yeast extract paste 0.3%, potassium primary phosphate 0.15%, all the other are water, and sour with its product of ammonia neutralization of mass concentration 25%, make its substratum PH be stable at 6.5.
To screen to such an extent that mutant strain lactobacterium casei LC-An226 is seeded on dull and stereotyped minimum medium under 50 ℃ of conditions and is inverted and cultivates after 48h, and be seeded to slant medium and cultivate, 50 ℃ of culture temperature, incubation time 24h.Slant culture is inoculated in fermention medium, 50 ℃ of culture temperature, 5L fermentor tank mixing speed is 80rpm, its product acid of ammonia neutralization with mass concentration 25%, makes PH be stable at 6.2.After fermentation culture 48h, with bio-sensing analyser, detect Pfansteihl ammonium content in fermented liquid, reached 206g/L, than the Pfansteihl ammonium of the original bacterium LC-N235 that sets out under equal culture condition, improved 58.46%.
embodiment 4
The present embodiment explanation mutant strain lactobacterium casei LC-An226 produces Pfansteihl ammonium in 5L fermentation cylinder for fermentation.
Culture medium prescription described in the present embodiment (% is mass percent):
Dull and stereotyped minimum medium: glucose 0.5%, Tryptones 1%, yeast extract paste 0.3%, bitter salt 0.05%, light calcium carbonate 0.3%, agar 1.8%, all the other are distilled water, PH6.5.
Slant medium is: glucose 0.5%, and Tryptones 1%, yeast extract paste 0.3%, bitter salt 0.05%, light calcium carbonate 0.3%, agar 1.8%, all the other are distilled water, PH6.5.
Fermention medium is: molasses 10%, and Tryptones 1%, yeast extract paste 0.3%, potassium primary phosphate 0.15%, all the other are water, and sour with its product of ammonia neutralization of mass concentration 25%, make its substratum PH be stable at 6.5.
To screen to such an extent that mutant strain lactobacterium casei LC-An226 is seeded on dull and stereotyped minimum medium under 50 ℃ of conditions and is inverted and cultivates after 48h, and be seeded to slant medium and cultivate, 50 ℃ of culture temperature, incubation time 24h.Slant culture is inoculated in fermention medium, 50 ℃ of culture temperature, 5L fermentor tank mixing speed is 80rpm, its product acid of ammonia neutralization with mass concentration 25%, makes PH be stable at 6.2.After fermentation culture 48h, with bio-sensing analyser, detect Pfansteihl ammonium content in fermented liquid, reached 170g/L, than the Pfansteihl ammonium output of the original bacterium LC-N235 that sets out under equal culture condition, improved 50%.
embodiment 5
The present embodiment explanation mutant strain lactobacterium casei LC-An226 produces Pfansteihl ammonium in 5L fermentation cylinder for fermentation.
Culture medium prescription described in the present embodiment (% is mass percent):
Dull and stereotyped minimum medium: glucose 0.5%, Tryptones 1%, yeast extract paste 0.3%, bitter salt 0.05%, light calcium carbonate 0.3%, agar 1.8%, all the other are distilled water, PH6.5.
Slant medium is: glucose 0.5%, and Tryptones 1%, yeast extract paste 0.3%, bitter salt 0.05%, light calcium carbonate 0.3%, agar 1.8%, all the other are distilled water, PH6.5.
Fermention medium is: glucose 20%, and corn steep liquor 1%, yeast extract paste 0.2%, potassium primary phosphate 0.15%, all the other are water, and sour with its product of ammonia neutralization of mass concentration 25%, make its substratum PH be stable at 6.2.
To screen to such an extent that mutant strain lactobacterium casei LC-An226 is seeded on dull and stereotyped minimum medium under 50 ℃ of conditions and is inverted and cultivates after 48h, and be seeded to slant medium and cultivate, 50 ℃ of culture temperature, incubation time 24h.Slant culture is inoculated in fermention medium, 50 ℃ of culture temperature, 5L fermentor tank mixing speed is 80rpm, its product acid of ammonia neutralization with mass concentration 25%, makes PH be stable at 6.2.After fermentation culture 48h, with bio-sensing analyser, detect Pfansteihl ammonium content in fermented liquid, reached 193g/L, than the Pfansteihl ammonium output of the original bacterium LC-N235 that sets out under equal culture condition, improved 48.46%.
embodiment 6
The present embodiment explanation mutant strain lactobacterium casei LC-An226 produces Pfansteihl ammonium in 50L fermentation cylinder for fermentation.
Culture medium prescription described in the present embodiment (% is mass percent):
Dull and stereotyped minimum medium: glucose 0.5%, Tryptones 1%, yeast extract paste 0.3%, bitter salt 0.05%, light calcium carbonate 3%, agar 1.8%, all the other are distilled water, PH6.5.
Slant medium is: glucose 0.5%, and Tryptones 1%, yeast extract paste 0.3%, bitter salt 0.05%, light calcium carbonate 0.3%, agar 1.8%, all the other are distilled water, PH6.5.
Seed culture medium is: glucose 10%, and Tryptones 1%, yeast extract paste 0.3%, potassium primary phosphate 0.15%, all the other are water, and sour with its product of ammonia neutralization of mass concentration 25%, make its substratum PH be stable at 6.5.
Fermention medium is: glucose 20%, and corn steep liquor 1%, yeast extract paste 0.2%, potassium primary phosphate 0.15%, all the other are water, and sour with its product of ammonia neutralization of mass concentration 25%, make its substratum PH be stable at 6.5.
To screen to such an extent that mutant strain lactobacterium casei LC-An226 is seeded on dull and stereotyped minimum medium under 50 ℃ of conditions and is inverted and cultivates after 48h, and be seeded to slant medium and cultivate, 50 ℃ of culture temperature, incubation time 24h.Slant culture is inoculated in 5L seed culture medium, 50 ℃ of culture temperature, 5L fermentor tank mixing speed is 80rpm, its product acid of ammonia neutralization with mass concentration 25%, makes PH be stable at 6.2.Inoculum size with 10% after cultivation 12h is inoculated in 50L fermention medium, and the mixing speed of 50L fermentor tank is 80rpm, with its product acid of ammonia neutralization of mass concentration 25%, makes PH be stable at 6.2 equally.With bio-sensing analyser, detect Pfansteihl ammonium content in fermented liquid, reached 189g/L, than the Pfansteihl ammonium of the original bacterium LC-N235 that sets out under equal culture condition, improved 57.35%.
Claims (6)
1. lactobacterium casei (Lactobacillus casei) LC-An226, has been preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC M 2013123.
2. the application of lactobacterium casei claimed in claim 1 (Lactobacillus casei) LC-An226 in L-lactic acid fermentation ammonium.
3. the application of lactobacterium casei according to claim 2 (Lactobacillus casei) LC-An226 in L-lactic acid fermentation ammonium, is characterized in that comprising the steps:
1) dull and stereotyped cultivation: lactobacterium casei LC-An226 is seeded on plate culture medium and is cultivated, and culture temperature is 45～55 ℃, and incubation time is 48 hours;
2) slant culture: the dull and stereotyped lactobacterium casei of cultivating of step 1) is seeded to slant medium and cultivates, culture temperature is 45～55 ℃, and incubation time is 24 hours;
3) slant culture of the lactobacterium casei L-lactic acid fermentation ammonium: by step 2) is inoculated in fermention medium, and culture temperature is 45～55 ℃, and mixing speed is 80 ~ 120 rpm, and pH is stabilized in 6 ~ 7, cultivates 48 ~ 96h.
4. the application of lactobacterium casei LC-An226 according to claim 3 in L-lactic acid fermentation ammonium, it is characterized in that: the component that described step 1) middle plateform substratum comprises following mass percent: carbon source 0.3%～0.6%, nitrogenous source 1.0%～3.0%, inorganic salt 0.1%～0.5%, neutralizing agent 0.2%～0.5%, agar 1.2%～1.8%, all the other are water, PH6.0～7.0; Wherein said carbon source is one or both the mixing in grape sugar and starch; Described nitrogenous source is one or more the mixing in Tryptones, extractum carnis, yeast extract paste, and described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and phosphoric acid salt; Described neutralizing agent is light calcium carbonate.
5. the application of lactobacterium casei LC-An226 according to claim 3 in L-lactic acid fermentation ammonium, it is characterized in that: the component that described step 2), slant medium comprises following mass percent: carbon source 0.3%～0.6%, nitrogenous source 1.0%～3.0%, inorganic salt 0.1%～0.5%, neutralizing agent 0.1%～0.5%, agar 1.5%～1.8%, all the other are water, PH6.0～7.0; Wherein said carbon source is one or both the mixing in grape sugar and starch; Described nitrogenous source is one or more the mixing in Tryptones, extractum carnis, yeast extract paste, and described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and phosphoric acid salt; Described neutralizing agent is light calcium carbonate.
6. the application of lactobacterium casei LC-An226 according to claim 3 in L-lactic acid fermentation ammonium, it is characterized in that: the component that in described step 3), fermention medium comprises following mass percent: carbon source 10%～25%, nitrogenous source 1.0%～3.0%, inorganic salt 0.1%～0.5%, neutralizing agent 20%～50%, all the other are water, PH6.0～7.0; Wherein said carbon source is one or both the mixing in grape sugar and starch; Described nitrogenous source is one or more the mixing in Tryptones, extractum carnis, yeast extract paste, and described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and phosphoric acid salt; Described neutralizing agent is the ammoniacal liquor of mass concentration 25%.
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| CN102653725A (en) * | 2012-05-30 | 2012-09-05 | 南京工业大学 | Bacillus coagulans and application thereof in mixed fermentation to produce L-lactic acid |
| CN102653724A (en) * | 2012-05-30 | 2012-09-05 | 南京工业大学 | Lactobacillus casei and application thereof to produce L-lactic acid by fermentation |
| CN102787087A (en) * | 2012-08-07 | 2012-11-21 | 江南大学 | Lactobacillus casei and application of lactobacillus casei to feed |
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| CN102653725A (en) * | 2012-05-30 | 2012-09-05 | 南京工业大学 | Bacillus coagulans and application thereof in mixed fermentation to produce L-lactic acid |
| CN102653724A (en) * | 2012-05-30 | 2012-09-05 | 南京工业大学 | Lactobacillus casei and application thereof to produce L-lactic acid by fermentation |
| CN102787087A (en) * | 2012-08-07 | 2012-11-21 | 江南大学 | Lactobacillus casei and application of lactobacillus casei to feed |
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