CN103305437A - L-ammonium lactate tolerant bacterium and application thereof - Google Patents
L-ammonium lactate tolerant bacterium and application thereof Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title abstract description 31
- 239000004251 Ammonium lactate Substances 0.000 title abstract 8
- 229940059265 ammonium lactate Drugs 0.000 title abstract 8
- 238000000855 fermentation Methods 0.000 claims abstract description 48
- 230000004151 fermentation Effects 0.000 claims abstract description 48
- 239000001963 growth medium Substances 0.000 claims abstract description 21
- 244000199866 Lactobacillus casei Species 0.000 claims abstract description 9
- 235000013958 Lactobacillus casei Nutrition 0.000 claims abstract description 9
- 229940017800 lactobacillus casei Drugs 0.000 claims abstract description 9
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 38
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 37
- 239000002609 medium Substances 0.000 claims description 37
- 238000002156 mixing Methods 0.000 claims description 35
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 30
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 claims description 28
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- 229940041514 candida albicans extract Drugs 0.000 claims description 25
- 239000012138 yeast extract Substances 0.000 claims description 25
- 108010046845 tryptones Proteins 0.000 claims description 23
- 239000003795 chemical substances by application Substances 0.000 claims description 21
- 230000003472 neutralizing effect Effects 0.000 claims description 21
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 20
- 229910052799 carbon Inorganic materials 0.000 claims description 20
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 19
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 18
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 18
- 229920001817 Agar Polymers 0.000 claims description 16
- 239000008272 agar Substances 0.000 claims description 16
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 9
- 229920002472 Starch Polymers 0.000 claims description 9
- 159000000003 magnesium salts Chemical class 0.000 claims description 9
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- 238000011534 incubation Methods 0.000 claims description 8
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 7
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- 229910052757 nitrogen Inorganic materials 0.000 abstract description 2
- 235000019286 ammonium lactate Nutrition 0.000 abstract 3
- RZOBLYBZQXQGFY-HSHFZTNMSA-N azanium;(2r)-2-hydroxypropanoate Chemical compound [NH4+].C[C@@H](O)C([O-])=O RZOBLYBZQXQGFY-HSHFZTNMSA-N 0.000 abstract 3
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 abstract 2
- NAOLWIGVYRIGTP-UHFFFAOYSA-N 1,3,5-trihydroxyanthracene-9,10-dione Chemical compound C1=CC(O)=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1 NAOLWIGVYRIGTP-UHFFFAOYSA-N 0.000 abstract 1
- -1 nitrogen ions Chemical class 0.000 abstract 1
- 239000001384 succinic acid Substances 0.000 abstract 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 28
- RZOBLYBZQXQGFY-UHFFFAOYSA-N ammonium lactate Chemical compound [NH4+].CC(O)C([O-])=O RZOBLYBZQXQGFY-UHFFFAOYSA-N 0.000 description 27
- 239000008103 glucose Substances 0.000 description 21
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 20
- 239000002253 acid Substances 0.000 description 20
- 239000000047 product Substances 0.000 description 18
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- 235000019341 magnesium sulphate Nutrition 0.000 description 10
- 238000012216 screening Methods 0.000 description 10
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- LZCLXQDLBQLTDK-UHFFFAOYSA-N ethyl 2-hydroxypropanoate Chemical compound CCOC(=O)C(C)O LZCLXQDLBQLTDK-UHFFFAOYSA-N 0.000 description 8
- 150000002500 ions Chemical class 0.000 description 7
- 229910019142 PO4 Inorganic materials 0.000 description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 6
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 6
- 239000006481 glucose medium Substances 0.000 description 6
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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 (Lactobacilluscasei) 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 strain Pfansteihl ammonium tolerance bacterial strain and the application in fermentation lactic acid producing ammonium thereof, belong to the 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 through Zeo-karb and obtain pure lactic acid solution.Can generate " green solvent " ethyl lactate with DL-Lactic acid ammonium salt and ethanol direct esterification, the purposes of the tool development prospect of ethyl lactate is to utilize the characteristics such as it is nontoxic, solvability good, not volatile, replace the many environmentally harmful solvent that is using at present, as: halo class, ethers and the chloro-fluorocarbon solvent that damages the ozone layer etc.Ethyl lactate is multiplex in 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, such as intermediate of medicine pindolol etc.As seen, the 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 microbial fermentation can specialty obtain Pfansteihl and pollution-free, so fermentation method is widely used in the production of Pfansteihl.Carry out the Pfansteihl fermentative production mainly with Rhizopus oryzae, moral formula Bacterium lacticum etc. 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 the tunning optical purity is not high.Lactic-acid-bacterium belongs to the homotype lactic fermentation, and transformation efficiency is high, and the product optical purity is also higher, belongs to photoheterotrophy type microorganism, must provide multiple nutrients material and somatomedin by the external world, such as amino acid, VITAMIN, nucleic acid base etc.
It is strong that occurring in nature produces the Pfansteihl ability, and can be applicable to industrial bacterial classification and only have lactobacillus, bacillus, streptococcus in the Sino-German Rhizopus of mould and the bacterium.In order to improve the output of L-lactic acid fermentation, reduce the manufacture cost, improve the quality of production, its production process has been carried out large quantity research both at home and abroad, mainly comprise: produce induction mutation of bacterium improvement, the development and use of low price raw material, the aspects such as optimization of zymotechnique.This shows, improve the study hotspot that Pfansteihl tunning optical purity, raising fermentation glucose acid invert ratio, the sugared ability of the raising anti-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 the fermentation to suppress owing to lactic acid generates the acid that causes, and regulates fermented liquid pH value and makes bacterial classification obtain higher lactic acid production at the pH value bottom fermentation that is fit to.Calcium carbonate is as neutralizing agent, and the fermented liquid main component is crude lactic acid calcium.Crude lactic acid calcium needs to 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 comes controlled fermentation liquid pH value take ammoniacal liquor as neutralizing agent, the product that directly obtains is DL-Lactic acid ammonium salt.But the accumulation of DL-Lactic acid ammonium salt can cause product to suppress, and the output of DL-Lactic acid ammonium salt is caused reducing to a certain extent.
Summary of the invention
The objective of the invention is to suppress for the accumulation that solves DL-Lactic acid ammonium salt in the fermenting process can cause product, the output of DL-Lactic acid ammonium salt is reduced, 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 kind of Pfansteihl ammonium tolerance bacterial strain, its Classification And Nomenclature was lactobacterium casei (Lactobacillus casei) LC-An226, had been preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC M 2013123 on April 3rd, 2013.
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 utilizes that high sugar is dull and stereotyped, succsinic acid is dull and stereotyped, the DL-Lactic acid ammonium salt plate screening obtains anti-high sugar, EMP Embden Meyerbof Parnas pathway is strengthened and do not decompose the bacterial strain that utilizes lactic acid after Low energy N+ ions mutagenesis.Obtained strains is cultivated domestication in being added with the kind liquid of threshold concentration DL-Lactic acid ammonium salt in the 3L fermentor tank, be that neutralizing agent comes controlled fermentation liquid pH value with ammoniacal liquor.The bacterium liquid of domestication after 72 hours is gone 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, the bacterium colony smooth surface is moistening
Gramstaining: the positive
The 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 anti-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 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 the aseptic plate, dries up with sterile wind, and be 20 KeV at Implantation Energy, implantation dosage is 2 * 10
15Ions/cm
2It is lower that it is carried out nitrogen is ion implantation.Ion implantation complete after, take out plate, under gnotobasis, with 1mL sterilized water wash-out, be applied on the high glucose medium, cultivated 48 hours 45～55 ℃ of lower inversions.
(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 are connected on the succsinic acid plate culture medium, cultivate 48 hours 45～55 ℃ of lower inversions.
(d) the dull and stereotyped multiple sieve of DL-Lactic acid ammonium salt: single bacterium colony that the ratio starting strain that step (c) is screened postpones to occur bacterium colony is chosen and is connected on the DL-Lactic acid ammonium salt plate culture medium, cultivates bacterium 48 hours down at 45～55 ℃.
(e) ammoniacal liquor domestication: the lactobacterium casei that step (d) is filtered out accesses slant medium, 45～55 ℃ of lower cultivations 24 hours, get slant culture access domestication substratum, in the 3L fermentor tank, spread cultivation, and sour with its product of ammonia neutralization of mass concentration 25%, the control pH value was tamed 96 hours in 6.2.Then the bacterium liquid after the domestication repeats above-mentioned steps until screen aimed strain as the starting strain of next round mutagenesis.
In above-mentioned screening method: the high glucose medium that step (b) adopts comprises the component of 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 the grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, the yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and the phosphoric acid salt; Neutralizing agent is light calcium carbonate.The succsinic acid plate culture medium that step (c) adopts is with the carbon source in 25%～35% the succsinic acid replacement high glucose medium, other components unchanged.The DL-Lactic acid ammonium salt plate culture medium that step (d) adopts is with the carbon source in 2%～3% the DL-Lactic acid ammonium salt replacement high glucose medium, other components unchanged.
In above-mentioned screening method: the slant medium that step (e) adopts comprises the component of 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 the grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, the yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and the phosphoric acid salt; Neutralizing agent is light calcium carbonate.
In above-mentioned screening method: the domestication substratum that step (e) adopts comprises the component of 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 the grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, the yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and the phosphoric acid salt; Neutralizing agent is the ammoniacal liquor of mass concentration 25%.
The application of lactobacterium casei (Lactobacillus casei) LC-An226 in the L-lactic acid fermentation ammonium may further comprise the steps:
1) the dull and stereotyped cultivation: lactobacterium casei LC-An226 is seeded on the plate culture medium cultivates, culture temperature is 45～55 ℃, and incubation time is 48 hours.
2) slant culture: the dull and stereotyped lactobacterium casei LC-An226 that cultivates of step 1) is seeded to slant medium 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: with step 2) is seeded in the 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 plate culture medium that step 1) adopts comprises the component of following mass percent: carbon source 1%～3%, nitrogenous source 1.0%～3.0%, inorganic salt 0.2%～0.8%, neutralizing agent 2%～4%, and 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 the grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, the yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and the phosphoric acid salt; Neutralizing agent is light calcium carbonate.
Step 2) slant medium that adopts comprises the component of 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%, and 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 the grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, the yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and the phosphoric acid salt; Neutralizing agent is light calcium carbonate.
The fermention medium that step 3) adopts comprises the component of 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 the grape sugar and starch; Nitrogenous source is one or more the mixing in Tryptones, extractum carnis, the yeast extract paste, and inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and the 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 a glucose acid invert ratio, and Pfansteihl ammonium that can enduring high-concentration is removed the product of general bacterial strain in L-lactic acid fermentation ammonium process and suppressed, and gained DL-Lactic acid ammonium salt purity is high; In the 5L fermentor tank, end product Pfansteihl ammonium content has reached 206g/L, has improved 128.9% than original starting strain L-lactic acid fermentation ammonium, 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 only are used for explanation the present invention, 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 anti-ammonium is carried out the lactobacterium casei starting strain in the present embodiment explanation.
The concrete steps of carrying out the domestication of the first step Low energy N+ ions mutagenesis screening and anti-ammonium are as follows:
(a) collecting cells: get 48 hours the fresh inclined-plane of lactobacterium casei LC-N235 of 45～55 ℃ of constant temperature culture and add sterilized water 10mL, scrape and wash bacterium colony and favour in the 250mL test tube with little granulated glass sphere, mixing on quick vortex mixer, break up bacterium colony, three pull-up fat filtered through gauze, filtrate simple staining microscopy is adjusted bacterial concentration, and evenly to coat the 100cm culture dish acellular overlapping until get the 0.1ml bacteria suspension.
(b) Low energy N+ ions mutagenesis: get the middle bacteria suspension of 0.1mL step (a) and evenly coat on the aseptic plate, microscopy is acellular, and overlapping person carries out Low energy N+ ions.This experiment Low energy N+ ions machine is the ion beam bioengineering center fixture.Under the energy of 20KeV, carry out ion implantation to lactobacterium casei.Implantation dosage is 2 * 10
15Ions/cm
2, target chamber vacuum tightness is 10
-3Pa injects with the 20S pulsed, and the control sample injected is not accepted in placement in the interval 15s, target chamber.Ion implantation complete after, take out plate, under gnotobasis, with 1mL sterilized water wash-out, be applied on the high sugared plate culture medium, cultivated 48 hours 45～55 ℃ of lower inversions.
(c) screening of mutagenic strain:
The dull and stereotyped primary dcreening operation of succsinic acid: the well-grown that step (b) is screened and the large single bacterium colony of transparent circle are chosen and are connected on the succsinic acid plate culture medium, 45～55 ℃ of lower cultivations 48 hours, filter 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 that screens on the succsinic acid flat board and the large single bacterium colony of transparent circle chosen be connected on the DL-Lactic acid ammonium salt plate culture medium, cultivated the bacterial strains that screening is not grown 48 hours 45～55 ℃ of lower inversions on the DL-Lactic acid ammonium salt flat board.
(e) determining of DL-Lactic acid ammonium salt threshold concentration: single colony inoculation that the primary dcreening operation finishing screen is chosen is to slant medium, 45～55 ℃ of lower cultivations 24 hours, the slant culture access has been added in the domestication substratum of 8% DL-Lactic acid ammonium salt, in the 3L fermentor tank, in 50 ℃, domestication is cultivated under the constant agitation rotating speed of 90rpm.It produces acid with ammonia neutralization, makes the nutrient solution pH value be stabilized in 6.5, tames 72 hours.With the concentration of DL-Lactic acid ammonium salt in the bio-sensing analysis-e/or determining domestication substratum, with the DL-Lactic acid ammonium salt concentration that the records critical DL-Lactic acid ammonium salt concentration as next round mutagenesis domestication.
(f) ammoniacal liquor gradient domestication: the single colony inoculation that will choose through above step finishing screen is to slant medium, 45～55 ℃ of lower cultivations 24 hours, the slant culture access is contained in the domestication substratum of threshold concentration DL-Lactic acid ammonium salt, in the 3L fermentor tank, in 45～55 ℃, domestication is cultivated under the constant agitation rotating speed of 90rpm.It produces acid with ammonia neutralization, makes the nutrient solution pH value be stabilized in 6.2.Tamed 72 to 96 hours.Then with the set out bacterium liquid of the kind liquid after the domestication as the next round mutagenesis screening, repeat the step of above mutagenesis, primary dcreening operation, multiple sieve and domestication, until filter out aimed strain.
Wherein, employed culture medium prescription (% is mass percent)
High sugared plate culture medium used in the 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 are that (wherein glucose 0.07MPa sterilized separately light calcium carbonate 0.1MPa 20 minutes to distilled water, 40min sterilizes separately), PH6.5.
Used succsinic acid plate culture medium replaces in the high glucose medium 30% glucose, other components unchanged in the step (c) with 25% succsinic acid.Used DL-Lactic acid ammonium salt plate culture medium replaces in the high glucose medium 30% glucose, other components unchanged in the step (d) with 2% DL-Lactic acid ammonium salt.
Used slant medium is in step (e) and the step (f): 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.
Used domestication substratum is in step (e) and the step (f): 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 the genetic stability experimental result as can be known, 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 the Pfansteihl ammonium in the 5L fermentation cylinder for fermentation.
The described culture medium prescription of 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 mutant strain lactobacterium casei LC-An226 be seeded on the dull and stereotyped minimum medium in be inverted under 50 ℃ of conditions cultivate 48h after, it is seeded to slant medium cultivates 50 ℃ of culture temperature, incubation time 24h.Slant culture is inoculated in the 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.Detect Pfansteihl ammonium content in the fermented liquid with the bio-sensing analyser behind the fermentation culture 48h, reached 206g/L, improved 58.46% than the Pfansteihl ammonium of the original bacterium LC-N235 that sets out under the equal culture condition.
Embodiment 4
The present embodiment explanation mutant strain lactobacterium casei LC-An226 produces the Pfansteihl ammonium in the 5L fermentation cylinder for fermentation.
The described culture medium prescription of 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 mutant strain lactobacterium casei LC-An226 be seeded on the dull and stereotyped minimum medium in be inverted under 50 ℃ of conditions cultivate 48h after, it is seeded to slant medium cultivates 50 ℃ of culture temperature, incubation time 24h.Slant culture is inoculated in the 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.Detect Pfansteihl ammonium content in the fermented liquid with the bio-sensing analyser behind the fermentation culture 48h, reached 170g/L, than the Pfansteihl ammonium output increased of the original bacterium LC-N235 that sets out under the equal culture condition 50%.
Embodiment 5
The present embodiment explanation mutant strain lactobacterium casei LC-An226 produces the Pfansteihl ammonium in the 5L fermentation cylinder for fermentation.
The described culture medium prescription of 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 mutant strain lactobacterium casei LC-An226 be seeded on the dull and stereotyped minimum medium in be inverted under 50 ℃ of conditions cultivate 48h after, it is seeded to slant medium cultivates 50 ℃ of culture temperature, incubation time 24h.Slant culture is inoculated in the 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.Detect Pfansteihl ammonium content in the fermented liquid with the bio-sensing analyser behind the fermentation culture 48h, reached 193g/L, than the Pfansteihl ammonium output increased of the original bacterium LC-N235 that sets out under the equal culture condition 48.46%.
Embodiment 6
The present embodiment explanation mutant strain lactobacterium casei LC-An226 produces the Pfansteihl ammonium in the 50L fermentation cylinder for fermentation.
The described culture medium prescription of 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 mutant strain lactobacterium casei LC-An226 be seeded on the dull and stereotyped minimum medium in be inverted under 50 ℃ of conditions cultivate 48h after, it is seeded to slant medium cultivates 50 ℃ of culture temperature, incubation time 24h.Slant culture is inoculated in the 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% behind the cultivation 12h is inoculated in the 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.Detect Pfansteihl ammonium content in the fermented liquid with the bio-sensing analyser, reached 189g/L, improved 57.35% than the Pfansteihl ammonium of the original bacterium LC-N235 that sets out under the equal culture condition.
Claims (6)
1. a 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 the L-lactic acid fermentation ammonium.
3. the application of lactobacterium casei according to claim 2 (Lactobacillus casei) LC-An226 in the L-lactic acid fermentation ammonium is characterized in that comprising the steps:
The dull and stereotyped cultivation: lactobacterium casei LC-An226 is seeded on the plate culture medium cultivates, culture temperature is 45～55 ℃, and incubation time is 48 hours;
Slant culture: the dull and stereotyped lactobacterium casei of cultivating of step 1) is seeded to slant medium cultivates, culture temperature is 45～55 ℃, and incubation time is 24 hours;
The slant culture of the lactobacterium casei L-lactic acid fermentation ammonium: with step 2) is inoculated in the 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 the L-lactic acid fermentation ammonium, it is characterized in that: described step 1) middle plateform substratum comprises the component of 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 the grape sugar and starch; Described nitrogenous source is one or more the mixing in Tryptones, extractum carnis, the yeast extract paste, and described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and the phosphoric acid salt; Described neutralizing agent is light calcium carbonate.
5. the application of lactobacterium casei LC-An226 according to claim 3 in the L-lactic acid fermentation ammonium, it is characterized in that: slant medium comprises the component of following mass percent described step 2): 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 the grape sugar and starch; Described nitrogenous source is one or more the mixing in Tryptones, extractum carnis, the yeast extract paste, and described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and the phosphoric acid salt; Described neutralizing agent is light calcium carbonate.
6. the application of lactobacterium casei LC-An226 according to claim 3 in the L-lactic acid fermentation ammonium, it is characterized in that: fermention medium comprises the component of following mass percent in the described step 3): 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 the grape sugar and starch; Described nitrogenous source is one or more the mixing in Tryptones, extractum carnis, the yeast extract paste, and described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and the 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 |
| 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 |
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| CN111235074A (en) * | 2020-03-30 | 2020-06-05 | 广东锦玛绿色食品科技有限公司 | Composite functional microbial inoculum of saccharomyces cerevisiae |
| CN111235074B (en) * | 2020-03-30 | 2020-09-18 | 广东锦玛绿色食品科技有限公司 | Composite functional microbial inoculum of saccharomyces cerevisiae |
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