CN102250800A - Oxalic acid degrading bacterium NJODL1 and application thereof - Google Patents
Oxalic acid degrading bacterium NJODL1 and application thereof Download PDFInfo
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Abstract
The invention discloses an oxalic acid degrading bacterium NJODL1, which is classified and named as Lc.lactis subsp.lactis with the collection number CGMCC No.4582. A strain has the effects of degrading oxalic acid, regulating oxalate metabolism and preventing and controlling calcium oxalate calculus of pets. The oxalic acid degrading bacterium NJODL1 has high tolerance on acids and bile salts, does not have antibiotic resistance on most antibiotics, is safe for mice and can be colonized in dogs. By adopting NJODL1, the absorption of oxalic acid in foods by a gastrointestinal tract can be reduced, the content of oxalic acid in urea is lowered, and the incidence rate of calcium oxalate calculus of dogs is further lowered. Regardless of being prepared into a food or a medicament or yoghourt or acidified milk, the strain has high acid resistance, high bile salt resistance, the effects of remarkably lowering urea oxalic acid and preventing and controlling calcium oxalate calculus of pets, and contains safe enterococcus.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of oxalic acid degradation bacteria NJODL1 and application thereof, particularly have degraded oxalic acid, regulate the oxalic acid metabolic function and prevent and treat the bacterial strain NJODL1 and the application in pet food, foodstuff additive or pharmaceutical production of calcinm oxalate calculus effect.
Background technology
Urinary stone sickness rate in global population is 6%～10%.And nearly 70%～80% in the lithangiuria, calcinm oxalate calculus.And up to the present, because the low-solubility of caoxalate, so the method for treatment calcinm oxalate calculus still mainly is to remove based on operation, this has brought great misery to the patient.The research report is arranged: in the forming process of calcinm oxalate calculus, oxalic acid absorption and metabolic disturbance role are big than calcium, and its slight variation just can cause the generation of calcium oxalate crystal.The source of oxalic acid is divided into endogenous and exogenous two portions in the body.Endogenous oxalic acid mainly is to produce as the end product of substance metabolisms such as vivo acid, VC, and exogenous oxalic acid mainly is to obtain by the food that absorption contains oxalic acid.Because vertebrates can not utilize oxalic acid to be carbon source, so most oxalic acid is got rid of by urine through kidney.Therefore the oxalic acid that absorbs in the food is the important source of urine mesoxalic acid.Colon is the main position that absorbs exogenous oxalic acid, has only 3%～5% oxalic acid to be absorbed by colon under the normal circumstances in the food.Yet in case the function of intestinal absorption oxalic acid gets muddled, the oxalic acid of intestinal absorption will increase, thereby produces hyperoxaluria.Although use the recipe of control oxalic acid intake can reduce the urine concentration of oxalic acid, in actual life, owing to oxalic acid is present in many foods, so this method can't realize.Concerning the normal people, the metabolism amount of endogenous oxalic acid is certain, therefore, reduces the absorption of enteron aisle to oxalic acid, is the comparatively feasible way of generation of prevention calcium oxalate nephrolithiasis.
It is reported that the calcinm oxalate calculus sickness rate only accounted for 5% of the whole urinary stone ratios of dog in 1981, and struvite calculus accounts for 78%.By 2007, the calcinm oxalate calculus sickness rate rose to 41%, and the sickness rate of struvite calculus then reduces to 40%.The reason that causes this phenomenon is in order to reduce the sickness rate of dog struvite calculus, often all can use souring agent to make animals urine maintain pH6.0-6.4 in the commodity food, and the main component of souring agent to be exactly oxalic acid or oxalate.The same with the mankind, the increase of food mesoxalic acid content has caused adult healthy dog urine oxalic acid and caoxalate supersaturation, thereby has increased the risk of suffering from calcinm oxalate calculus.
Oxalic acid is the simplest diprotic acid, low-yield territory, high reductibility, acidity and to the inhibition of some enzyme, and these chemical property make most of intestinal bacteria all can not utilize oxalic acid to be carbon source.Yet, in the complicated micro-ecological environment, also exist some can utilize the bacterium of oxalic acid.Can be the bacterium of sole carbon source with oxalic acid, we be called " specificity " oxalic acid degradation bacteria; And just with a kind of bacterium of oxalic acid as carbon source, we are called " facultative " oxalic acid degradation bacteria, and this bacterioid can utilize oxalic acid to earn a bare living under the environment condition of severe.
People such as Sidhu have set up the mouse model that utilizes bacterium to reduce urine oxalic acid, thereby utilize experimentation on animals to verify the symbiosis of Oxalobacter formigenes and the substantial connection between the oxalic acid metabolism.Murphy and Weese isolate the milk-acid bacteria of the oxalic acid of degrading and have carried out experimentation on animals in the dog enteron aisle respectively.Sanehlro Hokama also isolates the faecalis of the oxalic acid of degrading from the enteron aisle of rat, and external its relevant enzyme is purified.These experiment showed, because these bacteriums can utilize oxalic acid as the energy, enteron aisle absorbability oxalic acid content are lowered, so help preventing the formation of calcinm oxalate calculus.The bacterium that can decompose oxalic acid of report is arranged at present, comprise Oxalobacter formigenes, lactobacillus, enterococcus faecalis, eubacterium lentum, providencia rettgeri etc.
What separate in the animal intestinal is the bacterium of sole carbon source with oxalic acid though Oxalobacter formigenes is a unique strain, but because it is a strictly anaerobic bacterium, responsive especially to oxygen, place air will cause its death in 10 minutes, common laboratory is difficult to separation and obtains this bacterium.And this bacterium is responsive especially to cholate and microbiotic, and inflammatory bowel disease, microbiotic use, gallbladder cystic fibrosis (53%) can both cause the death of this bacterium in the enteron aisle.In case Oxalobacter formigenes death often is difficult to it be grown surely at enteron aisle again.Therefore, just be in conceptual phase before this Zoopagales, be difficult to be used on a large scale clinical treatment.
Yet also have the gallbladder cystic fibrosis patient of half nearly, although there is not the field planting Oxalobacter formigenes in the enteron aisle, the urine concentration of oxalic acid is still kept normally, also has other bacteriums that can utilize oxalic acid to exist in this explanation enteron aisle.Therefore, other " facultative " bacterium, particularly the ability of enteron aisle dominant bacteria degraded oxalic acid causes scientist's concern gradually.The milk-acid bacteria of Murphy and Weese report, the faecalis of Sanehlro Hokama report all has the ability of degraded oxalic acid.
Milk-acid bacteria is human and the main flora of animal intestinal, is usually used in the humans and animals probiotic products.Therefore, from the lower dog of calcinm oxalate calculus sickness rate, separate the probiotic lactobacillus of the oxalic acid of can degrading, oxalic acid metabolism in the oxalic acid that is used to degrade, the adjusting animal body, the fine biological control means of the dog calcinm oxalate calculus of can yet be regarded as.
Summary of the invention
The objective of the invention is to invent a kind of oxalic acid degradation bacteria NJODL1, this bacterial strain has degraded oxalic acid, regulates the effect of oxalic acid metabolic function and control pet calcinm oxalate calculus, in the hope of further development and use.
Another object of the present invention is to provide the screening method of above-mentioned oxalic acid degradation bacteria NJODL1.
Another purpose of the present invention is to provide described oxalic acid degradation bacteria NJODL1 to have application in pet food, foodstuff additive or the medicine of regulating the oxalic acid metabolic function in preparation.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of oxalic acid degradation bacteria NJODL1, this strain classification called after: Lactococcus lactis subsp.lactis (Lc.lactis subsp.lactis), (address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), culture presevation number is CGMCCNO.4582 to be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 25th, 2011.
This bacterial strain has excellent degraded oxalic acid ability, regulates the oxalic acid metabolism, acidproof and cholate ability, to antibiotic sensitive, to the small white mouse nontoxicity and have the good body milk-acid bacteria that grows the safety of effect decided at the higher level but not officially announced; By ne ar, physiology and cultural characteristic, identify and 16srDNA sequencing and comparing that in conjunction with the French Mei Liai VITEK compact of company 2 finally definite this bacterial strain is Lactococcus lactis subsp.lactis (Lc.lactis subsp.lactis).
The colonial morphology of Lactococcus lactis subsp.lactis NJODL1 of the present invention on the MRS substratum is: white, circular small colonies.Main biological characteristics is G
+, thalline is spherical, about 0.5～0.8 μ m of size becomes list or two existence, no gemma; The Genbank number of landing of this bacterial strain 16S rDNA is JF895186.
The screening method of above-mentioned oxalic acid degradation bacteria NJODL1 may further comprise the steps: (1) isolates milk-acid bacteria from the ight soil of the dog of less trouble calcinm oxalate calculus kind; (2), filter out and have the milk-acid bacteria that falls the oxalic acid ability by vitro detection strains for degrading oxalic acid ability; (3), bile tolerance ability acidproof, antibiotics sensitivity and to the security of mouse and prove that this bacterial strain can grow in that the dog body is decided at the higher level but not officially announced by the vitro detection bacterial strain; (4) have degraded oxalic acid ability by detection bacterial strain in the body and be NJODL1.
Described oxalic acid degradation bacteria NJODL1 has application in pet food, foodstuff additive or the medicine of regulating the oxalic acid metabolic function in preparation.
The described oxalic acid degradation bacteria NJODL1 that is applied as has the pet sour milk of regulating the oxalic acid metabolic function, the application in the fermented-milk in preparation.Have assisting therapy or prevent Hyperoxaluric effect.
Through experimentation on animals, the NJODL1 that the present invention screened has good acidproof and cholate ability, good colonization ability, by experimentation on animals, further confirms bacterial strain of the present invention to the experiment mice free of toxic effects, has the effect of obvious reduction urine concentration of oxalic acid.Finished product are that pet food, foodstuff additive or medicine all have assisting therapy or prevent Hyperoxaluric effect.
Microecology research normal microflora and its host's mutual relationship, little ecological therapy effect is special, direct, permanent, no obvious toxic-side effects.Probiotics---probiotic bacterium helps healthy number of bacteria and activity by improving some, rebuilds colony balance and the host is played beneficial effect.Common probiotic bacterium mainly comprises Bacterium lacticum, streptococcus acidi lactici, bifidus bacillus, faecium etc., and these bacteriums are the GI normal microfloras of healthy humans and animals.
Lactococcus lactis subsp.lactis NJODL1 provided by the present invention has excellent acidproof bile tolerance, to most of microbiotic antibiotic-free resistance; Confirm to have very strong degraded oxalic acid ability by external and experimentation on animals.
Beneficial effect of the present invention:
The oxalic acid degradation bacteria NJODL1 that the present invention screened can grow containing on the MRS nutrient agar of oxalic acid.Contain the MRS fermention medium of oxalic acid with oxalic acid degradation bacteria NJODL1 fermentation, the oxalic acid degradation rate is: 24.8%.The present invention also provides the enlarged culturing method of oxalic acid degradation bacteria NJODL1, and the thalline quantity in the product reaches 3.2 * 10
9CFU/mL.
Test shows, the oxalic acid degradation bacteria NJODL1 that the present invention screened has good tolerability to acid and cholate.According to the Standard Selection microbiotic of NCCLS (the stdn council of U.S. clinical labororatory), do the drug-resistant test of Lactococcus lactis subsp.lactis NJODL1, the result shows that Lactococcus lactis subsp.lactis NJODL1 is to most of microbiotic antibiotic-free resistance.
Further acute toxicity test proves that this Lactococcus lactis subsp.lactis NJODL1 does not have acute toxicity to small white mouse in the small white mouse body.And can grow in that the dog body is decided at the higher level but not officially announced.
The present invention further carries out interior therapeutic research with Lactococcus lactis subsp.lactis NJODL1 to Hyperoxaluric model dog, discover that probiotic lactic acid galactococcus lactic acid subspecies NJODL1 of the present invention can reduce the absorption of gi tract to the food mesoxalic acid, thereby reduce the content of urine mesoxalic acid, and then reduce the sickness rate of dog calcinm oxalate calculus.
Description of drawings
Fig. 1 is the aspect graph (gramstaining, 400 *) of Lactococcus lactis subsp.lactis NJODL1.
Fig. 2 is that the electrophoresis of the 16srDNA of Lactococcus lactis subsp.lactis NJODL1 is identified figure.
Wherein, M is DNAmarker (2000bp); Swimming lane 1 is the PCR product of NJODL1.
Fig. 3 is the kidney section (HE dyeing, 400 *) of 0.5% oxalic acid modeling group.
Fig. 4 is 0.5% oxalic acid+NJODL1 group kidney section (HE dyeing, 400 *).
Embodiment
The oxalic acid degradation bacteria NJODL1 of the present invention screening can grow containing on the MRS nutrient agar of oxalic acid.Contain oxalic acid MRS fermention medium with oxalic acid degradation bacteria NJODL1 fermentation, the oxalic acid degradation rate is: 24.8%.The present invention also provides the enlarged culturing method of oxalic acid degradation bacteria NJODL1, and the thalline quantity in the product reaches 3.2 * 10
9CFU/mL.
Further in-vitro simulated gastroduodenal environmental resistance discovers, Lactococcus lactis subsp.lactis NJODL1 be inoculated into pH be in 2.0 the sour environment behind the 120min amount of bacteria still can reach 10
4CFU/mL is inoculated into pH and is in 3.0 the sour environment that amount of bacteria still can reach 10 behind the 240min
6CFU/mL shows that Lactococcus lactis subsp.lactis NJODL1 is to the strong acidic environment well-tolerated; Simultaneously, Lactococcus lactis subsp.lactis NJODL1 amount of bacteria after being inoculated in the MRS liquid nutrient medium 24h that contains 0.3% cholate is 10
5CFU/mL is inoculated in that amount of bacteria is 10 behind the MRS liquid nutrient medium 12h that contains 1% cholate
3CFU/mL.Show that Lactococcus lactis subsp.lactis NJODL1 of the present invention has good tolerability to acid and cholate.
According to the Standard Selection microbiotic of NCCLS (the stdn council of U.S. clinical labororatory), do the drug-resistant test of Lactococcus lactis subsp.lactis NJODL1, the result shows that Lactococcus lactis subsp.lactis NJODL1 is to most of microbiotic antibiotic-free resistance.
Further acute toxicity test proves that this Lactococcus lactis subsp.lactis NJODL1 does not have acute toxicity to small white mouse in the small white mouse body.And can grow in that the dog body is decided at the higher level but not officially announced.
The present invention further carries out interior therapeutic research with Lactococcus lactis subsp.lactis NJODL1 to Hyperoxaluric model dog, discover that probiotic lactic acid galactococcus lactic acid subspecies NJODL1 of the present invention can reduce the absorption of gi tract to the food mesoxalic acid, thereby reduce the content of urine mesoxalic acid, and then reduce the sickness rate of dog calcinm oxalate calculus.
Oxalic acid degradation bacteria NJODL1 of the present invention can adopt the ordinary method of prior art to be prepared into pharmaceutical composition.This pharmaceutical composition contains the viable bacteria form of the Lactococcus lactis subsp.lactis NJODL1 of pharmacy effective dose.In addition, described pharmaceutical composition can also contain suitable pharmaceutical carrier.Described pharmaceutical composition can be capsule, solution or forms such as drinkable suspension, pockaged powder, and each single dose generally contains Lactococcus lactis subsp.lactis NJODL1 and is about 10
9～10
11Amount of bacteria.Described pharmaceutical composition can be used for the prevention and the treatment of pet hyperoxaluria and calcinm oxalate calculus, utilize unnecessary oxalic acid in the efficient bacterial strain metabolism animal body, not only can reduce the absorption of oxalic acid in the body, intestinal microflora in all right control agent, and have good application prospects.
Lactococcus lactis subsp.lactis NJODL1 of the present invention can also adopt ordinary method to prepare the form of food, healthcare products or foodstuff additive, and these food, healthcare products or foodstuff additive can be used for the prevention and the treatment of pet hyperoxaluria and calcinm oxalate calculus.Described food can be the form that contains the beverage of Lactococcus lactis subsp.lactis NJODL1 viable bacteria of the present invention, also can be forms such as the milk-product that contain this viable bacteria, fermented-milk.
Below by concrete example the present invention is done further detailed description.
Embodiment 1: the separation of Lactococcus lactis subsp.lactis and evaluation
1. culture medium preparation
The prescription of MRS liquid nutrient medium is to contain in the 1L distilled water: peptone 10g, yeast extract paste 5g, glucose 20g, extractum carnis 10g, sodium acetate 5g, Triammonium citrate 2g, tween 80 1mL, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.05g, K
2HPO
42g regulates pH=6.2～6.4; 121 ℃, high pressure steam sterilization 15min, standby.
2. sample source
Select the dog of 3 kinds that are difficult for trouble dog urinary calculus in certain dog field, each eight of the dogs of each kind, male and female half and half.All dogs all are the commodity dog grain (FOX dog grain, animal-derived food product factory of Science and Technology Development Center of Nanjing police dog institute) of feeding.Selected dog does not all have the history that changes dog grain, and does not use microbiotic in three months and diarrhoea did not take place.
Sample collecting with separate
The aseptic ight soil of taking above-mentioned dog.Before delivering to the laboratory,, finish the separation of bacterium in 4 hours with 4 ℃ of preservations of ight soil of taking.Be equipped with in the test tube of 4.5mL sterile saline 0.5g ight soil adds, fully shook 5 minutes, make 10 with the vortex instrument
-1Diluent, redilution obtains 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7Diluent.
Each ight soil diluent is got 0.1mL respectively be coated on the MRS flat board that contains 20 μ g/mL vancomycins, every sample repeats 3 plates.After placing 37 ℃ of anaerobism to cultivate 48h, the bacterium of the different colonial morphologies of picking carries out gramstaining and catalase test.The bacterium of Gram-positive, catalase test feminine gender is seeded to respectively on the inclined-plane, MRS basis carries out pure culture.It is standby in-70 ℃ of refrigerator cryopreservation to divide each pure strain bacterium can add behind the frostproofer.
4. the evaluation of Lactococcus lactis subsp.lactis NJODL1
(1) biochemical identification
Use French Mei Liai VITEK compact 2 to carry out biochemical identification.Bacteria Identification the results are shown in Table 1, by analysis, shows that the bacterial strain that the present invention screens is a Lactococcus lactis subsp.lactis.
The Bacteria Identification result of table 1 bacterial strain of the present invention
| AMY | - | PIPLC | - | dXYL | - | ADH1 | + | BGAL | - | AGLU | - |
| APPA | - | CDEX | - | AspA | + | BGAR | - | AMAN | - | PHOS | - |
| LeuA | - | ProA | - | BGURr | - | AGAL | - | PyrA | - | BGUR | - |
| AlaA | + | TyrA | + | dSOR | - | URE | - | POLYB | + | dGAL | + |
| dRIB | + | ILATk | - | LAC | - | NAG | + | dMAL | + | BACI | + |
| NOVO | + | NC6.5 | + | dMAN | - | dMNE | + | MBdG | - | PUL | - |
| dRAF | - | O129R | + | SAL | + | SAC | + | dTRE | + | ADH2s | + |
| OPTO | + |
Pairing Chinese write a Chinese character in simplified form in table 2 table 1 Chinese and English
| English is write a Chinese character in simplified form | Chinese | English is write a Chinese character in simplified form | Chinese |
| AMY | Amygdaloside | POLYB | The PXB tolerance |
| PIPLC | The phosphatidyl Phospholipase C | dGAL | The D-semi-lactosi |
| dXYL | The D-wood sugar | dRIB | D-ribose |
| ADH1 | Arginine dihydrolase 1 | ILATK | The L-lactic acid salt produces alkali |
| BGAL | The B-D-tilactase | LAC | Lactose |
| AGLU | Alpha-glucosidase | NAG | N-acetyl-D-amino glucose |
| APPA | L-Ala-phenylalanine-proline(Pro) arylamine enzyme | dMAL | D-maltose |
| CDEX | Cyclodextrin | BACI | The bacitracin tolerance |
| AspA | L-aspartic acid arylamine enzyme | NOVO | The Vulkamycin. PA-93 tolerance |
| BGAR | Beta galactose pyranoside enzyme | NV6.5 | The 6.5%NaCl growth |
| AMAN | Alpha-Mannosidase | dMAN | D-N.F,USP MANNITOL |
| PHOS | Phosphoric acid esterase | dMNE | The D-seminose |
| LeuA | Leucine arylamine enzyme | MBdG | Methyl-B-D-glucose pyrans glycosides |
| ProA | L-proline(Pro) arylamine enzyme | PUL | Amylopectin |
| BGURr | The B-glycuronidase | dRAF | The D-raffinose |
| AGAL | Alpha-galactosidase | O129R | The O/129 tolerance |
| PyrA | Pyrrolidonecarboxylic acid arylamine enzyme | SAL | Salicin |
| BGUR | β-D-glycuronidase | SAC | Sucrose |
| AlaA | L-Ala arylamine enzyme | dTRE | The D-trehalose |
| TyrA | Tyrosine arylamine enzyme | ADH2s | Arginine dihydrolase 2 |
| dSOR | The D-sorbyl alcohol | OPTO | The Ao Putuoxin tolerance |
| URE | Urease |
(2) the 16srDNA sequencing carries out the evaluation of bacterial strain
Adopt pillar faeces DNA out (sky, Beijing bounties Gene Tech. Company Limited) to extract bacterial genomes DNA.Primer: adopt the universal primer of 16srRNA gene to carry out the PCR reaction.Upstream primer: (27F) 5 '-AGAGTTTGATCCTGGCTCAG-3 ', downstream primer: (1492R) 5 '-ACGGCTACCTTGTTACGACTT-3 '.The PCR reaction system is formed: template DNA (concentration is 100ng/ μ l) 0.5 μ L, the PCR premixed liquid (contains the Taq enzyme, Buffer, dNTP, Mg
2+) 25 μ L, each 1 μ L of positive anti-primer, sterilized water is supplied system 50 μ L, when doing negative control with 1 μ LH
2O replaces template DNA.PCR reaction conditions: 94 ℃ of 5min, 94 ℃ of 30sec, 56 ℃ of 30sec, 72 ℃ of 7min after 72 ℃ of 1min circulation 35 times.1% agarose gel electrophoresis is checked the PCR product.Electrophoresis finishes to take pictures, and as seen the band of about 1500bp is target stripe, sees Fig. 2.
With 1% agarose gel electrophoresis, voltage is 100V with 50 μ L pcr amplification products, and electrophoresis time is 20min, and ultraviolet lamp is observed down, downcuts the purpose band of size about 1500bp with knife blade.Then, reclaim the test kit purify DNA with glue.Send the Beijing Liuhe Huada Genomics Technology Co., Ltd to check order the PCR product.Sequencing result: its length of nucleotides is 1444bp, through sequence alignment, shows that bacterial strain NJODL1 99% of the present invention is Lactococcus lactis subsp.lactis.
With this Lactococcus lactis subsp.lactis NJODL1 on January 25th, 2011, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number is CGMCC No:4582.
Embodiment 2: the enlarged culturing of embodiment 1 strain separated NJODL1
The prescription of seed culture medium is to contain in the 1L distilled water: peptone 10g, yeast extract paste 5g, glucose 20g, extractum carnis 10g, sodium acetate 5g, Triammonium citrate 2g, tween 80 1mL, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.05g, K
2HPO
42g regulates pH=6.2～6.4; 121 ℃, high pressure steam sterilization 15min, standby.
The bacterial strain NJODL1 that embodiment 1 is obtained is inoculated in the Erlenmeyer flask of the 250mL that the 50mL seed culture medium is housed, cultivates 24h under 37 ℃ of conditions of temperature, obtains bacterial strain NJODL1 liquid culture, and thalline quantity is 3.2 * 10
9CFU/mL.
Embodiment 3: utilize bacterial strain NJODL1 fermentative degradation oxalic acid in containing the MRS fermention medium of 20mmol/L oxalic acid
The prescription of oxalic acid MRS fermention medium is: contain in the 1L distilled water: peptone 10g, yeast extract paste 5g, glucose 20g, extractum carnis 10g, sodium acetate 5g, Triammonium citrate 2g, tween 80 1mL, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.05g, K
2HPO
42g, sodium oxalate 2.68g regulates pH=6.2～6.4; 121 ℃, high pressure steam sterilization 15min.
Get centrifugal 15 minutes of the NJODL1 liquid culture 12000r/min that embodiment 2 obtains, supernatant discarded is collected bacterial sediment, with stroke-physiological saline solution washing 3 times.Be inoculated in the 250mL Erlenmeyer flask that 100mL oxalic acid fermention medium is housed with 2% volume ratio then, and make blank (having oxalic acid not have bacterium).Bacterium do three parallel.Cultivate 72h under 37 ℃ of conditions of temperature.Centrifugal 15 minutes of bacterium liquid 12000r/min gets supernatant liquor, filters with 0.22 μ m filter, and it is to be measured to be stored in 4 ℃ of refrigerators.
Use ion chromatograph (U.S. wears peace (Dionex)) to measure: envrionment temperature is 35 ℃, and carrier gas is a nitrogen, pressure 200kPa, and the top pressure of setting pump is 21 * 10
3KPa (3000psi) is 11.72 * 10 during actual motion
3About kPa, background conductance is about 28 μ s, and flow velocity is 1mL/min, sampling volume 100 μ l.
Adopt Dionex I onPac AS23 (4 * 250mm) negatively charged ion guard columns, IonPacAG11 analytical column (4 * 50mm) the negatively charged ion guard columns of high-hydrophilic, high column capacity; Elutriant is the yellow soda ash of 16mmol/L and the sodium bicarbonate of 4mmol/L.
Prepare the oxalic acid stock solution of 1000mg/L earlier.With 0.2 μ m filtering with microporous membrane, being diluted to concentration of oxalic acid with ultrapure water is 16,8,4,2,1, the reference liquid of 0.5and 0.25mg/L.
Sample was through the low temperature ultracentrifugation and with 0.2 μ m filtering with microporous membrane, and getting a little after centrifugal dilutes mistake On-GuardH post and C according to 1: 100 usefulness ultrapure water
18Sample introduction analysis behind the pre-treatment pillar.
The result shows that fermented liquid mesoxalic acid content reduces by 24.8%.
Embodiment 4: external acidproof, the bile tolerance of Lactococcus lactis subsp.lactis NJODL1, antibiotic susceptibility test
1. acid resistance test
Take out frozen Lactococcus lactis subsp.lactis NJODL1 bacterial strain from-70 ℃ of refrigerators, be inoculated in the MRS liquid nutrient medium, 37 ℃ of anaerobism are cultivated 24h and are activated.Regulating MRS liquid nutrient medium pH value with 2mol/L sterilization physiology hydrochloric acid is 2.0 and 3.0.With the bacterial suspension for preparing is that 2% inoculum size (is adjusted into 1 * 10 with sterile saline with about bacterial concentration by volume
7CFU.mL
-1) be inoculated in the substratum of pH2.0 37 ℃ and cultivated 4 hours, respectively at 0,30,60,120,240min respectively takes a sample 1 time; With the bacterial suspension for preparing is that 2% inoculum size (is adjusted into 1 * 10 with sterile saline with about bacterial concentration by volume
7CFU.mL
-1) be inoculated in the substratum of pH3.0 37 ℃ and cultivated 4 hours, respectively at 0,60,120,240min respectively takes a sample 1 time.Be coated with flat band method with dilution and calculate viable count.Each tests triplicate.
Shown in result such as the table 3, table 4, Lactococcus lactis subsp.lactis NJODL1 inoculation is that amount of bacteria is 10 behind the 120min in 2.0 the sour environment to the pH value
4CFU.mL
-1Be inoculated into the pH value and be in 3.0 the sour environment, amount of bacteria is 10 behind the 240min
6CFU.mL
-1Show that Lactococcus lactis subsp.lactis NJODL1 bacterial strain is to the sour environment well-tolerated.
The screening (pH=2.0) of the acidproof ability of table 3 bacterial strain
Table 3Resistance of selected isolate in pH 2MRS medium
The screening (pH=3.0) of the acidproof ability of table 4 bacterial strain
Table 4Resistance of selected isolate in pH 3MRS medium.
2. bile tolerance test
Take out frozen Lactococcus lactis subsp.lactis NJODL1 bacterial strain from-70 ℃ of refrigerators, be inoculated in the MRS liquid nutrient medium, 37 ℃ of anaerobism are cultivated 24h and are activated.The Lactococcus lactis subsp.lactis NJODL1 that activation is good is respectively that 2% inoculum size (is adjusted into 1 * 10 with sterile saline with about bacterial concentration by volume
7CFU.mL
-1) to be inoculated in gallbladder salinity (mass percentage concentration) be 0.3%, 1% MRS liquid nutrient medium, respectively at 0,12,24h respectively takes a sample 1 time, is coated with flat band method with dilution and calculates viable count.Each tests triplicate.Be coated with flat band method with dilution and calculate viable count.Triplicate.
The test of table 5 bacterial strain bile tolerance
Table 5Resistance of selected isolate to bile salts.
The result is as shown in table 5, and Lactococcus lactis subsp.lactis bacterium NJODL1 bacterial strain is inoculated into gallbladder salinity respectively and is respectively 0.3% and 1% MRS substratum, gallbladder salinity be in 0.3% the MRS substratum behind the 24h amount of bacteria be 10
5CFU/mL, gallbladder salinity be in 1% the MRS substratum behind the 12h amount of bacteria be 10
3CFU/mL.Show that Lactococcus lactis subsp.lactis NJODL1 bacterial strain can tolerate high cholate environment.
3. antibiotic susceptibility test
According to the Standard Selection microbiotic of NCCLS, do the drug-resistant test of Lactococcus lactis subsp.lactis NJODL1 bacterial strain, observe the susceptibility of Lactococcus lactis subsp.lactis NJODL1 strains.
Table 6 test kind, drug sensitive test paper content and the antibacterial circle diameter criterion of antibacterials
Table 6 Antimicrobial agents and associated interpretative zone diameters for disc diffusionantibiotic susceptibility testing
The antibiotic susceptibility test result of table 7NJODL1
Table 7 Susceptibility of NJODL1 to antibiotics
Annotate: R represents insensitive; S represents sensitivity; M represents medium sensitivity.
The result is as shown in table 7, and Lactococcus lactis subsp.lactis NJODL1 bacterial strain is to the most antibiotics sensitivity.
Embodiment 5: the acute toxicity test of Lactococcus lactis subsp.lactis NJODL1 bacterial strain
In order to detect the security of bacterial strain of the present invention, carry out the acute toxicity test of mouse.Test is carried out according to GB159193-2003 Kou Shi (KORBOR) method, selects to use healthy kunming mice.Specifically be, from-70 ℃ of refrigerators, take out frozen Lactococcus lactis subsp.lactis NJODL1 bacterial strain, be inoculated in the MRS liquid nutrient medium, behind 37 ℃ of cultivation 24h, centrifugal 15 minutes of 12000r/min, supernatant discarded, collect the bacterial sediment postlyophilization, make viable count reach 10
10CFU/g.Get 20 of healthy mices, male and female half and half, body weight 20 ± 2g bought the back breeding observing back three.Given the mouse stomach administration with trying thing with the dosage of 15g/Kg, irritate stomach twice, midfeather 4h.After the administration promptly to the mental status of animal, hair color, autonomic activities, breathing, diet, two just, general situation such as mouth and nose secretory product and death condition observe, the observation period was 2 weeks.
Malaise symptoms does not appear in mouse after the administration, and mouse is movable normal, good in the whole viewing duration mental status, hair color is bright and clean, activity freely, no abnormal secretory product in the eupnea, mouth and nose, the animal diet followed amount is normal, and is just soft, it is no abnormal to urinate.Take off cervical vertebra and put to death animal after 2 weeks, and all animals are performed an autopsy on sb, internal organs such as the visual inspection heart, liver, spleen, lung, kidney there is no any unusual.
Embodiment 6: Lactococcus lactis subsp.lactis NJODL1 bacterial strain is grown test in that the dog body is decided at the higher level but not officially announced
Take out frozen Lactococcus lactis subsp.lactis NJODL1 from-70 ℃ of refrigerators, it is inoculated in the MRS liquid nutrient medium, 37 ℃ of anaerobism are cultivated 24h and are activated.To activate good bacterium bacterium liquid 200 μ L then and coat evenly that (MRS solid culture based formulas is: MRS liquid nutrient medium+20.0g/L agar), 37 ℃ of anaerobism are cultivated 72h on the MRS solid medium that contains 100 μ g/mL Rifampins.The bacterium that grows on this substratum then is the bacterial strain of anti-the Rifampin.Preserve in the liquid glycerine physiological saline of this bacterial strain of anti-Rifampin use 40% frozen in-80 ℃.Before using with inoculation on the MRS solid medium that contains 100 μ g/mL Rifampins, 37 ℃ of anaerobism are cultivated 48h and are activated.
Select 8 body weight 8 ± 2Kg, the local dog about 18 months for use, male and female half and half.All dog envrionment conditionss, basal diet, feeding and management are all identical before and after the test.Preliminary trial period is 7d, and the basic dog grain of feeding enters trial period after preliminary trial period finishes, control group fed basis dog grain, test group feed basic dog grain and supply 5mL 10
9The bacterial strain of anti-Rifampin of CFU/mL Lactococcus lactis subsp.lactis NJODL1, the time is 10d, Routine Management.
Respectively at experiment the 0th, 5,10,15,20 days, aseptic per rectum was taked dog ight soil.After the ight soil of collecting is weighed and used stroke-physiological saline solution vortex mixing, be inoculated on the MRS solid medium that contains 100 μ g/mL Rifampins 37 ℃ of anaerobism and cultivate 72h, to observe Lactococcus lactis subsp.lactis NJODL1 in the intravital situation of growing surely of dog.
Field planting experimental result in table 8 NJODL1 of anti-Rifampin body
Table 8 Gastric transit of Rif
R NJODL1
As shown in Table 8, at the 0th day of the bacterial strain of anti-Rifampin of feeding, do not have in the dog ight soil to find and to contain the bacterium that grows on the MRS substratum of Rifampin.Fed the 1st day, and can detect the NJODL1 bacterial strain of anti-Rifampin in all dog ight soil.The amount of bacteria that detected the NJODL1 of anti-Rifampin bacterial strain in the dog ight soil on the the 1st, 5,10 3 day between feed period, no significant difference (p＞0.05).Between feed period, bacterial strain amount of bacteria in animal body all can reach 10
6CFU/g.After stopping to feed, still can in dog ight soil, detect the NJODL1 bacterial strain of anti-Rifampin, but and there were significant differences between feed period (p＜0.05).
Embodiment 7: Lactococcus lactis subsp.lactis NJODL1 strains for degrading oxalic acid animal experiment
18 of local dogs are purchased in the food market, Nanjing.Be about 10Kg, male.Accept a health check-up, canine penton vaccine immunity before the test.The phase of raising was 2 weeks in advance, and the basic dog grain of feeding enters trial period after the phase of raising in advance finishes.All dog envrionment conditionss, basic dog grain (its trophic level sees Table 9 with prescription), feeding and management are all identical before and after the test.
The test dog is divided into two test group and control group at random, and 6 every group, the A group is blank group, the basic dog grain of feeding; B group is the oxalic acid control group, the basic dog grain and add oxalic acid by dog grain weight 0.5% of feeding every day; C group feed every day basic dog grain and add oxalic acid and supply 2g 5 * 10 by dog grain weight 0.5%
9The bacterial strain NJODL1 lyophilized powder of CFU/g.Time is 30d, Routine Management.
Basic dog grain prescription of table 9 and trophic level
Table 9 Ingredient composition and nutrient level of the basal diet
Test 0d, 15d and 30d, adopt the self-control urine acceptor to collect the dog urine.4 ℃ of preservations, wait to collect the 24h urine after, mixing, getting the 5ml urine, to be acidified to pH with concentrated hydrochloric acid be that 1 back is frozen, surveys the urine concentration of oxalic acid.The concentration of urine oxalic acid uses ion chromatograph to measure.Test 30d and put to death laboratory animal, adopt kidney, make the kidney section and observe the calcium oxalate crystal amount.
As shown in table 10, NJODL1 all can reduce the urine concentration of oxalic acid at experiment 15d and experiment 30d.As Fig. 3, shown in Figure 4,0.5% oxalic acid+NJODL1 group kidney crystallization obviously is less than 0.5% oxalic acid control group.
Table 10 oxalic acid degradation bacteria NLODL1 urinates oxalic acid content (concentration unit: influence mmol/L) to dog
Table10 Reduction of urine oxalate concentration by NLODL1 in vivo(n＝6，mean±S.D.)
| Time | The blank group | 0.5% oxalic acid group | 0.5% oxalic acid group+NJODL1 |
| 0d | 0.21±0.06 a | 0.21±0.05 a | 0.22±0.05 a |
| 15d | 0.2±0.07 c | 0.37±0.04 a | 0.34±0.04 ab |
| 30d | 0.21±0.07 c | 0.49±0.06 a | 0.42±0.05 ab |
Claims (4)
1. oxalic acid degradation bacteria NJODL1, this strain classification called after: Lactococcus lactis subsp.lactis (Lc.lactis subsp.lactis), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 25th, 2011, culture presevation number is CGMCC NO.4582.
2. the screening method of oxalic acid degradation bacteria NJODL1 as claimed in claim 1 is characterized in that may further comprise the steps:
(1) from the ight soil of the dog of less trouble calcinm oxalate calculus kind, isolates milk-acid bacteria;
(2), filter out and have the milk-acid bacteria that falls the oxalic acid ability by vitro detection strains for degrading oxalic acid ability;
(3), bile tolerance ability acidproof, antibiotics sensitivity and to the security of mouse and prove that this bacterial strain can grow in that the dog body is decided at the higher level but not officially announced by the vitro detection bacterial strain;
(4) be NJODL1 by detecting strains for degrading oxalic acid ability in the body.
3. the described oxalic acid degradation bacteria of claim 1 NJODL1 has application in pet food, foodstuff additive or the medicine of regulating the oxalic acid metabolic function in preparation.
4. the oxalic acid degradation bacteria NJODL1 that is applied as according to claim 3 has the pet sour milk of regulating the oxalic acid metabolic function, the application in the fermented-milk in preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107242350A (en) * | 2016-07-05 | 2017-10-13 | 南京农业大学 | A kind of selenium-rich lactobacillus preparation of degradable oxalic acid and preparation method and application |
| CN109402022A (en) * | 2018-12-05 | 2019-03-01 | 上海上药信谊药厂有限公司 | A kind of Oxalobacter formigenes and its application |
| CN114621904A (en) * | 2022-05-13 | 2022-06-14 | 中国科学院地理科学与资源研究所 | Micrococcus and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1409605A (en) * | 1999-09-22 | 2003-04-09 | 雀巢制品公司 | Method for increasing pet activity |
| JP2007259729A (en) * | 2006-03-28 | 2007-10-11 | Yotsuba Nyugyo Kk | Lactic acid bacterium belonging to genus lactococcus and viscous lactic acid bacterium belonging to genus lactococcus each having immunoactivating effect, and method for producing viscous fermented milk using their combination |
| CN102077904A (en) * | 2010-03-11 | 2011-06-01 | 哈尔滨龙猪科技开发有限公司 | Method for preparing biological feed additive of lactobacillus |
-
2011
- 2011-06-22 CN CN201110169482XA patent/CN102250800B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1409605A (en) * | 1999-09-22 | 2003-04-09 | 雀巢制品公司 | Method for increasing pet activity |
| JP2007259729A (en) * | 2006-03-28 | 2007-10-11 | Yotsuba Nyugyo Kk | Lactic acid bacterium belonging to genus lactococcus and viscous lactic acid bacterium belonging to genus lactococcus each having immunoactivating effect, and method for producing viscous fermented milk using their combination |
| CN102077904A (en) * | 2010-03-11 | 2011-06-01 | 哈尔滨龙猪科技开发有限公司 | Method for preparing biological feed additive of lactobacillus |
Non-Patent Citations (8)
| Title |
|---|
| 《ADVANCES IN APPLIED MICROBIOLOGY》 20101231 Abratt, VR "Oxalate-Degrading Bacteria of the Human Gut as Probiotics in the Management of Kidney Stone Disease" 第72卷, * |
| 《VETERINARY MICROBIOLOGY》 20110511 Ren, ZH "Oxalate-degrading capacities of lactic acid bacteria in canine feces" 第152卷, 第3-4期 * |
| 《上海交通大学学报(医学版)》 20091231 赵树田 "十种可制作酸奶的乳酸菌体外降解草酸能力评价" 第29卷, 第12期 * |
| 《中国优秀硕士学位论文全文数据库（农业科技辑）》 20080415 任普军 "犬源益生菌的筛选及其对犬细菌性腹泻的治疗试验" , 第4期 * |
| ABRATT, VR: ""Oxalate-Degrading Bacteria of the Human Gut as Probiotics in the Management of Kidney Stone Disease"", 《ADVANCES IN APPLIED MICROBIOLOGY》 * |
| REN, ZH: ""Oxalate-degrading capacities of lactic acid bacteria in canine feces"", 《VETERINARY MICROBIOLOGY》 * |
| 任普军: ""犬源益生菌的筛选及其对犬细菌性腹泻的治疗试验"", 《中国优秀硕士学位论文全文数据库（农业科技辑）》 * |
| 赵树田: ""十种可制作酸奶的乳酸菌体外降解草酸能力评价"", 《上海交通大学学报(医学版)》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107242350A (en) * | 2016-07-05 | 2017-10-13 | 南京农业大学 | A kind of selenium-rich lactobacillus preparation of degradable oxalic acid and preparation method and application |
| CN107242350B (en) * | 2016-07-05 | 2021-02-09 | 南京农业大学 | Selenium-rich lactobacillus preparation capable of degrading oxalic acid and preparation method and application thereof |
| CN109402022A (en) * | 2018-12-05 | 2019-03-01 | 上海上药信谊药厂有限公司 | A kind of Oxalobacter formigenes and its application |
| CN114621904A (en) * | 2022-05-13 | 2022-06-14 | 中国科学院地理科学与资源研究所 | Micrococcus and application thereof |
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