CN103667124B - One strain has milk-acid bacteria and the screening method thereof of degraded creatinine and urea ability - Google Patents
One strain has milk-acid bacteria and the screening method thereof of degraded creatinine and urea ability Download PDFInfo
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- CN103667124B CN103667124B CN201310625369.7A CN201310625369A CN103667124B CN 103667124 B CN103667124 B CN 103667124B CN 201310625369 A CN201310625369 A CN 201310625369A CN 103667124 B CN103667124 B CN 103667124B
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Abstract
The invention belongs to biological technical field, disclose a strain and screen the lactic bacterium strains in the Yoghourt of Inner Mongolia, this bacterial strain has higher creatinine and urea degradation capacity, is accredited as lactobacillus bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus).The invention also discloses the screening method of degraded creatinine bacterial strain.This bacterial strain is used for field of health care food, there is the effects such as assisting therapy renal failure, uremia, raising immunity of organisms.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a strain and can degrade the milk-acid bacteria of creatinine and urea ability, and relate to the screening method of this bacterial strain.The present invention carries out enrichment culture and separation and purification by the milk-acid bacteria in the yogurt milk of Inner Mongolia area, subsequently to domestication and rich this bacterial strain of sieve acquisition of milk-acid bacteria.
Background technology
Chronic renal failure, also known as chronic renal insufficiency, uremia, running down of the kidney Progressive symmetric erythrokeratodermia damage that to be various Primary Nephrosis or Secondary cases cause in other diseases and renal function.Clinical manifestation is mainly meta-bolites retention, water and eletrolytes disorder and acid base imbalance, renal function grievous injury.Up to the present, chronic renal failure also lacks effective treatment means, mainly relies on hemodialysis, renal transplantation, required somewhat expensive, and the annual required expense of dialysis treatment is huge, and renal transplantation kidney source supply and demand problem is serious, and required expense costly.These methods for the treatment of bring huge economic pressures to society and family, also make patient subject great misery.Except dialysis and the methods for the treatment of of renal transplantation, existing corynebacterium glutamicum is through external evoked generation urase and Creatininase at present, by making after its extraction and isolation, micro-capsule is oral decomposes the toxin such as creatinine, uric acid at enteron aisle, but this method is not because widely using clinical without effect urotoxin.In recent years, digestive tube bacterize method becomes the little therapy of the removing enterotoxin of the digestive enzyme therapy that continues, and its methods for the treatment of is: use nature bacterial classification, and as low toxicity soil bacteria, external evoked generation urease, orally enters enteron aisle, decomposes enteron aisle urotoxin; Or by another kind of for high yield enzyme channel genes bacterium, build engineering bacteria, this project bacterium is taken in enteron aisle, degradation of urine toxin.But there is potential safety hazard in the bacterium that these class methods are taken in, may cause alteration of intestinal flora, and bring out infection.
Probiotic bacterium is a kind of living microorganism useful to host, can be colonizated in human intestinal, improves intestinal microflora balance, controls intestinal tract infections.Activate intestinal lymphocyte and can start systemic immune system raising immunizing power, reduce the effect such as patients' blood, blood fat in addition.Suzuki(K.Suzuki, Y.Benno, T.Mitsuoka, S.Takebe, K.Kobashi, J.Hase.Urease-Producing Species of Intestinal Anaerobes and Their Activities. [J] Applied andEnvironmental Microbiology, Mar.1979, 379 ~ 382.) from the ight soil of Chronic Renal Failure Patients, be separated to the bifidus bacillus of a strain urease-producing, CHOW(Chow, Liu ZC, Prakash S, et al.Free and microencapsulatedLactobacillus and effects of metabolic induction on urea removal.Artif Cells Blood Substit ImmobilBiotechnol2003Nov, 31 (4): 425-34.), after etc. finding that moral formula Bacterium lacticum repeatedly goes down to posterity in high concentration urea substratum, the degradation capability to urea is shown.Research finds, oral Lactobacterium acidophilum can reduce dialysis patient uremic toxins level.Therefore exploitation has and the treatment of the active probiotic of creatinine and urea function for patients with chronic renal failure is significant.
Summary of the invention
The object of this invention is to provide milk-acid bacteria and screening method thereof that a strain has degraded creatinine and urea ability.
Bacterial strain provided by the present invention is lactobacillus, its deposit number is CGMCC No.7983, through 16S rDNA nucleotide sequence result and Physiology and biochemistry interpretation of result, be defined as lactobacillus bulgaricus (Lactobacillus delbrueckiisubsp.bulgaricus).
The present invention also provides a kind of screening to have the screening method of the Bacterium lacticum of degraded creatinine and urea ability.First be the enrichment culture of milk-acid bacteria, select the rich lactobacteria-containing yogurt milk in Inner Mongolia, join MRS liquid nutrient medium and carry out enrichment culture, 40 DEG C of quiescent culture 24h, inoculum size by 5% adds in the MRS substratum containing 0.1% creatinine and 0.05% urea, enrichment culture 24h; The initial gross separation purifying of following by milk-acid bacteria, gets above-mentioned enrichment culture liquid and carries out gradient dilution, get 10
-5, 10
-6, 10
-7diluent, the bacterium colony separation and purification of coat on purpurum bromocresolis skim milk plate, in 40 DEG C of Anaerobic culturel 48h, picking naked eyes are visible, producing acid (substratum its colour changed into yellow), and numbering is preserved; The domestication of following by milk-acid bacteria, by the lactobacillus inoculum kept in the MRS substratum containing 0.1% creatinine and 0.05% urea, 40 DEG C of Anaerobic culturel 48h, get above-mentioned enrichment culture liquid and carry out gradient dilution, get 10
-5, 10
-6, 10
-7diluent, coat in the MRS substratum containing 0.1% creatinine 0.05% urea, in 40 DEG C of Anaerobic culturel 48h, the strains separation purifying that picking upgrowth situation is good, and numbering preserve.By the milk-acid bacteria continuous domestication kept 20 times, final screening is obtained stablize the bacterial strain of surviving in the MRS substratum of 0.1% creatinine/0.05% urea, by strain number preservation; It is finally the richness sieve of milk-acid bacteria, the bacterial strain kept is activated by preceding method and coats in the MRS substratum containing 0.1% creatinine/0.05% urea, in 40 DEG C of Anaerobic culturel 48h, the good inoculation of picking upgrowth situation is in containing in the MRS substratum of 0.1% creatinine 0.05% urea, alkaline picrate method measures the degradation rate of creatinine, and Diacetylmonoxime determination of color blood urea nitrogen degradation rate, chooses the high strain strain number of degradation rate and preservation, its creatinine degradation rate reaches 25%, and degradation of urea rate reaches 30%.
Meanwhile, carried out Physiology and biochemistry qualification and 16S rDNA sequential analysis qualification to screening the degraded creatinine milk-acid bacteria obtained, its 16S rDNA sequence is as shown in sequence table SEQ ID No:1.
According to the sequencing result of this bacterial strain 16S rDNA sequence, itself and the lactobacillus delbruckii to have reported, subspecies bulgaricus (GenBank.No.CP000156) sequence homology reaches 98%, according to the above results, determine that this bacterial strain belongs to the lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) of lactobacillus, this bacterial strain has the ability reducing creatinine and urea, China Committee for Culture Collection of Microorganisms's common micro-organisms Bio-Centers is preserved on August 5th, 2013, deposit number is CGMCC No.7983, depositary institution address is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Another object of the present invention is to provide described bacterial strain and is preparing the application in protective foods or functional foodstuff.
Another object of the present invention is to provide the application of described bacterial strain in the medicine for the preparation for the treatment of chronic renal failure.
Accompanying drawing explanation
Fig. 1 is based on the phylogenetic tree based on the 16S rDNA sequence of lactobacillus bulgaricus CGMCC No.7983 bacterial strain.
The colonial morphology (figure A) of Fig. 2 lactobacillus bulgaricus CGMCC No.7983 bacterial strain and thalli morphology (figure B)
Embodiment
Embodiment 1. is degraded the screening of milk-acid bacteria of creatinine and urea
(1) the enrichment culture of milk-acid bacteria.Select the rich lactobacteria-containing yogurt milk in 10ml Inner Mongolia, join 100mlMRS liquid nutrient medium and carry out enrichment culture, 40 DEG C of quiescent culture 24h, get 10ml and add 200ml containing in the MRS substratum of 0.1% creatinine and 0.05% urea, 40 DEG C of quiescent culture 24h, obtain enrichment culture liquid.
Described MRS substratum: glucose 2%, peptone 1%, yeast powder 1%, extractum carnis 1%, sodium acetate 0.5%, Triammonium citrate 0.2%, tween-80 0.1%, MgSO
40.03%, MnSO
40.002%, K
2hPO
40.2%, pH6.8.
(2) the initial gross separation purifying of milk-acid bacteria.Get 1ml above-mentioned enrichment culture liquid PBS damping fluid and carry out gradient dilution, get 10
-5, 10
-6, 10
-7diluent, the bacterium colony separation and purification of coat on purpurum bromocresolis skim milk plate, in 40 DEG C of Anaerobic culturel 48h, picking naked eyes are visible, producing acid (substratum its colour changed into yellow), numbering is inoculated in 0.1% creatinine and 0.05% urea MRS culture medium slant is preserved.
Described PBS damping fluid: Na
2hPO
40.12%, NaH
2pO
42H
2o0.03%, NaCl0.9%, tween-80 0.05%.
Described purpurum bromocresolis degreasing milk medium is dull and stereotyped: purpurum bromocresolis 0.01%, skimming milk 10%.
0.1% creatinine and 0.05% urea MRS solid medium flat board: glucose 2%, peptone 1%, yeast powder 1%, extractum carnis 1%, sodium acetate 0.5%, Triammonium citrate 0.2%, tween-80 0.1%, MgSO
40.03%, MnSO
40.002%, K
2hPO
40.2%, creatinine 0.1%, urea 0.05%, pH6.8.
(3) the domestication of milk-acid bacteria.Go bail for the milk-acid bacteria inclined-plane kept, with transfering loop picking 1 ring, be inoculated in 50ml containing in the MRS substratum of 0.1% creatinine and 0.05% urea, 40 DEG C of Anaerobic culturel 24h, get the above-mentioned enrichment culture liquid of 1ml and carry out gradient dilution, get 10
-5, 10
-6, 10
-7diluent, coat in 0.1% creatinine and 0.05% urea MRS solid medium, in 40 DEG C of Anaerobic culturel 48h, the good mono-clonal inoculation of picking upgrowth situation is in 0.1% creatinine and 0.05% urea MRS culture medium slant, and numbering is preserved.By milk-acid bacteria repeating step 3 continuous domestication kept 20 times, final screening being obtained can the bacterial strain of stable survival in the MRS substratum of 0.1% creatinine and 0.05% urea, by inoculation in 0.1% creatinine and 0.05% urea MRS solid slope numbering preservation.
(4) the richness sieve of milk-acid bacteria, the milk-acid bacteria inclined-plane kept of going bail for, with transfering loop picking 1 ring, be inoculated in 50ml and contain in the MRS substratum of 0.1% creatinine and 0.05% urea, 40 DEG C of Anaerobic culturel 24h, get the above-mentioned enrichment culture liquid of 1ml and carry out gradient dilution, get 10
-5, 10
-6, 10
-7diluent, coat in 0.1% creatinine and 0.05% urea MRS solid medium, in 40 DEG C of Anaerobic culturel 48h, observe the good bacterial strain of upgrowth situation and number, number in 50ml is containing the MRS substratum of 0.1% creatinine and 0.05% urea by transfering loop picking numbering inoculation, Simultaneous vaccination is numbered in 0.1% creatinine and 0.05% urea MRS culture medium slant and preserves, alkaline picrate method measures the degradation rate of creatinine, the creatinine detection reagent box (picric acid method) that alkaline picrate method uses German Roche Diagnostics GmbH to produce, operate by method shown in test kit specification sheets.Adopt Diacetylmonoxime determination of color blood urea nitrogen degradation rate, concrete grammar is as follows: it is working fluid that acid reagent 5ml adds 0.5ml Diacetylmonoxime, working fluid adds 0.02ml sample determination sample concentration, and add 0.02ml water for blank, adding 0.02ml urea reference liquid is standard.After mixing, boiling water bath 15min, takes out in placement cold water and cools 5min, under 540nm, measure absorbancy.Method of calculation: urea concentration=A
sample/ A
standard× 5
Detected result is as shown in table 1:
Table 1. creatinine and urea measurement result
Wherein No. 15 strains for degrading rates are higher, its creatinine degradation rate reaches 25%, degradation of urea rate reaches 30%, this bacterial strain has the ability reducing creatinine and urea, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms Bio-Centers on August 5th, 2013, deposit number is CGMCC No.7983.
The qualification of embodiment 2. bacterium
(1) morphology describes
On MRS solid medium, bacterium colony is circular, smooth, oyster white, edge clear, and in mucus shape, thalline is easy to be provoked.Thalline under the microscopic examination of amplification 400 times, gram-positive, in rod-short, as shown in Figure 2.
(2) Physiology and biochemistry qualification
Gram-positive, nonfermented lactic acid salt, not liquefy gelatin, do not decompose casein, edwardsiella hoshinae and hydrogen sulfide, and catalase is negative, acellular pigment, and benzidine reaction is negative, and sugar fermentating test is as shown in table 2:
The Physiology and biochemistry qualification result of table 2. bacterial strain
(2) 16S rDNA sequential analysis qualification
Check order to the 16S rDNA of this bacterial strain, its sequence is as shown in SEQ ID No.1, and itself and the lactobacillus delbruckii to have reported, subspecies bulgaricus (GenBank.No.CP000156) sequence homology reaches 98%.According to the above results, this bacterial strain is accredited as lactobacillus bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus).
Above embodiment further illustrates content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
SEQUENCE LISTING
<110> Inner Mongolia Medical University
<120> mono-strain has milk-acid bacteria and the screening method thereof of degraded creatinine and urea ability
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1443
<212> DNA
<213> Lactobacillus delbrueckii subsp.bulgaricus
<400> 1
tctcgagcgg cgatctatga ggttatccca ccgactttgg gcattgcaga cttccatggt 60
gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcgtgct gatccgcgat 120
tactagcgat tccagcttcg tgcaggcgag ttgcagcctg cagtccgaac tgagaacagc 180
tttaagagat ccgcttaccc tcgcgggttc gcttctcgtt gtactgccca ttgtagcacg 240
tgtgtagccc aggtcataag gggcatgatg acttgacgtc atccccacct tcctccggtt 300
tgtcaccggc agtctcttta gagtgcccaa cttaatgatg gcaactaaag acaagggttg 360
cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacag ccatgcacca 420
cctgtctctg cgtccccgaa gggaaccacc tatctctagg tgtagcacag gatgtcaaga 480
cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccry tkgtgcgrsc 540
cccccgtcaa ktcstttgag tttcaacctt gcggtcgtac tccgccaggc ggagcgcwta 600
atgcgtttgm tgcggcactg aggaccggaa agtccccaac acctagcgct catcgtttac 660
ggcatggact acccagggta tmtaatcctg ttcgctaccc atgctttcga gcctcagcgt 720
cagttgcaga ccagagagcc gcsttcgcca ctggtgttct tccatatatc tacgcattcc 780
accgctacac atggaattcc actctcctct tctgcactca agaatgacag tttccgatgc 840
agttccacgg ttgagccgtg ggctttcaca tcagacttat cattccgcct gcgctcgctt 900
yacgcccaat aaatccggam aacgcttgcc acctacgtat taccgcggct gctggcacgt 960
agttagccgt gactttctgg ttgattaccg tcaaataaag accagttact gcctctatcc 1020
ttcttcacca acaacagagc tttacgatcc gaaaaccttc ttcactcacg cggcgttgct 1080
ccatcagact tgcgtccatt gtggaagatt ccctactgct gcctcccgta ggagtttggg 1140
ccgtgtctca gtcccaatgt ggccgatcag tctctcaact cggctacgca tcattgcctt 1200
ggtaggcctt taccccacca actagctaat gcgccgcggg ctcatcctaa agtgacagct 1260
tacgccgcct ttcaaacttg aatcatgcga ttcatgttgt tatccggtat tagcacctgt 1320
ttccaagtgg tatcccagtc tttagggcag attgcccacg tgttactcac ccatccgccg 1380
ctatacgttt catacaaatg caccccgaag ggatctgtga gtctggtcgc aaactgctgc 1440
aca 1443
Claims (4)
1. a strain lactobacillus acidophilus strains, it is characterized in that: described bacterial strain belongs to the lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) of lactobacillus, this bacterial strain has the ability reducing creatinine, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms Bio-Centers on August 5th, 2013, deposit number is CGMCC No.7983.
2. the bacterial strain described in claim 1 is preparing the application in functional foodstuff.
3. the bacterial strain described in claim 1 is preparing the application in protective foods.
4. the application of the bacterial strain described in claim 1 in the medicine for the preparation for the treatment of chronic renal failure.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733095A (en) * | 2020-06-10 | 2020-10-02 | 云南皇氏来思尔乳业有限公司 | Lactobacillus delbrueckii subspecies bulgaricus for high yield of gamma-aminobutyric acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106479922B (en) * | 2016-10-19 | 2019-05-17 | 江南大学 | The lactobacillus plantarum of one plant of degrade simultaneously arginine and urea |
| CN108048342B (en) * | 2017-10-26 | 2021-06-04 | 中国农业大学 | Pressure-resistant probiotics and food and preparation method thereof |
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| CN1893964A (en) * | 2003-12-15 | 2007-01-10 | D·P·佩内瓦 | A synbiotic product and its application as a carrier of natural bioactive substances in functional food additives |
| CN102747004A (en) * | 2007-11-29 | 2012-10-24 | 株式会社明治 | Lactic acid bacteria with effect of uric acid level reducing in blood |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1893964A (en) * | 2003-12-15 | 2007-01-10 | D·P·佩内瓦 | A synbiotic product and its application as a carrier of natural bioactive substances in functional food additives |
| CN102747004A (en) * | 2007-11-29 | 2012-10-24 | 株式会社明治 | Lactic acid bacteria with effect of uric acid level reducing in blood |
Non-Patent Citations (2)
| Title |
|---|
| "Free and microencapsulated Lactobacillus and effects of metabolic induction on urea removal";Kai Ming Chow 等;《Artificial cells,blood substitutes,and biotechnology》;20031130;第31卷(第4期);第425-434页 * |
| 焦闻文."保伽利亚乳杆菌对尿素,肌酐降解能力的定向诱导与诱变".《中国优秀硕士学位论文全文数据库(电子期刊)-医药卫生科技辑》.2007,(第6期),E067-21. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733095A (en) * | 2020-06-10 | 2020-10-02 | 云南皇氏来思尔乳业有限公司 | Lactobacillus delbrueckii subspecies bulgaricus for high yield of gamma-aminobutyric acid |
| CN111733095B (en) * | 2020-06-10 | 2022-05-24 | 云南皇氏来思尔乳业有限公司 | Lactobacillus delbrueckii subsp bulgaricus strain for high yield of gamma-aminobutyric acid |
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