CN1888051B - Plant lactobacillus strain and its application - Google Patents
Plant lactobacillus strain and its application Download PDFInfo
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Abstract
The present invention discloses one plant lactobacillus strain and its application. The plant lactobacillus, Lactobacillus plantarum L323 CGMCC No. 1329, is separated from Chinese pregnant woman's vaginal secretion, and has relatively high bacteriotasis on common vaginal pathogens, such as staphylococcus aureus, candida albicans, colibacillus and vaginal Gardnar bacillus, and acid producing and H2O2 producing capacity higher than other lactobacillus. The plant lactobacillus, Lactobacillus plantarum L323 CGMCC No. 1329, may be used in preparing vaginal microbial preparation for preventing and/or treating vaginal infectious diseases.
Description
Technical field
The present invention relates to a strain lactobacillus and an application thereof, particularly a lactobacillus plantarum and application thereof.
Background technology
One, lactobacillus and woman vagina disease
Lactobacillus (Lactobacillus) be the healthy women intravaginal main, also be most important normal microflora, ability of Lactobacillus in human vagina is no pathogenicity not only, and can self-cleaning microecology in vaginas environment, restricts other pathogenic bacterium field planting growth.Therefore, clinically with vaginal smear visible a large amount of (under the high power lens each visual field several) lactobacillus, do not examine and see or a small amount of gram negative bacillus is only arranged as normal, the standard that absence of vagina infects, and ability of Lactobacillus in human vagina disappearance or minimizing are important indicator (the Robert P.Nugent of vaginal infection, Marijane A.Krohn, and Sharon L.Hillier Reliability of diagnosing bacterial vaginosisis improved by a standardized method of Gram stain interpretation Journalof Clincal Microbiology 1991,29 (2): 297-301).
Women's bacterial vaginosis (Bacterial Vaginosis, be called for short BV) mainly be meant by bacterium, mould, the vaginitis that microorganisms such as trichomonad cause, clinical general use antibiotic therapy, but antibiotic therapy has higher recurrent rate, especially for some senile vaginitiss and multiple vaginitis patient, antibiotic therapy is poor effect often, because when using microbiotic to kill pathogenic microorganism, also destroyed intravital normal microflora, especially suppressed the growth of ability of Lactobacillus in human vagina, thereby lost restrictive function, made other pathogenic microorganism field planting growth pathogenic microorganism in the micro-ecological environment.In addition, women's gestation time is because immunoregulatory disorder is easily sent out vaginal infection, and pregnant woman's singularity makes it can not use antibiotic therapy.
Two, the applicable cases of lactobacillus vagina preparation
Lactobacillus is as probiotics treatment woman vagina disease be used widely abroad (Nigel W.Mclean and Isobel J.Rosenstein Characterisation and selection of aLactobacillus species to re-colonise the vagina of women with recurrentbacterial vaginosis L Med Microbiol 2000,49:543-552; Elmer GM.Probiotics: " living drugs " Am J Health Syst Pharm 2001,58 (12): 1101-9), this medicine belongs to the OTC (over-the-counter) preparation, and the patient can buy voluntarily.Lactobacillus in the preparation generally separates in the normal women vagina, and common have Lactobacterium acidophilum (Lactobacillus acidophilus), lactobacterium casei (Lactobacillus casei) and a plant lactobacillus (Lactobacillus plantarum) etc.Because the not necessarily suitable Aisa people who comprises Chinese of ethnic group and individual difference, external similar preparation, domesticly begun to develop similar preparation Bacterium lacticum.
Using probiotics treatment disease, is the another magnificent bright spot after the microbiotic splendid age certainly, is the another pharmacy direction that microbiotic can't replace, and is containing huge economic benefit and social benefit.
Lactobacillus is suitable for following crowd as the intravaginal probiotics:
(1) senile vaginitis patient
Senile vaginitis is atrophic vaginitis both, it is one of elderly woman common disease, at the domestic 30-50% that is reported as of the scorching sickness rate of menopausal women medial vagina, be reported as 30%. experts abroad and think that the main diseases of senile vaginitis is because of being that lactobacillus lacks in the vagina microenvironment, due to other pathogenic microorganism field planting. antibiotic therapy senile vaginitis poor effect, can suppress other pathogenic bacterium growth and regularly implant lactobacillus preparation, be to improve gerontal patient's vaginitis symptom, make it be in state of health, thereby improve fundamental way (Reid G, the Bruce AW.Urogenital infections in women:can probiotics help of elderly woman quality of life? PostgradMed is Aug J.2003; 79 (934): 428-32).
(2) pregnant woman
Pregnant woman and normal women relatively because change of hormone contents causes that T inhibition lymphocyte is hyperfunction, thereby cause hypoimmunity, Genito-urinary system easily takes place infect.The treatment pregnant woman infects and does not generally use microbiotic, because most antibiotics is influential to fetus, for example be used for the treatment of the colpitic medicine of trichomonad and anerobe property " Metronidazole plain BP.98 99 ", show carcinogenic and teratogenesis in the experimentation on animals, the Macrocyclolactone lactone kind medicine that is used for the treatment of nonspecific vaginitis influences fetal bone and grows.And lactobacillus is a normal microflora, and no pathogenicity has self-cleaning action to the vagina microenvironment.Therefore implant lactobacillus preparation for suffering from colpitic pregnant woman's intravaginal, can make it ride out pregnancy period and perinatal period.The pregnant woman infects BV and causes endometritis and premature labor easily, and the up-to-date investigation of Britain shows that gestation is during 20 weeks, and the intravaginal clone has product H
2O
2The pregnant woman of lactobacillus, its danger of suffering from BV is significantly less than does not have the H of producing
2O
2The pregnant woman of lactobacillus, the morbidity of its endometritis and early productive rate also be significantly less than the latter (Wilks M, Wiggins R, Whiley A, Hennessy E, Warwick S, Porter H, CorfieldA, Millar M.Identification and H (2) O (2) production of vaginal lactobacillifrom pregnant women at high risk of preterm birth and relation with outcome.J Clin Microbiol.2004 Feb; 42 (2): 713-7).Therefore, the someone utilizes and produces H
2O
2Lactobacillus bacterium liquid be 22 and suffer from BV, the age washes 1 time at 20-34 year, pregnant woman's washing vagina of 3 months of gestation every day, washing 7 days is 1 course of treatment, begins for the 2nd course of treatment after 1 week.The result treats after 3 days all, and pregnant woman's symptoms improve, 90% pregnant woman's vagina situation is recovered normal (Tasdemir M after 2 courses of treatment, Tasdemir I, Tasdemir S, Tavukcuoglu S.Alternative treatment for bacterial vaginosisin pregnant patients; Restoration of vaginal acidity and flora.Arch AIDSRes.1996; 10 (4): 239-41).
(3) chronic
For the chronic, as the diabetic subject, the normal microflora imbalance usually can appear in long-term use immunosuppressor such as lupus erythematosus, rheumatosis, organ transplantation and the antibiotic crowd of broad-spectrum; Some physical disabilities, the long-term vagina that crouches of sitting infect anerobe easily; For this class women, implant the lactobacillus vagina preparation, will remove misery for them, play effectively " keeping a public place clean " effect.
Three, as the source of lactobacillus vagina preparation bacterial strain and the condition that should have
1, is used as the source of lactobacillus vagina preparation bacterial strain
Bacterial strain as lactobacillus microecology in vaginas preparation generally all derives from healthy women vagina normal microflora, and the lactobacillus that separates from the fabulous pregnant woman's vagina normal microflora of vagina cleanness degree will have better effect.Britain's report was used to develop the microecology in vaginas preparation from pregnant woman's separating lactic acid bacillus in 1999.
The pregnant woman especially in pregnant early stage, owing to change of hormone contents causes the body immune system wadding disorderly, usually was that T suppresses the hyperfunction hypoimmunity that causes of cell, thereby the infection of Genito-urinary system easily takes place in the pregnancy period.But under the state of hypoimmunity, some pregnant woman still can keep vagina cleanness degree preferably, and its vagina normal microflora is described, both lactobacillus had the ability that suppresses the pathogenic bacterium growth more by force.
The condition that 2, generally should have the following aspects as the bacterial strain of lactobacillus microecology in vaginas preparation:
(1) sticks occupancy
Lactobacillus is extremely important for being used as probiotics to the adhesion of vaginal epithelial cell, in vitro tests shows, lactobacillus is in the silica gel surface growth, can disturb absorption (the Velraeds MM Interference in initial adhesion of uropathogenic bacteriaand yeasts to silicone rubber by a Lactobacillus acidophilus J MedMicrobiol 1998,47 (12): 1081-5) of urethra pathogenic bacterium on this silica gel surface.
Lactobacillus in process of growth constantly to a kind of non-pili adhesin of exocytosis, the visible bacterium periphery of morphological observation one deck is thick, fine and close, uneven, spination material, be called hair sample projection (fibritlae) again, its chemical ingredients is lipoteichoicacid (lipoteichoic acid, LTA), play the effect of sticking with host cell.The mechanism of the effect of sticking of LTA and host cell is considered to, combine with host cell receptor as part on the one hand, on the other hand by electrically charged and host cell interphase interaction (the Otero MC of LTA free fraction institute, Ocana VS, Elena Nader-Macias M.Bacterial surfacecharacteristics applied to selection of probiotic microorganisms.MethodsMol Biol.2004; 268:435-40).Therefore, the hair sample projection of gram positive bacterium is many more, and is strong more to the effect of sticking of host cell.
Lactobacillus utilizes non-pili adhesin, and promptly hair sample projection can be well attached on the vaginal wall, and formed biological barrier makes other pathogenic bacterium form the clone in vagina, has played the occupy-place effect.The lactobacillus volume is big more, and the occupy-place effect is good more.Plant lactobacillus (Lactobacillus plantarum) is the kind of volume maximum in lactobacillus, thereby has better occupancy.About plant lactobacillus be used for probiotics with and vaginal epithelial cell had adhesion and occupancy preferably, the existing report of foreign literature and patent (Bonetti A, Morelli L, Campominosi E, Ganora E, Sforza F.Adherenceof Lactobacillus plantarum P 17630 in soft-gel capsule formulation versusderlein ' s bacillus in tablet formulation to vaginal epithelial cells.Minerva Ginecol.2003 Jun; 55 (3): 279-84,284-7).
(2) produce acid
Because the acidophilia of lactobacillus makes it be suitable for growing in sour environment, the product of thanking that its fermenting carbohydrate produced all is an acetate almost, and intravaginal environment pH is reduced, thereby is unfavorable for other pathogenic bacterium growth.The year women is because its vagina epidermic cell glycogen degree reduces, and a large amount of lactobacilluss are lost, so intravaginal microenvironment pH rising, makes elderly woman suffer from vaginitis easily.Therefore, many researchs are thought, lactobacillus have a formed acid micro-ecological environment, be the main factor (E.R.Boskey that prevents vaginosis, K.M.Telsch Acid production gyvaginal flora in vitro is consistent withthe rate and extent of vaginal acidification Infection and Immunity 1999,67 (10): 5170-5).
(3) produce hydrogen peroxide (H
2O
2)
The entozoic lactobacillus quantity of some woman vagina is not few, but still has inflammation to take place, and this is because these lactobacilluss do not produce H
2O
2, and the big voluminous H of lactobacillus of the good woman vagina parasitism of degree of cleaning
2O
2As add the situations that Leah was investigated 40 healthy womens and 72 infection BV woman vagina floras in 2003 respectively, find that the clone has product H in healthy women 92.5% (37/40) vaginal microbial flora
2O
2Lactobacillus, only have that the clone has product H in 4.1% (3/72) vaginal microbial flora and infect BV women
2O
2Lactobacillus.The author thinks, H that lactobacillus produces
2O
2Play effect (Shopova E.Hydrogenperoxide-producing Lactobacillus species in healthy women and in women withbacterial vaginosis Akush Ginekol (Sofiia) .2003 of protection vagina microenvironment non-specificly; 42 Suppl 1:12-5).H
2O
2Can suppress the growth of some aerobic bacteria and mould.Bibliographical information lacks and produces H
2O
2Lactobacillus, be principal element (the Juarez Tomas MS of the vaginitis that causes of intestinal bacteria, urethritis recurrence, Ocana VS, WieseB, Nader-Macias ME.Growth and lactic acid production by vaginalLactobacillus acidophilus CRL 1259, and inhibition of uropathogenicEscherichia coli.J Med Microbiol.2003 Dec; 52 (Pt 12): 1117-24).Someone uses H
2O
2Liquid washing vagina treatment BV infects, and compares with Metronidazole plain BP.98 99 and clindamycin treatment: 58 ages are at the clinical 30ml of the using 3%H of female patient that infects BV in 18-42 year
2O
2The liquid washing vagina, washed 7 days altogether at every day 1 time.Compare with Metronidazole plain BP.98 99 and clindamycin treatment after 3 months, the cardinal symptom of BV is removed, and comprises that 98% patient eliminates stench, and 98% vagina pH＜4.5,97.7% amine test feminine genders does not detect clues cell and sets up and contains product H in 100% vaginal secretions
2O
2The normal microflora of lactobacillus (Cardone A, Zarcone R, Borrelli A, Di Cunzolo A, Russo A, Tartaglia E.Utilisation of hydrogenperoxide in the treatment of recurrent bacterial vaginosis.Minerva Ginecol.2003 Dec; 55 (6): 483-92).
(4) bacteriostatic action
Some lactobacillus is in process of growth, can be to external secretion lactobacillus rhzomorph, these rhzomorphs can produce the effect that suppresses its growth to many vagina pathogenic microorganisms, the composition and the mechanism of action of these rhzomorphs are not clear, there are some researches prove that the lactobacillus rhzomorph is a kind of material with protein component of anti-microbial effect, also the someone thinks so-called lactobacillus rhzomorph, promptly is the acidic metabolite of lactobacillus.But no matter the lactobacillus rhzomorph is the material of which kind of composition, lactic bacilli strains as the microecology in vaginas preparation must have bacteriostatic action (Aroutcheva AA.Simoes JA, Faro S.Antimicrobial protein produced byvaginal Lactobacillus acidophilus that inbibits Gardnerella vaginalisInfect Dios Obstet gynecol 2001,9 (1): 33-9; Tomas MS, Claudia Otero M, OcanaV, Elena Nader-Macias M.Production of antimicrobial substances by lacticacid bacteria I:determination of hydrogen peroxide.Methods Mol Biol.2004.268:337-46).
(5) growth conditions and stability
Lactobacillus growth is very big to the demand difference of nutrition, and most of lactobacillus nutritional needs harshnesses need expensive nutritional additive, and poor growth, are not suitable for preparation production.Want to adapt to the ordinary student elongate member as the lactic bacilli strains of microecology in vaginas preparation, under the ordinary student elongate member, can breed in a large number; In addition, this bacterial strain can long-term surviving also be the essential condition that is used as microecology in vaginas preparation bacterial strain in suitable preservation liquid.
Summary of the invention
The purpose of this invention is to provide the new plant lactobacillus of a strain.
Plant lactobacillus provided by the present invention is plant lactobacillus (Lactobacillus plantarum) L323, this bacterial strain has been preserved in Chinese common micro-organisms culture presevation management committee common micro-organisms center (being called for short CGMCC) on 03 14th, 2005, deposit number is CGMCC No.1329.
Plant lactobacillus (Lactobacillus plantarum) L323CGMCC No.1329 separates from vagina cleanness degree height, human body that microenvironment is good, pathogenic bacterium such as streptococcus aureus, Candida albicans, intestinal bacteria and the gardnerella vaginalis common to woman vagina show stronger bacteriostatic action, produce acid and produce H
2O
2Ability all be higher than other lactobacillus, cultivated 96 hours, the pH of nutrient solution can reach 3.96; This bacterial strain surface has thick non-pili adhesin, and vaginal epithelial cell is had the effect of sticking preferably, and room temperature is placed half a year in suitable preservation liquid, and viable count increases by 3 * 10
4Doubly.Plant lactobacillus (Lactobacillus plantarum) L323CGMCC No.1329 can be used for preparing the microecology in vaginas preparation, in order to prevent and/or treat the woman vagina infectious diseases.
Description of drawings
Fig. 1 is the gramstaining photo of plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329
Fig. 2 A is the bacteriostatic action of plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 to streptococcus aureus ATCC6035
Fig. 2 B is the bacteriostatic action of plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 to Candida albicans ATCC1023
Fig. 2 C is the bacteriostatic action of plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 to intestinal bacteria ATCC25922
Fig. 2 D is the bacteriostatic action of plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 to gardnerella vaginalis ATCC14018
Fig. 3 is that plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 produces H
2O
2The dull and stereotyped photo that is the blue-greenish colour bacterium colony
Fig. 4 is the electromicroscopic photograph of plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Separation and the evaluation of embodiment 1, plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329
1, the separation of plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329
Be captured at first that 301 Hospital's Obstetric and Gynecologic Department carry out regularly that the pregnancy period checks 300 surplus healthy Chinese pregnant woman's posterior fornix position, position secretory product, carrying out the isolating while of bacterium with the MRS substratum, carry out the test of following several respects: (1) direct smear, according to the quantity of lactobacillus and gram negative bacterium carry out the degree of cleaning evaluation (the 0-2 branch, normal; The 4-6 branch, mild inflammation; The 8-10 branch, hyperphlogosis), wherein, judgement criteria reference literature (the Robert P.Nugent of vagina cleanness degree, Marijane A.Krohn, and Sharon L.HillierReliability of diagnosing bacterial vaginosis is improved by a standardizedmethod of Gram stain interpretation Journal of Clincal Microbiology1991,29 (2): 297-301); (2) vaginal secretions pH measures; (3) quantity of lactobacillus in the quantitative assay vaginal secretions.The result obtains strain separation and is evaluated as the 0-2 branch from vagina cleanness degree, and lactobacillus quantity is greater than 1 * 10 in the vaginal secretions
8CFU/ml, the pH value is the lactobacillus of 3.25 vaginal secretions, bacterial strain number is L323.
2, Bacteria Identification
Thalli morphology is observed routinely method and is carried out gram stain microscopy.The visible L323 strain of gramstaining lactobacillus is the thick bacillus of Gram-positive, and size is about 1 * 3-8um (Fig. 1).
Biochemical reaction adopts the French Mei Liai API of biotech firm 50 Bacteria Identification systems, comprises API 50 substratum, test bar, McFarland standard point, mineral oil, cultivation box and API Plus software etc.API 50 Bacteria Identification system qualification results show that L323 strain lactobacillus is plant lactobacillus (Lactobacillusplantarum), and the result is as shown in table 1 for its biochemical reaction.
The biochemical reaction of table 1 plant lactobacillus (Lactobacillus plantarum) L323
| Polychrom ESC | + | Xylitol XLT | - |
| Glycerine GLY | - | Gentiobiose GEN | + |
| Red tinea alcohol ERY | - | D-turanose TUR | + |
| D-pectinose DARA | - | D-lyxose LYX | - |
| L-arabinose LARA | + | D-tagatose TAG | - |
| Ribose RIB | + | D-rock sugar DFUC | - |
| D-wood sugar DXYL | - | L-rock sugar LFUC | - |
| L-wood sugar LXYL | - | D-arabitol DARL | + |
| Adonose ADO | - | L-arabinose alcohol LARL | - |
| Beta-methyl-D-xyloside MDX | - | Gluconate GNT | + |
| Semi-lactosi GAL | + | 2-ketone group-gluconate 2KG | - |
| Glucose GLU | + | 5-ketone group-gluconate 5KG | + |
| Fructose FRU | + | Glycogen GLYG | - |
| Seminose MNE | + | Starch AMD | - |
| Sorbose SBE | - | Raffinose RAF | + |
| Rhamnosyl RHA | - | Pine three sugared MLZ | + |
| Melampyrin DUL | - | Synanthrin INU | - |
| Inositol INO | - | Trehalose TRE | + |
| N.F,USP MANNITOL MAN | + | Sucrose SAC | + |
| Sorbyl alcohol SOR | + | Melibiose MEL | + |
| Alpha-Methyl-D-sweet dew glucoside MDM | + | Lactose LAC | + |
| Polychrom ESC | + | Xylitol XLT | - |
| Alpha-Methyl-D-glycoside MDG | - | Maltose MAL | + |
| N-acetyl-grape amine NAG | + | Cellobiose CEL | + |
| Vitamin B17 AMY | + | Salicin SAL | + |
| Ursin ARB | + |
Annotate: "+" is reacting positive, and "-" is reaction negative.
The characteristic of embodiment 2, plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329
Material
Strain golden look staphylococcus A TCC6035, Candida albicans ATCC1023, intestinal bacteria ATCC25922, gardnerella vaginalis ATCC14018
Reagent
MRS substratum, LM broth culture are available from French Mei Liai biotech firm;
Tryptones, yeast extract, agar powder are available from Oxide;
Tetramethyl benzidine (TMB), dimethyl formamide, horseradish peroxidase (HRP) are available from Sigma company.
1, bacteriostatic test
With plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 inoculation in the MRS substratum, 37 ℃, 5%CO
2Cultivated 24 hours, and washed lawn, than turbid and be diluted to 1 * 10 with stroke-physiological saline solution
8CFU/ml concentration bacterium liquid, it is standby that diameter is that the circle sterilization filter paper of 5mm soaks behind the full bacterium liquid.
Streptococcus aureus ATCC6035, Candida albicans ATCC1023, intestinal bacteria ATCC25922 are inoculated in ordinary nutrient agar substratum (extractum carnis 3.0 gram/L respectively, peptone 10.0 gram/L, NaCl 5.0 gram/L, agar 15 gram/L, pH 7.0) on, cultivated 24 hours for 37 ℃, gardnerella vaginalis ATCC14018 is inoculated in the GC blood agar, 37 ℃ of 5%CO
2Cultivated 24 hours, and washed thalline with stroke-physiological saline solution respectively, than turbid and be diluted to 1 * 10
8CFU/ml concentration bacterium liquid, respectively get 0.1ml coating ordinary nutrient agar flat board, the above-mentioned circular filter paper sheet that is soaked with plant lactobacillus (Lactobacillus plantarum) L323CGMCC No.1329 is placed on the dull and stereotyped mid-way that is coated with above-mentioned bacterium respectively, be adjacent to, carry out mark, the flat board that scribbles streptococcus aureus ATCC6035, Candida albicans ATCC1023, intestinal bacteria ATCC25922 is put into common incubator respectively, the flat board that scribbles gardnerella vaginalis ATCC14018 is put into 5%CO respectively
2Incubator was cultivated 48 hours for 37 ℃, and the survey record antibacterial circle diameter is also taken a picture.
The result of bacteriostatic test shows that plant lactobacillus (Lactobacillusplantarum) L323 CGMCC No.132 has bacteriostatic action to streptococcus aureus, Candida albicans, intestinal bacteria and gardnerella vaginalis (streptococcus aureus, Candida albicans, intestinal bacteria and gardnerella vaginalis all are to cause the vaginitis common pathogen) shown in table 2 and Fig. 2 A-Fig. 2 D.Illustrate that plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.132 has fungistatic effect preferably to the common pathogenic bacterium of woman vagina.
Table 2 L323 strain lactobacillus is to the fungistatic effect of common pathogen
2, produce acidacidity and composition analysis test
Plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 is inoculated in the MRS substratum, 37 ℃ of 5%CO
2Cultivate after 24 hours, wash lawn, make 1 * 10 with physiological saline
8The CFU/ml bacteria suspension is got the 0.1ml bacterial suspension inoculation in 5ml LM broth culture, 37 ℃ of 5%CO
2Cultivated respectively 24 hours, 48 hours, 72 hours, 96 hours, and measured the pH value that different time is cultivated bacterium liquid with the pH acidity tester.
The changing conditions of the pH value of the different incubation time nutrient solutions of plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 is as shown in table 3.
Table 3. plant lactobacillus L323 different time produces sour test-results
Get 24 hours lactobacillus LM broth culture 10ml of above-mentioned cultivation, centrifugal 15 minutes of 8000rpm abandons bacterial sediment, collects supernatant, and to the short carbon organic acid in the opsonigenous substances: acetate, propionic acid, butyric acid, valeric acid carry out gas chromatographic analysis.The result is as shown in table 4, shows that the short carbon organic acid that plant lactobacillus (Lactobacillusplantarum) L323 CGMCC No.1329 produces is mainly acetate, cultivates 24 hours production acetate and can reach 1160ug/ml.
Table 4. plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 produces the composition and the volume analysis of acid
| The organic acid composition | Output (ug/ml) |
| Acetate | 1160 |
| Propionic acid | ＜50 |
| Butyric acid | ＜50 |
| Valeric acid | ＜20 |
3, produce hydrogen peroxide (H
2O
2) test
Take by weighing tetramethyl benzidine (TMB) 12.5mg and be dissolved in the 1ml dimethyl formamide, with 50ml MRS substratum mixing, pave plate after 121 ℃ of sterilizations in 15 minutes, obtain the TMB-MRS culture medium flat plate, 4 ℃ of lucifuges are placed standby.
Press 0.01mg/ml concentration preparation horseradish peroxidase (HRP) solution, 0.45 μ m membrane filtration degerming is got 0.1ml and evenly is coated with the TMB-MRS culture medium flat plate before the inoculation, dry, and 4 ℃ of lucifuges are placed.
Plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 is inoculated in the TMB-MRS substratum of the above-mentioned HRP of being coated with, 37 ℃ of 5%CO
2The incubator lucifuge was cultivated 24 hours, took out culture plate, exposed light after 3 minutes, observed colony colour variation and record result on the culture plate.Interpretation as a result: H is being arranged
2O
2Under the situation about existing, HRP can form the blue-greenish colour oxide compound by oxidation TMB.Therefore, if milk-acid bacteria produces H
2O
2, the bacterium colony that grows on the substratum that contains TMB and be coated with HRP will become blue-greenish colour, and does not produce H
2O
2The bacterium colony of lactobacillus strain be canescence.The result as shown in Figure 3, plant lactobacillus (Lactobacillusplantarum) L323 CGMCC No.1329 produces H
2O
2Be the blue-greenish colour bacterium colony.Lactobacillus produces H
2O
2Can make vagina microenvironment anoxic, thereby suppress aerobic pathogenic bacterium growth.There is test to show, lacks in the recurrent vaginitis that causes by intestinal bacteria and urethritis patient vagina and the urethra and produce H
2O
2Lactobacillus (Wilks M, Wiggins R, Whiley A, Hennessy E, Warwick S, Porter H, Corfield A, Millar M.Identification and H (2) O (2) production of vaginal lactobacilli frompregnant women at high risk of preterm birth and relation with outcome.J Clin Microbiol.2004 Feb; 42 (2): 713-717).
4, the Ultrastructural electron microscopic observation of bacterial strain
37 ℃ of 5%CO of plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 inoculation MRS substratum
2Cultivate 24h, wipe away with aseptic cotton swab and get lawn and be inoculated in 5ml LM broth culture, 37 ℃ of 5%CO
2Cultivated 24 hours, centrifugal 10 minutes of 5000rpm/min, abandon supernatant, the normal mouse eyeball is got behind the blood separation of serum immediately, 1 of mice serum getting fresh separated splashes in the bacterial sediment, room temperature is placed and the bacterium piece was fixed in 10 minutes, adds glutaraldehyde stationary liquid 1ml, places 4 ℃ of preparations that are used for electron microscope specimen.The electron microscopic observation result as shown in Figure 4, plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 thalline surface has thick hair sample projection, promptly non-pili adhesin, this material are the epithelial bases of bacterial adhesion.
5, the stability test of bacterial strain preservation
Observe the survival condition of lactobacillus bacterium in difference preservation liquid.
Preserve the preparation of liquid:
Preserve liquid 1:MRS substratum+5% (quality percentage composition) skimmed milk
Preserve liquid 2:MRS substratum+5% (quality percentage composition) skimmed milk+trehalose 3.78g
Preserve liquid 3:MRS substratum
With plant lactobacillus (Lactobacillus plantarum) the L323 CGMCC No.1329 that cultivated in 24 hours centrifugal 5 minutes with 4800rpm/min, preserve the liquid precipitation thalline that suspends with difference, the thalline counting is 2.2 * 10
5CFU/ml, packing is preserved pipe 0.5ml/ and is propped up respectively, places respectively in differing temps (37 ℃, the room temperature) environment and keeps in Dark Place, and goes bail in every month and to deposit 1 of pipe, and sucking-off bacterium liquid 0.1ml is diluted to different concns with 1ml LB meat soup, is coated with the MRS culture medium flat plate, 37 ℃ of 5%CO
2Cultivate counting after 24 hours, observe and different liquid preservation effect, i.e. the bacteria living situations of preserving of record.The survival condition of plant lactobacillus (Lactobacillus plantarum) L323 CGMCCNo.1329 is as shown in table 5.
The different survival numbers (CFU/ml) of preserving plant lactobacillus (Lactobacillus plantarum) L323 CGMCCNo.1329 in the liquid of table 5.
Claims (2)
1. plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329.
2. plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 prevents and/or treats application in the woman vagina infectious disease medicament in preparation.
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|---|---|---|---|
| CN200510080072A CN1888051B (en) | 2005-06-28 | 2005-06-28 | Plant lactobacillus strain and its application |
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| CN200510080072A CN1888051B (en) | 2005-06-28 | 2005-06-28 | Plant lactobacillus strain and its application |
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| CN1888051B true CN1888051B (en) | 2010-05-05 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200510080072A Expired - Fee Related CN1888051B (en) | 2005-06-28 | 2005-06-28 | Plant lactobacillus strain and its application |
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Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974449B (en) * | 2010-09-13 | 2012-06-06 | 郑州大学 | Lactobacillus plantarum and application thereof |
| CN101974468B (en) * | 2010-10-26 | 2012-04-18 | 郑州大学 | Lactobacillus plantarum and application thereof |
| CN101974467B (en) * | 2010-10-26 | 2012-10-24 | 郑州大学 | Lactobacillus plantarum and application thereof |
| FR2992861B1 (en) * | 2012-07-09 | 2014-10-17 | Probionov | USE OF THIOSULFATE FOR POTENTIATING THE ANTI-PATHOGENIC EFFECT OF LACTOBACILLES |
| CN103911306B (en) * | 2013-01-05 | 2016-08-24 | 欣力菲生物医药技术（天津）有限公司 | Lactobacillus gasseri bacterial strain and application thereof |
| CN103911309B (en) * | 2013-01-05 | 2016-08-24 | 欣力菲生物医药技术（天津）有限公司 | Lactobacillus gasseri bacterial strain and application thereof |
| CN106167786A (en) * | 2016-09-05 | 2016-11-30 | 北京蓝色美城科技有限公司 | One strains of lactic acid bacteria and application thereof |
| CN106166163A (en) * | 2016-09-05 | 2016-11-30 | 北京蓝色美城科技有限公司 | A kind of feminine wash of the fermentation supernatant containing lactic acid bacteria |
| CN107299065B (en) * | 2017-06-20 | 2020-02-14 | 广东强基药业有限公司 | Lactobacillus plantarum and application thereof in preparation of vagina bacteriostatic drugs |
| CN108295096B (en) * | 2017-12-21 | 2020-12-25 | 天津科睿恒生物科技有限公司 | Application of bacterial strain in preparation for preventing and treating mycotic and bacterial vaginosis |
| KR101930438B1 (en) * | 2018-10-12 | 2018-12-18 | (주) 에이투젠 | Novel Lactobacillus plantarum strain ATG-K2, ATG-K6 or ATG-K8, and composition comprising thereof for preventing or treating vaginosis |
| CN109439592A (en) * | 2018-12-06 | 2019-03-08 | 青岛东海药业有限公司 | Lactobacillus plantarum preparation and its application |
| CN109504630B (en) * | 2018-12-17 | 2022-03-11 | 吉林中粮生化有限公司 | Lactobacillus plantarum strain produced by recombinant D-lactic acid and method for producing D-lactic acid by using strain |
| CN109628354A (en) * | 2019-01-30 | 2019-04-16 | 江南大学 | The lactobacillus plantarum of one plant height bacteriostatic activity and application |
| CN111000873A (en) * | 2019-09-03 | 2020-04-14 | 江苏中天药业有限公司 | Lactobacillus vaginal effervescent capsule and preparation method thereof |
| CN112798370A (en) * | 2020-12-24 | 2021-05-14 | 桂林优利特医疗电子有限公司 | Quality control material for detecting visible components of vaginal secretion and preparation method thereof |
| CN114717150B (en) * | 2022-04-20 | 2023-01-17 | 河北农业大学 | Lactobacillus plantarum CRS33 and application thereof |
| CN114869917B (en) * | 2022-07-07 | 2022-10-28 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus plantarum N13 in preparation of medicine for preventing and treating vaginitis or inhibiting pathogenic bacteria |
| CN116004457B (en) * | 2022-12-28 | 2024-05-07 | 内蒙古双奇药业股份有限公司 | Humanized lactobacillus plantarum and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1185111A (en) * | 1995-03-23 | 1998-06-17 | 普罗比公司 | Eipthelial adhesive lactobacilli |
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2005
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1185111A (en) * | 1995-03-23 | 1998-06-17 | 普罗比公司 | Eipthelial adhesive lactobacilli |
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| CN1888051A (en) | 2007-01-03 |
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