CN1322111C - Plant lactobacillaceae and use thereof - Google Patents
Plant lactobacillaceae and use thereof Download PDFInfo
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- CN1322111C CN1322111C CNB200410093213XA CN200410093213A CN1322111C CN 1322111 C CN1322111 C CN 1322111C CN B200410093213X A CNB200410093213X A CN B200410093213XA CN 200410093213 A CN200410093213 A CN 200410093213A CN 1322111 C CN1322111 C CN 1322111C
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- Coloring Foods And Improving Nutritive Qualities (AREA)
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Abstract
The present invention relates to a novel lactobacillus bacterial strain and an application thereof, particularly to novel lactobacillus plantarum (LP-Onlly), CGMCC No. 1258 and an application of the lactobacillus plantarum (LP-Onlly) to the intestinal flora regulation and the intestinal mucosal barrier function enhancement. Particularly, after being combined with conventional carriers, the lactobacillus plantarum (LP-Onlly) can be used for preventing and/or treating the alteration of intestinal flora and preventing and/or treating the translocation infection of intestinal bacteria.
Description
Invention field
The present invention relates to a kind of new bacterial strain of lactobacillus, more precisely, the new bacterial strain LP-Onlly, 16S rRNA gene GeneBank (NCBI) the sequence accession number that the invention describes plant lactobacillus (Lactobacillus plantarum) are the AY590777 sequence, and regulate the intestinal flora function, strengthen the intestinal mucosal barrier function and to the bacteriostatic action of pathogen enterobacteria, food spoilage bacterium.The invention still further relates to lactobacterium plantarum strain LP-Onlly and conventional carrier combinations, can be used for preventing and/or treating alteration of intestinal flora and intestinal bacterial translocation and infect.
Background technology
People's gi tract are being perched various bacteriums, can be divided into probiotics, harmful bacterium and neutral bacterium according to the influence to human body.Probiotics is preponderated in the human body, will show state of health, and wherein bifidus bacillus and Bacterium lacticum are the most representative probioticss.
Milk-acid bacteria is that Frenchman's pasteur was found first in 1857.The application of milk-acid bacteria on food and medicine is very extensive, and milk-acid bacteria and goods thereof mainly contain the effect of human body: (1) regulates host's intestinal microflora, and enhance immunity power strengthens resistance against diseases; (2) lactic acid of metabolism generation, the digestion that can improve food in the stomach promotes the discharge of gastric content, promotes digestion, the ferment of generation can promote the decomposition of food nutrition, the promotion dietetic alimentation; (3) milk-acid bacteria reduces lactose, and produces lactic acid, can effectively suppress the breeding of enteron aisle spoilage organism and the generation of objectionable impurities, promotes intestinal peristalsis, prevents constipation; (4) milk-acid bacteria decomposing protein makes it become micromolecular peptide and amino acid etc., is easy to digest and assimilate etc.
From (Ann Surg, 1987 such as the mid-80 Detich in last century; 205 (6): 681～692) proposed since the notion of " bacterial translocation ", intestinal mucosal barrier destroys and causes that bacterium or endotoxin translocation have become common recognition, intestinal mucosal barrier destroys and produces pyemia, shock, MOFE, has increased patient with severe symptoms's mortality ratio significantly.So a lot of scholars propose should be at its " source "---enteron aisle to the treatment infected, rather than target organ.For this reason, adopt beneficial bacteria of intestinal tract to improve intestinal microecology, the enhancing intestinal epithelial cells closely connects and intestinal barrier function receives publicity gradually thereby reach.
Plant lactobacillus (Lactobacillus plantarum) is a human body ancestral home bacterium, has biological action in the body, over the past thousands of years, plant lactobacillus is used to the human food prods always, it is traditional lactic acid fermented critical strain, also be the basis that food such as sauerkraut can long storage, human in edible a large amount of plant lactobacillus leavened foods, also evolved a large amount of plant lactobacillus that adapted to field planting of human intestinal.Phenotype analytical studies show that in conjunction with hereditary feature, the symbiotic dominant bacteria of healthy people's digestive tube is mainly plant lactobacillus (L.plantarum), lactobacillus rhamnosus (L.rhamnosus) and lactobacterium casei (L.paracasei), its verification and measurement ratio accounts for all 52%, 26% and 17% (The normal Lactobacillus flora of healthy human rectal and oralmucosa that separate Bacterium lacticum respectively, Journal of Applied Microbiology, 1998,85:88～94).In plant latic acid-fermented foods such as sauerkraut, contain a large amount of plant lactobacilluss, traditional plant lactobacillus viable bacteria product is the beverage " togwa " of Tanzania, with cassava or wheat is raw material, plant lactobacillus is that starter is made plant lactobacillus viable bacteria product, usually be used to prevent and treat intestinal tract disease, improve intestinal mucosal barrier function (Probiotics in foods not containingmilk or milk constituents, with special reference to Lactobacillus plantarum 299v, American Journal of Clinical Nutrition, 2001,73 (suppl): 380S～5S).
Summary of the invention
A new bacterial strain LP-Onlly who the purpose of this invention is to provide a kind of plant lactobacillus, it has regulates the intestinal flora function, strengthens the intestinal mucosal barrier function and to the bacteriostatic action of pathogen enterobacteria, food spoilage bacterium.The invention still further relates to lactobacterium plantarum strain LP-Onlly and conventional carrier combinations, can be used for preventing and/or treating alteration of intestinal flora and prevent and/or treat the intestinal bacterial translocation infection.It has the intestinal microflora of adjusting and strengthens the intestinal mucosal barrier function.
The solution that realizes the foregoing invention purpose is:
A kind of lactobacterium plantarum strain LP-Onlly, this lactobacterium plantarum strain on December 6th, 2004 at China Committee for Culture Collection of Microorganisms common micro-organisms center, be called for short: CGMCC (address: No. 13, one of Zhong Guan-cun, Haidian District, BeiJing, China city, Institute of Microorganism, Academia Sinica) preservation, and receive preservation registration number CGMCC NO 1258.Its called after of classifying: plant lactobacillus, latin name: Lactobacillus plantarum.Lactobacterium plantarum strain LP-Onlly, its 16S rRNA gene GeneBank (NCBI) sequence accession number is AY590777.
Lactobacterium plantarum strain LP-Onlly has the intestinal microflora of adjusting function.
Lactobacterium plantarum strain LP-Onlly has the intestinal mucosal barrier of enhancing function.
Lactobacterium plantarum strain LP-Onlly has the function that suppresses pathogen enterobacteria and food spoilage microorganism.
More than said pathogen enterobacteria be enterobacteria, listeria bacteria, Salmonellas, streptococcus aureus, faecalis, dysentery bacterium, clostridium perfringens.
More than said food spoilage microorganism mainly refer to subtilis, bacillus cereus.
Plant lactobacillus LP-Onlly and conventional carrier combinations can be used for preventing and/or treating alteration of intestinal flora.
Plant lactobacillus LP-Onlly and conventional carrier combinations can be used for preventing and/or treating intestinal bacterial translocation and infect.
Description of drawings
Fig. 1: abdominal cavity infection rat intestine flora dna fingerprinting.
Among the figure: M is Marker, and Lane 1～8 is the plant lactobacillus group, and Lane 9～10 is a normal rat, and Lane11～17 are the PN group.
Fig. 2: PN organizes small intestine epithelium occludin protein expression (* 400).
Fig. 3: probiotic group small intestine epithelium occludin protein expression (* 400).
Fig. 4: PN organizes big enteric epithelium occludin protein expression (* 400).
Fig. 5: the big enteric epithelium occludin of probiotic group protein expression (* 400).
Fig. 6: PN group small intestine epithelium perspective Electronic Speculum (2 * 10
4).
Fig. 7: the big enteric epithelium perspective of probiotic group Electronic Speculum (2 * 10
4).
Fig. 8: PN group small intestine epithelium perspective Electronic Speculum (2 * 10
4).
Fig. 9: the big enteric epithelium perspective of probiotic group Electronic Speculum (2 * 10
4).
Embodiment
Lactobacterium plantarum strain LP-Onlly of the present invention directly separates acquisition in healthy babies ight soil, its form is shaft-like, and Gram-positive does not form gemma, produces lactic acid, amphimicrobian.It can utilize L-arabinose, sorbyl alcohol, melibiose, D-turanose, D-arabitol, Sunmorl N 60S, Alpha-Methyl-D-mannoside, can not utilize sorbose, rhamnosyl, D-tagatose, Alpha-Methyl-D-glycoside.
Its fermenting process: in the MRS liquid nutrient medium, cultivate 72h for 37 ℃.
This lactobacterium plantarum strain LP-Onlly on December 6th, 2004 at China Committee for Culture Collection of Microorganisms common micro-organisms center (address: No. 13, one of Zhong Guan-cun, Haidian District, BeiJing, China city, Institute of Microorganism, Academia Sinica) preservation, and receive preservation registration number CGMCC NO 1258.
The lactobacterium plantarum strain that the present invention relates to, its 16S rna gene sequence is put on record in GeneBank (NCBI), and its accession number is AY590777.
Further specify the present invention below in conjunction with embodiment and Tu:
Embodiment 1: the bacterial strain microscopy
Choose lactobacterium plantarum strain LP-Onlly with transfering loop under the aseptic condition, be applied on the slide glass, film-making and gramstaining are examined under a microscope thalli morphology.
The dull and stereotyped purifies and separates of embodiment 2:MRS
Choose lactobacterium plantarum strain LP-Onlly with transfering loop under the aseptic condition, rule on the MRS flat board, flat board places incubator to cultivate 72h for 37 ℃, selects single bacterium colony and carries out microscopy, reaches the purebred separation of bacterial strain.
Embodiment 3: strain fermentation
With transfering loop lactobacterium plantarum strain LP-Onlly picking two from the inclined-plane is encircled to being equipped with the 50ml MRS liquid nutrient medium, cultivate 24h for 37 ℃, insert in the 50ml MRS liquid nutrient medium with 5% inoculum size again, cultivate 72h, promptly get the plant lactobacillus LP-Onlly fermented liquid for 37 ℃.
Embodiment 4: lactobacterium plantarum strain LP-Onlly bacteriostasis property
Lactobacterium plantarum strain LP-Onlly puts this bacterium liquid on the MRS solid plate in 37 ℃ of aerobic cultivation 24h of MRS liquid nutrient medium, and parallel three points of every plate are in 37 ℃ of aerobic cultivation 24h.Indicator liquid (intestinal bacteria with various 200 μ l overnight incubation, streptococcus aureus, Lactobacterium acidophilum, subtilis, plant lactobacillus, enterococcus faecalis, salmonella typhi, dysentery bacterium, Listeria monocytogenes, bacillus cereus, clostridium perfringens and bifidobacterium adolescentis) respectively with 7ml soft agar mixing, be poured on (soft agar remains on 45～50 ℃) on the MRS of the lactobacterium plantarum strain LP-Onlly flat board a little arranged, aerobic or anaerobism (according to indicator) is cultivated 24h (aerobic) or 48h (anaerobism) for 37 ℃, takes pictures and measures inhibition zone size (table 1) around the indicator spot.
Table 1 is the result show, lactobacterium plantarum strain LP-Onlly all has restraining effect to most of harmful intestinal tract bacteria, wherein the restraining effect to subtilis, bacillus cereus, Listeria monocytogenes and salmonella typhi is the strongest, a little less than the restraining effect summary to intestinal bacteria, streptococcus aureus, enterococcus faecalis, dysentery bacterium, clostridium perfringens, and to probiotic bacterium such as the equal unrestraint effect of Lactobacterium acidophilum, plant lactobacillus and bifidobacterium adolescentis.
Table 1 lactobacterium plantarum strain LP-Onlly bacteriostasis property
| Indicator | Antibacterial circle diameter (mm) | |||
| 1 | 2 | 3 | On average | |
| Escherichia coli and staphylococcus aureus lactobacillus acidophilus bacillus subtilis Lactobacillus plantarum enterococcus faecalis salmonella typhi Shigella shigae Listeria monocytogenes bacillus cereus C.perfringens bifidobacterium adolescentis | 10.0 12.0 - 20.6 - 12.9 20.7 11.4 18.7 26.3 10.7 - | 10.6 12.2 - 30.4 - 13.7 19.1 11.5 20.8 21.5 6.7 - | 12.6 10.3 - 29.4 - 10.2 19.8 13.9 25.4 19.9 9.6 - | 11.1 11.5 - 26.8 - 12.3 19.9 12.3 21.6 22.6 9.0 - |
Annotate: "-" expression lactobacterium plantarum strain LP-Onlly does not have restraining effect to indicator.
Embodiment 5: lactobacterium plantarum strain LP-Onlly regulates the experimentation on animals of intestinal microflora function
1. lactobacterium plantarum strain LP-Onlly regulates the experimentation on animals of mouse intestinal flora function
1.1 lactobacterium plantarum strain LP-Onlly regulates the experimentation on animals of normal mouse intestinal microflora function
40 normal mouses are divided into two groups, and one group is plant lactobacillus LP-Onlly group (15), irritate every day and feed plant lactobacillus LP-Onlly fermented liquid 1ml (＞1 * 10
8Cfu/ml), continuous 6d, another group is control group (25), detects mouse intestinal microorganism changing conditions, data represent that with x ± s applied statistics software SPSS 10.0 carries out t check and correlation analysis.(table 2)
Table 2 is the result show, compares with control group, irritates hello plant lactobacillus LP-Onlly fermented liquid the bifidus bacillus of mouse intestinal is obviously increased, and significant difference (P＜0.05) is arranged.This shows that lactobacterium plantarum strain LP-Onlly has the effect of regulating the intestinal microflora function aspects.
Table 2 plant lactobacillus is to regulating effect (x ± s, the lg10 of normal mouse intestinal flora
n/ ml)
| Enteric microorganism | Control group (n=25) | Plant lactobacillus group (n=15) |
| Faecalis intestinal bacteria Bacterium lacticum bifidus bacillus | 6.376±0.409 7.704±0.303 9.280±0.354 8.698±0.234 | 6.215±0.401 7.683±0.282 9.138±0.500 8.900±0.228 * |
*Compare plant lactobacillus group P＜0.05 with the normal control group.
1.2 lactobacterium plantarum strain LP-Onlly regulates the intestinal microflora function experimentation on animals of flora imbalance mouse
59 of mouse are divided into following three groups, carry out the intestinal microflora function regulating effect research of plant lactobacillus LP-Onlly to the alteration of intestinal flora mouse.
25 mouse of control group; 17 mouse of rheum officinale group are irritated the big yellow liquor 1ml of stomach every day, and successive administration 8d causes alteration of intestinal flora, and 9d puts to death; 17 mouse of plant lactobacillus group are irritated the big yellow liquor 1ml of stomach every day, continuous 8d, but at the 3d time-out of giving big yellow liquor, more in addition stomach plant lactobacillus LP-Onlly fermented liquid 1ml (＞1 * 10 that irritate more
8Cfu/ml), continuous 6d, 9d puts to death; Get the ight soil of respectively organizing mouse respectively and detect mouse intestinal microorganism changing conditions, data represent that with x ± s applied statistics software SPSS 10.0 carries out t check and correlation analysis.(table 3)
Table 3 plant lactobacillus is to intestinal microflora regulating effect (x ± s, the lg10 of mouse intestinal flora imbalance
n/ ml)
| Enteric microorganism | Control group (n=25) | Rheum officinale group (n=17) | Plant lactobacillus group (n=17) |
| Faecalis intestinal bacteria Bacterium lacticum bifidus bacillus | 6.376±0.409 7.704±0.303 9.280±0.354 8.698±0.234 | 6.087±0.406 * 8.194±0.387 *** 9.010±0.346 ** 8.362±0.560 * | 6.334±0.396 * 7.888±0.328 * 9.208±0.302 * 8.587±0.329 * |
Compare with control group,
*Be P＜0.05,
*Be P＜0.01,
* *Be P＜0.001.
By table 3 as seen, rheum officinale group and control group are relatively, four kinds of main flora quantity of enteron aisle all have considerable change, show as faecalis and bifidus bacillus quantity obviously descend (P＜0.05), Bacterium lacticum digital display work reduces (P＜0.01), and coliform count extremely significantly increases (P＜0.001), and this shows mouse after being irritated the stomach rheum officinale, its alteration of intestinal flora.And the plant lactobacillus group compared with the control, adds plant lactobacillus LP-Onlly again behind the 3d of the big yellow liquor of clothes, though rheum officinale is still obeyed 8d, four kinds of bacterial numbers in the enteron aisle all can remain on normal range, with the rheum officinale group significant difference are arranged more all.This shows that after alteration of intestinal flora lactobacterium plantarum strain LP-Onlly can regulate flora imbalance, keeps the normal intestinal flora balance.
2. lactobacterium plantarum strain LP-Onlly is to the regulating effect of abdominal cavity infection rat intestinal microflora
15 rats are after caecum ligation and perforation method and jugular vein jejunum are put pipe to make the abdominal cavity infection model (table 4), give parenteral nutrition (PN group) and PN+ plant lactobacillus LP-Onlly fermented liquid (plant lactobacillus group) respectively and continue 5d, 6d puts to death, get the interior ight soil of caecum and carry out intestinal microflora counting (table 5), and to its dna fingerprinting analysis, to inquire into the regulating effect of plant lactobacillus LP-Onlly to abdominal cavity infection rat intestinal microflora.
Table 4 is the result show, plant lactobacillus LP-Onlly group abdominal cavity infection rats death rate is starkly lower than the PN group.This shows the plant lactobacillus LP-Onlly fermented liquid by adjusting abdominal cavity infection rat intestinal microecology, thereby reduces its mortality ratio.
Table 4PN group compares with plant lactobacillus group abdominal cavity infection rats death rate
| Group | Experiment number (only) | Death toll (only) | Mortality ratio (%) |
| PN group plant lactobacillus group | 33 16 | 26 8 | 78.7 50 |
Table 5 intestinal microflora analysis of accounts (x ± s, lg10
n/ ml)
| Intestinal bacteria | PN organizes (n=7) | Plant lactobacillus group (n=8) |
| Enterobacteria bifidus bacillus Bacterium lacticum Clostridium perfringens | 7.061±0.191 8.006±0.272 5.416±0.304 5.702±0.373 | 7.231±0.171 8.313±0.303 * 5.713±0.178 * 5.056±0.355 * |
Annotate:
*Compare P＜0.05 with the PN group
Can find obviously in the table 5 that plant lactobacillus group rat enteron aisle Bacterium lacticum and bifidus bacillus quantity are many than the PN group, and the potentially pathogenic organism clostridium perfringens quantity in the enteron aisle is few than the rat of PN group, and significant difference is all arranged.This has shown that the lactobacterium plantarum strain LP-Onlly among the present invention has the intestinal microflora of adjusting function.
Is primer with the ITS universal primer to PS2 (5 '-TG (C/T) ACA CACCGC CCG T-3 '), PL2 (5 '-GGGT (G/C/T) CCC CAT TC (A/G) G-3 '), above-mentioned rat faeces DNA is carried out pcr amplification, and make agarose gel electrophoresis and identify (Fig. 1).
Can find out obviously that from Fig. 1 the collection of illustrative plates band obviously is less than normal group Lane9-10 and plant lactobacillus group Lane1～8 in PN group Lane11～17, and Lane11,14,15 there is a very bright band (as marking among the figure).This shows, the flora imbalance of PN group rat intestine, and the minimizing of normal microflora kind, non-intestinal microflora increase in the enteron aisle, and plant lactobacillus group band is comparatively approaching with normal group Lane9～10 collection of illustrative plates bands obviously more than PN group Lane.The result also shows among the figure, and plant lactobacillus group rat flora band and normal rat have higher consistence, and the PN group then has notable difference.This results suggest, the intestinal microflora disorder when lactobacterium plantarum strain LP-Onlly can correct abdominal cavity infection P of Rats N has the function of the intestinal microflora of adjusting.
3. conclusion
Above-mentioned experimental result shows that the lactobacterium plantarum strain LP-Onlly among the present invention has the effect of regulating the intestinal microflora function, with conventional carrier combinations, can prevent and/or treat alteration of intestinal flora.
Embodiment 6: lactobacterium plantarum strain LP-Onlly strengthens the intestinal barrier function experimentation on animals
Animal model experiment is used more and more widely in the functional evaluation experimental of probiotic bacterium.
The present invention adopts male SD rat, give parenteral nutrition (PN group) and PN+ plant lactobacillus LP-Onlly fermented liquid (probiotic group) respectively and continue 5 days, put to death in the 6th day, and got Vena cava blood, lung, liver and the homogenate of mesentery Lymphoid tissue and make microbial culture survey bacterial translocation rate; Get latter end ileum and colon, the electron microscopic observation intestinal epithelial cells closely connects ultrastructure, and the immunohistochemical methods method is measured it and striden embrane-associated protein (Occludin) expression, thereby judges whether this bacterial strain has the intestinal mucosal barrier of enhancing function.
The foundation of 1 rat abdominal cavity infection model
Male SD rat, body weight 250～320g (Medical Center of Fudan University's animal center provides).After 2% hydrochloric acid chlore-ammonia ketone injection liquid (30mg/g) is done intraperitoneal injection anesthesia, rat is fixed in dorsal position, neck and belly unhairing, with the sterilization of 0.1% bromogeramine tincture, make the 4cm otch along the belly center and open the abdominal cavity, adopt the cecal ligation and perforation method to make model, be about to 1/3rd places ligation of caecum far-end, and run through with No. 12 syringe needles in the top, put one of the wide skin graft of 2mm, and give fixing with anti-slip.Jejunum at distance caecum 30-40cm is made pocket, inserts external diameter and is the soft silicone tube of 1.2mm and make jejunum fistulization pipe, and silicone tube is drawn by nape portion is subcutaneous, and abdomen is closed in layering.Insert the soft silicone tube that external diameter is 1.2mm with reference to methods such as Steiger through external jugular vein, silicone tube is drawn from nape portion is subcutaneous through the subcutaneous abdomen tunnel, pass spiral protection spring and link to each other with swivel arrangement.
2. animal grouping and the configuration of PN liquid
The animal of 15 modeling successes is divided into two groups: (1) PN organizes (n=7), and the 1st～5d gives PN and supports that PN liquid is kept 24h with microinfusion pump, evenly input, the about 80ml/d of average every rat transfusion quantity; (2) beneficial puffball group (n=8), the 1st～5d gives the support of PN liquid, and (viable bacteria content is 10 in conjunction with the plant lactobacillus LP-Onlly fermented liquid
8Cfu/ml), wherein PN liquid amount of infusion is organized with PN, and probiotics fermention liquid divides through jejunum fistulization pipe slowly inject about altogether 10ml/d three times every day.Above-mentioned two groups of rats detect or observe in 6d execution of modeling success back and sampling.
PN liquid formula (table 6), two groups of impartial nitrogen, heat provide non-nitrogen heat 348kJ (about 240kcal/kg) every day, confession nitrogen 504mg.
Table 6PN liquid formula (content of every 100ml solution)
| Prescription | Dosage |
| The non-albumen calorie of 50%Glucose (ml) 8.5%Novamin (ml) 20%Intralipid (ml) Soluvit (ml) Addamel Injection (ml) 10%kCl (ml) 10%NaCl (ml) RI (u) heparin (u) (KJ) total amount of heat (Kcal) nitrogen pool (mg) | 33 45 17 1 1 1 1 4 40 435 101 630 |
3. observation index
(1) mucous membrane occludin protein immunization group mensuration:
Get terminal ileum and colon's frozen section, adopt indirect immunofluorescence to detect small intestine and mucous membrane of colon occludin protein content.One anti-is the mouse occludin of goat Chinese People's Anti-Japanese Military and Political College antibody (Santa Crus), dilution in 1: 300; The two anti-anti-goat IgG of ABC test kit (Santa Crus) that adopt.Use HPIAS1000 high-definition color picture and text pathological replacement analytical system, under identical magnification (* 400), in each time group section, select 5 visuals field at random, adopt optical densitometric method to measure every visual field corresponding cell occludin density, and the positive colour developing of calculating area account for the per-cent in the measurement window.
(2) intestinal mucosa form Electronic Speculum detects
Get apart from ileocecus 5cm place's ileum and each about 0.5cm of colonic segment, stringer is cut open, cleans, and (1 * 1mm is cut into small pieces
2) glutaraldehyde fixes, ultra-thin film-making, the transmission electron microscope observing epithelial cell closely connects.
(3) Vena cava blood, mesentery Lymphoid tissue, liver and lung tissue bacteria content are measured
Getting Vena cava blood 1ml, mesentery Lymphoid tissue (5g), liver middle period (5g) and inferior lobe of right lung tissue (5g) pours in the heart and brain leach liquor pipe in refiner respectively after the homogenate, cultivate 18～24h for 35 ℃, place Yihong methylene blue flat board then, count behind 35 ℃ of cultivation 18～24h.
(4) statistical procedures
Data represent that with x ± s applied statistics software spss10.0 carries out x
2Check, variance analysis, t check and correlation analysis, P＜0.05 is a significant difference.
4. interpretation of result
(1) enteric epithelium is striden embrane-associated protein (occludin) content
The enteric epithelium barrier function with closely be connected (Tight Junctions, TJ) complex body is closely related, in vitro study shows that the opening of intestinal epithelial cells TJ causes the increase of bacterial translocation probability, the material of regulating TJ comprises immunomodifier and TJ albumen etc.Wherein stride embrane-associated protein occludin and claudin is the special conformity membrane albumen of topmost TJ for two kinds.Under intestinal microflora race conditio, intestines source property pathogenic bacteria can be sticked with enteric epithelium, influences by the membrane bone frame system that changes cell and strides embrane-associated protein.The detection of striding embrane-associated protein occludin level of enteric epithelium can reflect that not only pathogenic bacterium also can be reflected the recovery situation of intestinal microflora to the destruction situation of gut barrier under the intestinal microflora race conditio.
Can find obviously that from Fig. 2～5 surface epithelial cell of probiotic group small intestine and large intestine, iuntercellular and kytoplasm all have more occludin protein expression, and the occludin protein expression of PN group is very low.Compare with the PN treated animal, the positive area of the occludin protein expression of probiotic group small intestine, large intestine accounts for the interior per-cent of measurement window and all has significant difference (table 7).This shows that than the PN group, lactobacterium plantarum strain LP-Onlly can significantly improve enteric epithelium and stride embrane-associated protein content, thereby strengthens the intestinal mucosal barrier function.
The small intestine of table 7PN group and probiotic group, the comparison of intestinal epithelial cells occludin protein content greatly (A, x ± s)
| Group | n | Intestinal epithelial cell occludin protein content (%) | Big intestinal epithelial cells occludin protein content (%) |
| PN organizes probiotic group | 7 8 | 1.207±0.587 1.981±0.513 * | 1.254±0.203 2.276±0.526 ** |
Compare with the PN group,
*Be P＜0.05,
*Be P＜0.01.
(2) electron microscopic examination
Can find out that from Fig. 6～9 the probiotic group small intestine epithelium has slight microvillus to take off mistake, plastosome endoplasmic reticulum vacuolar degeneration, but closely connect more obvious; Closely connect normal between the large intestine intestinal epithelial cells.And PN group small intestine epithelium microvillus takes off mistake, and plastosome endoplasmic reticulum vacuolar degeneration closely connects unclear; Closely connect between the large intestine intestinal epithelial cells, but wide and lack.This shows that lactobacterium plantarum strain LP-Onlly can promote enteric epithelium closely to connect significantly, thereby strengthens the intestinal mucosal barrier function.
(3) Vena cava blood, mesentery Lymphoid tissue, liver and lung tissue bacterial translocation
PN group rat mesentery Vena cava blood, mesentery Lymphoid tissue, liver and lung tissue bacterial translocation rate are 60.7%, and probiotic group bacterial translocation rate only is 34.3%, significantly reduces (table 8) than the PN group.This shows that lactobacterium plantarum strain LP-Onlly can obviously reduce the bacterial translocation rate, thereby strengthens the intestinal mucosal barrier function.
Table 8 Vena cava blood and abdominal viscera bacterial translocation number
| Group | n | Vena cava blood and abdominal viscera bacterial translocation number | Easy bit rate (%) | |||
| Vena cava blood | The mesentery Lymphoid tissue | Liver | Lung tissue | |||
| PN organizes probiotic group | 7 8 | 4 2 | 6 4 | 4 3 | 3 2 | 60.7 34.3 |
5. conclusion
The above results shows that lactobacterium plantarum strain LP-Onlly of the present invention can increase abdominal cavity infection rat intestine epithelium and stride embrane-associated protein content, keeps enteric epithelium and closely connects the normal intestinal mucosa form of maintenance, reduces bacterial translocation, thereby strengthens the intestinal mucosal barrier function.Above-mentioned lactobacterium plantarum strain LP-Onlly and conventional carrier combinations can prevent and/or treat intestinal bacterial translocation and infect.
Claims (7)
1, a kind of lactobacterium plantarum strain LP-Onlly, its preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC NO.1258.
2, lactobacterium plantarum strain LP-Onlly according to claim 1, its 16S rRNA gene order is AY590777.
3, lactobacterium plantarum strain LP-Onlly according to claim 1 and 2, it has the intestinal microflora of adjusting function.
4, lactobacterium plantarum strain LP-Onlly according to claim 1 and 2, it has the intestinal mucosal barrier of enhancing function.
5, lactobacterium plantarum strain LP-Onlly according to claim 1 and 2, it has the function that suppresses pathogen enterobacteria and food spoilage microorganism.
6, lactobacterium plantarum strain LP-Onlly according to claim 5, described pathogen enterobacteria is enterobacteria, listeria bacteria, Salmonellas, streptococcus aureus, faecalis, dysentery bacterium, clostridium perfringens.
7, lactobacterium plantarum strain LP-Onlly according to claim 5, described food spoilage microorganism is subtilis, bacillus cereus.
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| CN102106463B (en) * | 2011-01-31 | 2012-12-05 | 杨永浩 | Microbial feed additives and preparation method thereof |
| CN103805547B (en) * | 2013-03-07 | 2016-02-17 | 练冬娣 | A kind of plant lactobacillus LP-12 and the application in the efficient animal intestinal conditioning agent of preparation thereof |
| CN104531587B (en) * | 2014-12-30 | 2017-12-01 | 上海交大昂立股份有限公司 | One lactobacillus plantarum and its application in active fermentation breast |
| CN105112334B (en) * | 2015-04-27 | 2018-08-17 | 南昌大学 | Probiotics containing lactobacillus plantarum |
| CN105255752B (en) * | 2015-04-27 | 2018-08-17 | 南昌大学 | High acid plant lactobacillus ZDY2013 |
| CN104974963A (en) * | 2015-07-09 | 2015-10-14 | 香港益盟生物科技有限公司 | Lactic acid bacteria composition for protecting gastrointestinal mucosa and preparation method thereof |
| CN105861399B (en) * | 2016-06-17 | 2019-11-05 | 浙江大学 | Lactobacillus plantarum and its application for preventing chicken necrotizing enterocolitis |
| CN107099492B (en) * | 2017-07-05 | 2019-12-17 | 重庆第二师范学院 | Lactobacillus plantarum YS4 and its application in preparing food for preventing constipation |
| CN109182220A (en) * | 2018-10-22 | 2019-01-11 | 东北农业大学 | One plant of inhibition C.perfringens proliferation growing plants lactobacillus and its application |
| CN110616173B (en) * | 2019-10-09 | 2021-04-06 | 吉林农业大学 | A kind of Lactobacillus plantarum for effectively lowering cholesterol level in vivo and its application |
| CN112812158B (en) * | 2021-02-02 | 2022-07-26 | 上海珑欣生物医学科技有限公司 | Polypeptide fragment C and application thereof |
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| US6132710A (en) * | 1997-03-17 | 2000-10-17 | Probiotix, Inc. | Preventing/treating neonatal NEC by administering lactobacillus salivarius and lactobacillus plantarum or a combination thereof |
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| CN1190346A (en) * | 1995-05-09 | 1998-08-12 | 普罗比公司 | A pharmaceutical composition comprising Lactobacillus plantarum and arginine |
| US6132710A (en) * | 1997-03-17 | 2000-10-17 | Probiotix, Inc. | Preventing/treating neonatal NEC by administering lactobacillus salivarius and lactobacillus plantarum or a combination thereof |
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