CN105567586A - Lactobacillus plantarum having anti-diabetic function and application thereof - Google Patents
Lactobacillus plantarum having anti-diabetic function and application thereof Download PDFInfo
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- CN105567586A CN105567586A CN201510982300.9A CN201510982300A CN105567586A CN 105567586 A CN105567586 A CN 105567586A CN 201510982300 A CN201510982300 A CN 201510982300A CN 105567586 A CN105567586 A CN 105567586A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Lactobacillus plantarum having an anti-diabetic function and application thereof are disclosed, the strain is Lactobacillus plantarum NCU116, and the accession number is CGMCC 11885. The strain has a function to alleviate symptoms of diabetes, and the composition is in a form of food or a pharmaceutical composition. The present invention also discloses a method for screening and identification of the lactic acid bacteria strain as well as a method to achieve the anti-diabetic effect by regulating blood sugar, blood lipids and hormone levels and regulating metabolic pathways in the body. The daily-intake lactic acid bacteria or a composition thereof is used to replace a medicine to achieve the anti-diabetic effect; and the healthy daily-intake lactic acid bacteria or the composition thereof is free of toxic and side effects to human body is used as a new choice to alleviate the symptoms of diabetes.
Description
Technical field
The invention belongs to biological technical field, relate to plant lactobacillus and application thereof.
Background technology
Probiotic bacterium refers to that a class produces the living microorganism of beneficial effect to host, by being colonizated in host intestine, reproductive system, definite health efficacy can be produced to host, thus improve microecological balance in host, play the general name of living microorganism of beneficial effect.Milk-acid bacteria as the representational Pseudomonas of most in probiotic bacterium, physiologically actives such as there is helping digest, promote the absorption of nutritive substance, alleviate lactose intolerance, prevention and therapy diarrhoea, regulating intestinal canal flora and relief of constipation, conditioner body immunity function, reduce the total cholesterol of blood, improve anaphylactic disease that food causes, delay senility and be antitumor.
Type ii diabetes is that a class lacks the metabolism disorder disease causing hyperglycemic symptoms relatively with insulin resistant or Regular Insulin.Chronic hyperglycemic symptoms may cause complication at multiple organ (as eyes, kidney, nerve, heart and blood vessel), thus increases the mortality ratio of diabetic subject.By gene and environment acting in conjunction, many factors (as age, diet, shortage motion and obesity) can induce generation type ii diabetes.In the past few decades, diabetic subject's number worldwide increases fast.Current, developed the medicine of multiple treatment diabetes, such as biguanides, thiazolidinediones and α glucoside enzyme, these medicines can control glucose level.But their side effect (flatulence, abdominal discomfort and diarrhoea etc.) also can not be ignored.In recent years, researchist recognizes gradually, and intestinal microflora may as a kind of important means of intervention diabetes of novelty.As the important component part of intestinal microflora, probiotic bacterium can improve body health level when taking in doses.In addition, recent studies have found that, probiotic bacterium can not only promote that intestinal microflora balances, and may have function of blood sugar reduction.
At present, what research was both at home and abroad many has the bacterial strains such as bifidus bacillus, lactobacterium casei, Lactobacterium acidophilum, lactobacillus rhamnosus, lactobacterium helveticus.But because the prebiotic activity of milk-acid bacteria has strain specificity, the prebiotic active difference between different strains is larger.Therefore, the bacterial strain, particularly China that need screening more targetedly to have special physiological activity have a large amount of traditional fermented food, wherein have the urgently research and development of a large amount of milk-acid bacterias.
Summary of the invention
The object of this invention is to provide the plant lactobacillus that a strain has anti-diabetic function, bacterial strain of the present invention is plant lactobacillus (Lactobacillusplantarum) NCU116, to solve the DOMESTICATION PROBLEM of the bacterial strain that lactic acid bacteria product uses in Chinese market.
Plant lactobacillus provided by the invention is from the old liquid of Chinese conventional Kimchi, be separated the plant lactobacillus NCU116 obtained, and this bacterial strain has anti-diabetic function.This bacterial strain is separated and preservation with technology National Key Laboratory by University Of Nanchang's Foodstuffs Academy Food science, numbering NCU116; This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 16th, 2015, address, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, culture presevation is numbered: CGMCCNo.11885, and suggestion Classification And Nomenclature is plant lactobacillus (Lactobacillusplantarum).
Invention also provides a kind of composition, said composition is the physiologically acceptable vehicle or the thinner that comprise described plant lactobacillus NCU116 bacterial strain, can be food or medicine.
Plant lactobacillus NCU116 of the present invention is the derivative of viable bacteria or inactivated bacteria or this bacterial strain.
Food of the present invention can be fermentation fruits and vegetables, fermented-milk, cheese, milk-contained drink, milk powder or other contain any one of the food of this bacterial strain or this strain derivative.
Medicine of the present invention is any one of capsule, pulvis, tablet or other pharmaceutically acceptable vehicle or thinner.
Composition provided by the present invention may be used for diabetes-alleviating symptom, is oral form.Specifically comprise:
For regulating body blood sugar, blood fat, hormonal readiness, raise short chain fatty acid levels, repair oxidativestress damage, pancreas and Pathological to damage, regulate the genetic expression of low density lipoprotein receptor, cholesterol 7α-hydroxylase, GLUT4, Peroxisome proliferator-activated receptorγ α and γ;
Or, play antidiabetic effect for regulating the pathways metabolisms such as glucose in body, lipid acid, cholic acid and amino acid.
In addition, present invention also offers and utilize the food of plant lactobacillus NCU116 bacterial strain for the preparation of diabetes-alleviating symptom or the method for medicine.
The purposes of food of the present invention or medicine broad sense can comprise a kind of method of diabetes-alleviating symptom, and described method comprises the composition giving Mammals and can reach this bacterial strain of diabetes-alleviating effect dosage.
The in vitro tests confirmation provided is being provided, plant lactobacillus NCU116 growth performance is excellent, and possess good acid resistance and bile tolerance characteristic, survival rate is higher in the gastrointestinal tract, and possess stronger enteron aisle Adhesion property, effectively can suppress the breeding of harmful bacteria in gi tract.
Those skilled in the art of the invention can utilize the present invention to the widest degree according to the above description.Therefore, below illustrate and only exemplarily illustrate, not limit remaining disclosure by any way.If of the present invention and clear and definite integer when there is known coordinator in the correlation technique relevant with the present invention, these known coordinators will be considered as independent item and be incorporated herein.
The invention provides the plant lactobacillus NCU116 that a strain has anti-diabetic function, this bacterial strain or its composition can play antidiabetic effect by pathways metabolisms such as glucose, lipid acid, cholic acid and amino acid in adjustment body.In addition, plant lactobacillus NCU116 also has good gastrointestinal toleration energy and Adhesion property, effectively can suppress the propagation of the pathogenic bacteria such as intestinal bacteria in enteron aisle.A target of the present invention carrys out alternative medicine with daily absorption milk-acid bacteria or its composition exactly, reaches antidiabetic effect; Using to the new selection as diabetes-alleviating symptom of human non-toxic, side effect and wholesome milk-acid bacteria or its composition.Bacterial strain of the present invention can be the composition of food or drug form, and large fruits and vegetables that can ferment produce acid fresh good to eat, nutritious, natural fruit and vegetables taste is strong and have the probiotic bacterium high-efficiency fermenting fruit and vegetable food (pickles, beverage, slurry) of good nourishing function, without the need to adding the foodstuff additive such as any essence and flavoring agent, pigment, acidic flavoring agent, stablizer, sanitas in product, all local flavors are produced by probiotics fermention fruit and vegetable materials.
Accompanying drawing explanation
Figure 1 shows that isolated strains plant lactobacillus NCU116 of the present invention and fermentation Hu trailing plants juice thereof are on the impact of blood glucose in diabetic rats, FBG: fasting blood sugar; NDM: normal group, gavage physiological saline; DM: diabetic model group, gavage physiological saline; NCU: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; FCJ: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); NFCJ:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.Result represents (n=10) with mean number ± standard deviation, has significant difference (P<0.05) between different Superscript letters expression group.
After Figure 2 shows that Wistar rat fills with hello isolated strains plant lactobacillus NCU116 of the present invention and fermentation Hu trailing plants juice thereof, the changing conditions of faecal short chain fatty acid level, ordinate zou is short-chain fat acid concentration (concentration); In X-coordinate, Aceticacid represents acetic acid, and Propionicacid represents propionic acid, and Butyricacid represents butyric acid, and TotalSCFA represents total short chain fatty acid; NDM: normal group, gavage physiological saline; DM: diabetic model group, gavage physiological saline; NCU: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; FCJ: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); NFCJ:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.Result represents (n=10) with mean number ± standard deviation, has significant difference (P<0.05) between different Superscript letters expression group.
After Figure 3 shows that Wistar rat fills with hello isolated strains plant lactobacillus NCU116 of the present invention and fermentation Hu trailing plants juice thereof, pancreas (A) and kidney (B) changes in histopathology situation (100X), NDM: normal group, gavage physiological saline; DM: diabetic model group, gavage physiological saline; NCU: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; FCJ: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); NFCJ:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.
Trailing plants juice is on diabetes rat lipid metabolism and glycometabolic impact recklessly to Figure 4 shows that isolated strains plant lactobacillus NCU116 of the present invention and fermentation thereof, and ordinate zou is related gene expression level (RelativemRNAlevel); In X-coordinate, LDLreceptor represents low density lipoprotein cholesterol acceptor, CYP7A1 represents cholesterol 7α-hydroxylase, GLUT4 represents GLUT4, PPAR-α represents Peroxisome proliferator-activated receptorγ-α, and PPAR-γ represents Peroxisome proliferator-activated receptorγ-γ; NDM: normal group, gavage physiological saline; DM: diabetic model group, gavage physiological saline; NCU: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; FCJ: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); NFCJ:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.Result represents (n=10) with mean number ± standard deviation, has significant difference (P<0.05) between different Superscript letters expression group.
Figure 5 shows that isolated strains plant lactobacillus NCU116 of the present invention and fermentation Hu trailing plants juice thereof feed the possible mechanism of action setting up type ii diabetes Wistar rat model in conjunction with low dose of streptozotocin induction to high-sugar-fat-diet.TC in figure: total cholesterol; TG: triglyceride level; LDL-C: low density lipoprotein cholesterol; LDLreceptor: low density lipoprotein cholesterol acceptor; CYP7A1: cholesterol 7α-hydroxylase; GLUT4: GLUT4; GLP-1: glucagon-like peptide 1; PYY: Peptide YY; SCFA: short chain fatty acid; Oxidativestress: oxidative stress; Glucose: glucose; Isletcells: islet cells; Insulinsensitivity: insulin sensitivity; Gutmicrobiota: intestinal microflora; Intestinaltract: enteron aisle; L.plantarumNCU116: plant lactobacillus NCU116; Fermentedcarrotjuice: ferment carrot juice.
Figure 6 shows that type II diabetes Wistar rat blood serum sample total ion current figure, (A) positive ion mode, (B) negative ion mode, ordinate zou is ionic strength; X-coordinate is the time.
After Figure 7 shows that Wistar rat fills with hello isolated strains plant lactobacillus NCU116 of the present invention and fermentation Hu trailing plants juice thereof, each group 3DPCA shot chart: (A) positive ion mode, (B) negative ion mode, a: normal group, gavage physiological saline; B: diabetic model group, gavage physiological saline; C: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; D: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); E:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.n＝10。
After Figure 8 shows that Wistar rat fills with hello isolated strains plant lactobacillus NCU116 of the present invention and fermentation Hu trailing plants juice thereof, often organize 3DPLS-DA shot chart: (A) positive ion mode, (B) negative ion mode, a: normal group, gavage physiological saline; B: diabetic model group, gavage physiological saline; C: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; D: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); E:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.n＝10。
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in more detail.
Embodiment 1: the screening of bacterial strain and preservation.
Plant lactobacillus NCU116 of the present invention is that separation screening obtains in the old liquid of Chinese tradition pickles, and concrete isolation identification step is as follows:
(1) separation and ientification of bacterial strain.
The old liquid of Chinese tradition pickles that source of fetching is different, the inoculum size by 3% is inoculated in vegetables juice enrichment medium, 37 DEG C of Anaerobic culturel 24h.Pipette vegetables juice enrichment culture liquid 1mL respectively, adopt 10 times of gradient dilution methods, select acceptable diluent degree, be coated on primary dcreening operation plate culture medium, 37 DEG C of Anaerobic culturel 48h, compare colonial morphology in culture dish, the bacterial strain that growth selection is fast, bacterium colony is large, yellow bacterium circle is large.Primary dcreening operation being selected the bacterial strain obtained is transferred on multiple sieve plate culture medium, 37 DEG C of Anaerobic culturel.Observe 12h, 24h, 36h, 48h rear plate respectively with or without calcium circle, calcium circle size and variable color speed.To select on flat board calcium circle more greatly, comparatively obviously and flavescence look bacterial strain faster, be transferred to that pig cholate content is 1.0%, pH value is in the multiple sieve liquid nutrient medium of 2.0 respectively, cultivate 20h for 37 DEG C.Get respectively and above-mentionedly sieve liquid nutrient medium again, adopt dull and stereotyped gradient dilution method, be coated on multiple sieve plate culture medium and repeat screening, 37 DEG C of Anaerobic culturel 48h, select yellow, that bacterium circle is larger single bacterium colony in culture dish, being transferred to sugar degree is respectively in the multiple sieve test-tube culture medium of 10%, then from multiple sieve test-tube culture medium, pick out that variable color is fast, the good bacterial strain of growing way to the preservation of MRS solid slope numbering, the bacterial classification obtained has the characteristic of resistance to high sugar, salt tolerant, resistance to peracid and bile tolerance.
Leavening property measures: sieve above-mentioned the strain excellent and contrast lactobacillus inoculation CICC6092 that obtain again, be seeded in MRS liquid nutrient medium respectively, cultivate 12h for 37 DEG C, then being transferred to leavening property by 2.0% (v/v) inoculum size measures in substratum, cultivate 24h, sample 1 time every 4h for 37 DEG C, measure viable count, acidity and the pH value in leavening property substratum.By comparing the change of viable count, acidity and pH value, show that the leavening property of plant lactobacillus NCU116 is best.
(2) preliminary evaluation of bacterial strain.
Bacterial classification Morphological Identification: cultivate coating after actication of culture on MRS solid plate, picking list bacterium colony, film-making microscopy, observes colonial morphology; The bacterial strain of institute's screening and separating is carried out gramstaining and examines under a microscope the cellular form of thalline; By strain inoculation to be identified on test tube slant, after 37 DEG C of cultivation 7d, carry out spore staining, oily sem observation.
Colonial morphology on table 1 plate culture medium and microscopy result
| Strain number | Colonial morphology | Microscopy result |
| NCU116 | Circle, smooth, projection is milky white, opaque | G+ is carefully short, shaft-like, and two ends are round and smooth |
Physio-biochemical characteristics are identified: carry out biochemical identification with milk-acid bacteria trace fermentation tube to bacterial strain, according to morphological specificity and the physiological and biochemical property of bacterial strain NCU116, and about the standard of perfection of milk-acid bacteria is analyzed it in reference uncle Jie Shi Bacteria Identification handbook, this strain bacterium is initially identified as plant lactobacillus (Lactobacillusplantarum).
Table 2 bacterial strain NCU116 biochemical identification result
(3) molecular biology identification of bacterial strain.
The strain excellent screened is carried out the order-checking of 16SrDNA complete sequence, by the 16SrDNA complete sequence input GenBank recorded, application BLAST software carries out homology search, choose the sequence that source is different, and choose the 16SrDNA sequence of some conventional bacteriums, by these sequences after ClustalX software carries out Multiple Sequence Alignment, software MEGA4.1 is utilized to draw evolutionary tree, and do pheS (phenylalanine tRNA synthetase alpha subunit) gene order Phylogenetic Analysis, draw evolutionary tree, and homology analysis is carried out to test strains.
1) sequencing result of 16SrDNA.
Bacterial strain NCU116 is after order-checking, and obtain NCU11616SrDNA gene fragment 1321bp, gene order has submitted GenBank (the gene order number of logging in: pheS:JQ278710 and 16SrRNA:JQ278711) to.
2) the 16SrDNA sequential system developmental analysis of bacterial strain NCU116.
By 14 relevant to milk-acid bacteria for the 16SrDNA sequence of surveyed bacterial strain NCU116 type strain 16SrDNA sequences together, adopt MEGA4.1 software, constructing system evolutionary tree, ortho position connection method display NCU116 and the 16SrDNA phylogenetic tree of relevant kind, carry out the similarity double counting of 1000 times.
16SrDNA sequential system developmental analysis shows: test strain NCU116 and L.plantarumsubsp.plantarum, L.plantarumsubsp.argentoratensis, L.pentosus, L.paraplantarum homology are all greater than 99.6%, with other associative mode bacterial strain homologys lower than 97.0%, determine that this bacterial strain is plant lactobacillus group (L.plantarumgroup).
3) the pheS gene order Phylogenetic Analysis of bacterial strain NCU116.
Adopt MEGA4.1 software, ortho position connection method display bacterial strain NCU116 and the pheS gene order phylogenetic tree of relevant kind, carry out the similarity double counting of 1000 times.
PheS gene sequencing shows: NCU116 and type strain L.plantarumsubsp.plantarumLMG6907T homology are 100%, is all less than 91% with other 3 kinds of pattern bacterium homologys of plant lactobacillus group (L.plantarumgroup).Determine that this bacterial strain is plant lactobacillus plant subspecies (L.plantarumsubsp.Plantarum).
Embodiment 2: the gastrointestinal tolerability test of plant lactobacillus NCU116.
By simulation human body alimentary canal environment, inquire into plant lactobacillus NCU116 survival ability in the gastrointestinal tract, thus provide reference for can this strains of lactic acid bacteria play health-promoting effect in human body alimentary canal environment.
(1) artificial gastric juice resistance's property.
Get the freeze-dried vaccine powder of plant lactobacillus NCU116, it is in the simulated gastric fluid of 1.5,2.5,3.5 and 4.5 that inoculum size by 1% is seeded to pH value respectively, mixing, the rotating speed shaking table pressing 100r/min under 37 DEG C of conditions is cultivated, sample respectively at after 0,1,2 and 3h, carry out viable lactic acid bacteria counting, and measure its transmittance at 600nm wavelength place.
(2) simulated intestinal fluid tolerance.
Get the freeze-dried vaccine powder of plant lactobacillus NCU116, the inoculum size by 1% is seeded in simulated intestinal fluid, mixing, the rotating speed shaking table pressing 100r/min under 37 DEG C of conditions is cultivated, sample respectively at after 0,1,2,3 and 4h, carry out viable lactic acid bacteria counting, and measure its transmittance at 600nm wavelength place.
(3) bile tolerance experiment.
Add bovine bile in MRS liquid nutrient medium, and make the mass concentration of bovine bile be respectively 0,0.03,0.10,0.20,0.30,0.50,0.75g/100mL and 1.00g/100mL, 121 DEG C of sterilizing 30min, cool for subsequent use, by the seed liquor of plant lactobacillus NCU116 by 5% inoculum size be seeded in above-mentioned cooled substratum, be placed in 37 DEG C of constant temperature culture 24h, then carry out viable lactic acid bacteria counting.
(4) resistance to high salt experiment.
Add NaCl solid in MRS liquid nutrient medium, and make the mass concentration of NaCl be respectively 0,1,2,3,4,5,6,7g/100mL and 8g/100mL, 121 DEG C of sterilizing 30min, cool for subsequent use, by the seed liquor of plant lactobacillus NCU116 by 5% inoculum size be seeded in above-mentioned cooled substratum, be placed in 37 DEG C of constant temperature culture 24h, then carry out viable lactic acid bacteria counting.
Result and conclusion: (1) plant lactobacillus NCU116 has stronger tolerance in simulated gastric fluid, be in the simulated gastric fluid of 1.5 and 2.5 in pH value, after effect 3h, thalline survival rate reaches 32.62% and 45.76% respectively, be in the simulated gastric fluid of 3.5 and 4.5 in pH value, viable count variation tendency is mild, and thalline survival rate all keeps very high, and the transmittance change of the simulated gastric fluid of each pH value at 600nm wavelength place also all tends to be steady, and slightly increases.(2) tolerance of plant lactobacillus NCU116 in simulated intestinal fluid is comparatively strong, and after effect 4h, residual viable bacteria concentration is still 10
8on the order of magnitude, thalline survival rate is still up to 49.63%, and the transmittance change at 600nm wavelength place also tends to be steady, and slightly declines.(3) plant lactobacillus NCU116 has stronger tolerance to cholate, after cultivating 24h respectively, its viable bacteria concentration progressively reduces, thalline well-grown in the substratum of 0.30g/100mL bovine bile with the mass concentration increase of bovine bile in substratum, and its viable bacteria concentration is still 10
8on the CFU/mL order of magnitude, even if when bovine bile concentration is up to 1.00g/100mL, thalline also can grow, and viable bacteria concentration is still 10
7more than CFU/mL.(4) plant lactobacillus NCU116 ferments after 24h in the substratum of different N aCl concentration, its viable bacteria concentration slowly reduces with the mass concentration increase of NaCl, thalline still can grow under NaCl mass concentration is the hypersaline environment of 8g/100mL, and its viable bacteria concentration still reaches 10
8more than CFU/mL, therefore it has stronger resistance to osmotic pressure ability.Comprehensively show that plant lactobacillus NCU116 has good tolerance to simulated gastric fluid, simulated intestinal fluid, cholate and high salt, good survival rate can be kept in human body alimentary canal, there is good gastrointestinal tolerability.
Embodiment 3: the Adhesion property test of plant lactobacillus NCU116.
Simulation human intestinal environment, adopts In vitro cell model human colon cancer cell system HT-29 to investigate the adhesion of plant lactobacillus NCU116, and studies Different factor sticks HT-29 impact on NCU116.Human intestinal environment partial neutral, therefore the sterile PBS buffer simulation intestinal environment selecting pH7.30, and to adjust cell concentration be 1.0 × 10
8cFU/mL, hatches 2.0h altogether with HT-29 cell in 37 DEG C of anaerobic culture boxes, and the bacterial count sticked is 4.78 ± 0.21CFU/cell (adhering to bacterium number/HT-29), and the Adhering capacity of plant lactobacillus NCU116 is stronger.
(1) adjust cell concentration with sterile PBS buffer (pH7.30) and be respectively 1 × 10
5, 1 × 10
6, 1 × 10
7, 1 × 10
8, 1 × 10
9cFU/mL, hatches 2.0h altogether with cell, calculates the bacterium number sticked, studies the impact of different cell concentration on adhesion, and result shows: along with the raising of cell concentration, the bacterial count sticked increases; When cell concentration reaches 1 × 10
8during CFU/mL, the bacterial count average out to 4.78CFU/cell sticked, when bacteria concentration increases again, the growth of sticking bacterium number tends towards stability, and sticks and is tending towards saturated; Deducibility, when thalline reaches finite concentration, the most of specific receptors of adhesin in cell of phage surface is combined, and when cell concentration improves further, the bacterial count sticked is substantially constant.
(2) growth phase is studied on the impact of adhesion, select retardation latter stage (8h), logarithmic phase (12h) respectively, stablize the initial stage (16h), stablize latter stage (20h), the NCU116 thalline of decline phase (24h) carries out sticking experiment, experimental result shows: when incubation time is less than 16h, the bacterial count sticked significantly increases along with the increase of cell age; Stick bacterium number average out to 4.78CFU/cell when cultivating 16h, reach maximum; During stationary phase, the bacterial count sticked slowly declines; During decline phase, the bacterial count sticked significantly declines; Illustrate that the Adhesion property of plant lactobacillus NCU116 when stationary phase is better, supposition may be because somatic cells during stationary phase starts to accumulate reserve substance, and the synthesis such as adhesin and related substances increases, and stationary phase thalline surface tissue comparatively stable caused by.
(3) study action time to the impact of adhesion, it is 1.0 × 10 that the plant lactobacillus NCU116 sterile PBS buffer (pH7.30) cultivating 16h is adjusted to cell concentration
8cFU/mL, more respectively with co-culture of cells 0.5,1.0,1.5,2.0, carry out sticking experiment after 2.5h, experimental result shows: when being less than 2.0h action time, sticking bacterium number significantly increases along with the prolongation of action time; Action time, when being 2.0h, sticking bacterium number mean value was 4.78CFU/cell, and when being greater than 2.0h action time, sticking the increase of bacterium number and tending to be steady.The adhesion of milk-acid bacteria is the combined effect of adsorption and desorption, sticks process lifetime effect, just can reach running balance after needing effect certain hour.
(4) research environment pH value is on the impact of adhesion, by the plant lactobacillus NCU116 cultivating 16h respectively with pH3.0,4.0,5.0,6.0,7.0,8.0, to be adjusted to cell concentration be 1.0 × 10 for the damping fluid of 9.0
8cFU/mL, then carry out sticking experiment, result shows, and when pH of cushioning fluid is less than 5.0, sticking bacterium number increases along with the rising of pH value; During pH5.0, stick bacterium number and reach maximum, on average can reach 6.11CFU/cell; And when pH value is greater than 6.0, sticks bacterium number and reduce along with the rising of pH value on the contrary.Foregoing study results display plant lactobacillus NCU116 acid-fast ability is better, therefore deducibility, its adhesion is in acid condition than good in the basic conditions, and this may also combine more relevant with the specific receptors in the adhesin on acidic conditions hypothallus surface and cell.
(5) study salt ionic concentration to the impact of adhesion, it is 1.0 × 10 that the plant lactobacillus NCU116 cultivating 16h is adjusted its concentration by the salt ion solution of different concns respectively
8cFU/mL, then carry out sticking experiment, result shows, and with the increase of CaCl2 concentration, have and slightly increase, but trend is not obvious; And along with the increase of MgCl2 concentration, the bacterial count that each HT-29 cell sticks is almost unchanged.This demonstrate, Ca2+ and Mg2+ on the adhesion of plant lactobacillus NCU116 and HT-29 cell without impact.Comprehensively show that plant lactobacillus NCU116 has good adhesion in host.
Embodiment 4: the bacteriostasis property test of plant lactobacillus NCU116.
Using 9 kinds of encountered pathogenic bacterias and nonpathogenic bacteria as indicator, extracorporeal bacteria inhibitor test is carried out to plant lactobacillus NCU116 fermented liquid, thalline and meta-bolites.
(1) first the bacteriostatic activity of plant lactobacillus NCU116 is measured, result shows it to lactobacillus strain unrestraint effect, but all have good fungistatic effect to other pathogenic bacterias and non-pathogenic strain, by the fungistatic effect of more known zymocyte liquid comparatively meta-bolites and thalline stronger.The zymocyte liquid of plant lactobacillus NCU116, meta-bolites and thalline all have fungistatic effect, and it is the strongest to the restraining effect of colon bacillus ATCC25922, the flourish of pathogenic bacteria in gi tract can be suppressed, wherein the antibacterial circle diameter of zymocyte liquid and meta-bolites is all greater than 19mm, and fungistatic effect is strong; The antibacterial circle diameter of thalline is slightly little, and 15 ± 0.67mm, fungistatic effect is better.Deducibility thus, the bacteriostatic action of plant lactobacillus NCU116 depends on its meta-bolites, but thalline also has good fungistatic effect, and it is the strongest to the restraining effect of colon bacillus ATCC25922, therefore follow-up test selects colon bacillus ATCC25922 to be indicator.
(2) measure the bacteriostatic activity of different concns sample, result shows, the fungistatic effect no significant difference of different cell concentration sample, and fungistatic effect is better, but wherein 10
9the zymocyte liquid bacteriostatic action of CFU/mL is the strongest, and its antibacterial circle diameter is greater than 19mm, and fungistatic effect is strong, analyzes the dual bacteriostatic effect that major cause is meta-bolites and thalline; Sample cell concentration has no significant effect its fungistatic effect.
(3) different organic acids bacteriostatic activity is measured, acetic acid, propionic acid and the lactic acid restraining effect to colon bacillus ATCC25922 is better, and fungistatic effect: acetic acid > propionic acid > lactic acid, the hydrochloric acid soln of pH4.0 does not produce bacteriostatic action containing organic acid, describes acetic acid in zymocyte liquid, propionic acid and lactic acid bacteriostatic action at low ph conditions.And acetic acid, propionic acid and lactic acid are not antibacterial substance unique in fermentation using bacteria product, these three kinds of organic acid biocidal properties all do not reach the antibacterial level of former zymocyte liquid, and this explanation may also exist other antibacterial substance, as bacteriocin class etc.
(4) organic acid in fermented liquid and hydrogen peroxide interference are got rid of, found that, after getting rid of organic acid interference, the fungistatic effect of fermented liquid weakens, illustrate that the antibacterial substance that plant lactobacillus NCU116 produces also has other active substances except organic acid, and the bacteriostatic activity of other active substances may be better than organic acid.After hydrogen peroxide ferment treatment, the bacteriostatic activity of fermented liquid, without considerable change, illustrate that in the antibacterial substance that NCU116 produces, possibility is without hydrogen peroxide, or the concentration of hydrogen peroxide contained by it is not enough to produce fungistatic effect, eliminates the interference of hydrogen peroxide.
(5) study the impact of different pH on sample bacteriostatic activity, result shows, and when fermented liquid pH is equal to or less than 4.0, antibacterial circle diameter is greater than 19mm, and fungistatic effect is strong; When pH is 5.0,6.0 and 7.0, fungistatic effect weakens.Illustrate that low pH is the prerequisite producing fungistatic effect, pH reduces, and fungistatic effect strengthens.
(6) study different heat treatment method to the impact of sample bacteriostatic activity, result shows, after four kinds of temperature (50 DEG C, 80 DEG C, 100 DEG C, 121 DEG C) condition processing samples, and bacteriostatic activity and blank no significant difference, and fungistatic effect is strong.Illustrate that the antibacterial substance that plant lactobacillus NCU116 produces is insensitive to heat, get rid of the possibility of macro-molecular protein as antibacterial substance.
(7) study different proteolytic enzyme pre-treatment to the impact of sample bacteriostatic activity, result shows, and after papoid, Proteinase K, trypsinase and stomach en-process respectively, fungistatic effect has and more obviously declines.Once studies have reported that the thermally-stabilised peptide of small molecules was a large bacterioid element, the scope of antimicrobial spectrum is widely different because of the difference of bacteriocin kind.This research show that antibacterial substance is to protease-sensitive, has eliminated heat sensitive macro-molecular protein in addition, therefore antibacterial substance may containing micromolecule polypeptide bacterioid element.
Based on the above results, illustrate that main antibacterial substance that NCU116 produces is various organic acid in thalline and meta-bolites thereof and micromolecule polypeptide bacterioid element etc.
Embodiment 5: plant lactobacillus NCU116 and the blood sugar of ferment carrot juice to diabetes rat improve Mechanism Study.
(1) diabetes model is set up and grouping.
After rat adapts to 1 week, normal group is fed with normal diet, and each diabetic groups is fed with high-sugar-fat-diet.The each group of free diet drinking-water of rat, monitor a body weight weekly, fasting 12h after 8 weeks, experimental group rat is by body weight tail vein injection 30mg/kgSTZ normal saline solution, and normal group injects Isodose physiological saline.Fasting 12h after 1 week, tail venous blood sampling, detects fasting plasma glucose, and with continuous 3 fasting blood sugar >=11.1mmol/L and with many foods, drink, diuresis symptom is type ii diabetes rat model more.
The diabetes rat be successfully established by model is divided at random: (B) diabetic model group (DM), gavage physiological saline; (C) NCU116 group (NCU), gavage plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; (D) ferment carrot juice group (FCJ), gavage is containing plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); (E) Radix Dauci Sativae juice group (NFCJ), the non-ferment carrot juice of gavage.Separately establish (A) normal group (NDM), gavage physiological saline.Often organize 10 animals.Press 10mL/kg successive administration every day 5 weeks.Animal freely ingests, and freely drinks water.
(2) glucose level.
As shown in Figure 1, compared with normal group (4.55mmol/L), diabetic model group (26.72mmol/L) fasting blood sugar significantly improves (P<0.05).Plant lactobacillus NCU116 group (19.37mmol/L) and fermentation Hu trailing plants juice group (17.19mmol/L) blood glucose value comparatively diabetic model group significantly reduce (P<0.05).Radix Dauci Sativae juice group (23.34mmol/L) blood glucose value also has and to a certain degree reduces.
(3) hormonal readiness.
Diabetic model group serum insulin, hyperglycemic-glycogenolytic factor and leptin level are significantly higher than normal group (P<0.05).After gavage plant lactobacillus NCU116 and ferment carrot juice, three kinds of hormones decrease, and wherein ferment carrot juice group hyperglycemic-glycogenolytic factor and leptin level are more close to normal group.In trailing plants juice group, These parameters and diabetic model group do not have significant difference (table 3) recklessly.The horizontal compared with normal group of diabetic model group GLP-1 and PYY significantly reduces, and after gavage plant lactobacillus NCU116 and ferment carrot juice, these two kinds of hormonal readinesses increase (P<0.05).
Table 3 plant lactobacillus NCU116 and fermentation Hu trailing plants juice are on the impact (n=10) of diabetes rat hormonal readiness
Note: GLP-1: glucagon-like peptide 1; PYY: Peptide YY; NDM: normal group, gavage physiological saline; DM: diabetic model group, gavage physiological saline; NCU: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; FCJ: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); NFCJ:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.Result represents (n=10) with mean number ± standard deviation, has significant difference (P<0.05) between different Superscript letters expression group.
(4) blood lipid level.
Table 4 shows, diabetic model group TC (8.08mmol/L), TG (9.86mmol/L) and LDL-C (2.86mmol/L) compared with normal group significantly improve, HDL-C level significantly reduces, plant lactobacillus NCU116 group and ferment carrot juice group TC, TG and LDL-C decreases, but the horizontal difference of each diabetic groups HDL-C not obvious (0.74-0.85mmol/L).
Table 4 plant lactobacillus NCU116 and fermentation Hu trailing plants juice are on the impact of diabetes rat blood lipid level
Note: TC: total cholesterol; TG: triglyceride level; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; NDM: normal group, gavage physiological saline; DM: diabetic model group, gavage physiological saline; NCU: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; FCJ: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); NFCJ:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.Result represents (n=10) with mean number ± standard deviation, has significant difference (P<0.05) between different Superscript letters expression group.
(5) oxidative stress.
Diabetic model group SOD (156.94U/mL), GSH-Px (289.28U/mL), CAT (98.65U/mL) vigor and T-AOC (6.67U/mL) level are significantly lower than normal group (P<0.05, table 5).This four indices in ferment carrot juice group (be respectively 200.35,361.72,150.32 and 13.35U/mL) increases.Plant lactobacillus NCU116 group (3.73nmol/mL) and ferment carrot juice group (3.70nmol/mL) MDA content are lower than diabetic model group (5.36nmol/mL) and Radix Dauci Sativae juice group (5.74nmol/mL).
Table 5 plant lactobacillus NCU116 and fermentation Hu trailing plants juice are on the impact of diabetes rat oxidative stress level
Note: SOD: superoxide-dismutase; GSH-Px: glutathione peroxidase; MDA: mda; T-AOC: total antioxidant capacity; CAT: catalase; NDM: normal group, gavage physiological saline; DM: diabetic model group, gavage physiological saline; NCU: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; FCJ: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); NFCJ:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.Result represents (n=10) with mean number ± standard deviation, has significant difference (P<0.05) between different Superscript letters expression group.
(6) renal function.
High lipid food is fed and is set up type ii diabetes rat model meeting increasing serum blood urea nitrogen, uric acid and creatinine level in conjunction with STZ.After gavage plant lactobacillus NCU116 and ferment carrot juice, blood urea nitrogen and creatinine level obviously reduce (P<0.05), uric acid level also decreases, but result does not have significant difference (P>0.05).
Table 6 plant lactobacillus NCU116 and fermentation Hu trailing plants juice are on the impact of diabetes rat renal function
Note: NDM: normal group, gavage physiological saline; DM: diabetic model group, gavage physiological saline; NCU: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; FCJ: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); NFCJ:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.Result represents (n=10) with mean number ± standard deviation, has significant difference (P<0.05) between different Superscript letters expression group.
(7) faecal short chain fatty acid content.
Diabetic model group contains acetic acid (120.54 μm of ol/g), propionic acid (49.15 μm of ol/g), butyric acid (16.44 μm of ol/g) compared with normal group low (P<0.05).Plant lactobacillus NCU116 group, ferment carrot juice group and Radix Dauci Sativae juice group short-chain fat acid content increase, wherein plant lactobacillus NCU116 group and ferment carrot juice group comparatively diabetic model group have significant difference (P<0.05).Plant lactobacillus NCU116 group and the total short chain fatty acid levels of ferment carrot juice group are close to normal group (see accompanying drawing 2).
(8) pancreas and Renal Paphology.
Normal group pancreatic tissue acinous cell is arranged in around islet cells regularly, pancreas islet form is normal; Diabetic model group beta Cell of islet significantly reduces, part pancreas islet degeneration atrophy; Plant lactobacillus NCU116 group and the damage of ferment carrot juice group islet cells alleviate, vacuolation reduces, and pancreas islet quantity increases (see accompanying drawing 3A).
Similarly, compare with normal group, the mesentery of Renal of Diabetic Rats be broadening, renal interstitial fibrosis, the sex change of glomerular epithelium cell cavity sample, the symptom such as glomerular sclerosis atrophy.Plant lactobacillus NCU116 group and ferment carrot juice group renal glomerulus interstitial fibrosis degree reduce, epithelial cell cavity symptom alleviates, renal glomerulus atrophy symptom is alleviated (see accompanying drawing 3B) to some extent.
(9) lipid metabolism and glucose metabolism genes are expressed.
Compare with normal group, diabetes rat hepatic LDL receptor and PPAR-α and skeletal muscle GLUT4 genetic expression reduce (P<0.05), gavage plant lactobacillus NCU116 and ferment carrot juice are after 5 weeks, and said gene expression level significantly improves; Meanwhile, two administration group CYP7A1 and PPAR-γ gene expression dose are significantly higher than diabetic model group (see accompanying drawing 4).
Plant lactobacillus NCU116 and fermentation recklessly trailing plants juice are shown in accompanying drawing 5 to the possible mechanism of action that high-sugar-fat-diet nursing sets up type ii diabetes rat model in conjunction with low dose of streptozotocin induction.
Embodiment 6: plant lactobacillus NCU116 and the blood serum metabolic group of ferment carrot juice to diabetes rat are probed into.
(1) diabetes model is set up and grouping.
After rat adapts to 1 week, Normal group is fed with normal diet, and experimental group is fed with high-sugar-fat-diet.The each group of free diet drinking-water of rat, monitor a body weight weekly, fasting 12h after 8 weeks, experimental group rat is by body weight tail vein injection 30mg/kgSTZ normal saline solution, and normal group injects Isodose physiological saline.Fasting 12h after 1 week, tail venous blood sampling, detects fasting plasma glucose, and with continuous 3 fasting blood sugar >=11.1mmol/L and with many foods, drink, diuresis symptom is type ii diabetes rat model more.
Mould diabetes rat is become to be divided at random by 40: (B) diabetic model group (DM), gavage physiological saline; (C) NCU116 group (NCU), gavage plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; (D) ferment carrot juice group (FCJ), gavage is containing plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); (E) Radix Dauci Sativae juice group (NFCJ), the non-ferment carrot juice of gavage.Separately establish (A) normal group, gavage physiological saline.Often organize 10 animals.Press 10mL/kg successive administration every day 5 weeks.Animal freely ingests, and freely drinks water.
(2) sample collection and pre-treatment.
After gavage terminates, rat through anesthesia, Culling heart blood, centrifugal serum.Getting 200 μ L serum adds in 800 μ L acetonitrile-deionized waters (4:1, v/v), and vortex mixes, centrifugal, after film crossed by supernatant liquor, is placed in sample injection bottle.
(3) serum protein moteblites atlas analysis.
UPLC-Q-TOF/MS is a kind of strong metabonomic analysis instrument.In this research, UPLC-Q-TOF/MS is utilized just to carry out (bearing) pattern ion scan to monitor the difference of each group of metabolite to serum sample.Accompanying drawing 6 represents the total ions chromatogram under positive ion scan pattern (A) and negative ion scan pattern (B), and display negative ions pattern all has good resolution and ion abundance.In addition, research adopts the reference ion of positive ion (m/z value is 121.0508 and 922.0097) and negative ion (m/z value is 112.39855 and 1034.9881) to guarantee the mass accuracy that negative ions scans and circulation ratio.
(4) metabolite multivariate analysis.
On the basis that UPLC-Q-TOF/MS analyzes, in order to embody the Difference of Metabolism of five groups of administration compositions better, first PCA analysis is carried out to experimental result.Accompanying drawing 7 is shown as the 3D shot chart that PCA analyzes positive ion (A) and negative ion (B), in figure, each point represents a sample, the composition of each sample small molecule metabolites and concentration determine this sample position in the drawings, the sample of composition and concentration comparable, the position on shot chart is also more close.Result shows, and compared with normal group, diabetic model group distributing position is comparatively far away, and point out diabetic model group rat sample compound component and concentration and normal group to have larger difference, in its body, Biochemical processes have had obvious change.Compared with diabetic model group, plant lactobacillus NCU116 and the close trend of the oriented normal group of ferment carrot juice group, illustrate that plant lactobacillus NCU116 and ferment carrot juice can regulate component and the content of diabetes rat blood small molecular compound.In addition, under positive and negative ion pattern, PCA eight kinds of principal constituents are respectively 68.7% and 71.38% through MPP software analysis.
On the basis that PLS-DA can be separated at PCA, the one expressing group difference more clearly supervises multivariable statistical method.PLS-DA analytical results shows, plant lactobacillus NCU116, difference more obvious (accompanying drawing 8) between ferment carrot juice and Radix Dauci Sativae juice group sample.Therefore, plant lactobacillus NCU116 and ferment carrot juice change the blood serum metabolic type of diabetes rat.Ferment carrot juice group sample, more near normal group, shows that ferment carrot juice group is to improve in diabetes serum metabolism performance the most obvious.
Cross validation is used for assessing the accuracy of PLS-DA Modling model.Empirical tests matrix model is analyzed known, and under positive ion scan pattern, accuracy rate is 98% (plant lactobacillus NCU116 group is 90%, and other group is 100%); Under negative ion scan pattern, accuracy rate is 100% (each group is 100%).This validation matrix model value when close to 100%, represent the success of this Mathematical Models, can predict the outcome quite exactly.In addition, these data also show each group and have good resolution; Represent that diabetes rat is after plant lactobacillus NCU116, ferment carrot juice and Radix Dauci Sativae juice group gavage, serum small molecule metabolite composition there occurs larger change.Gavage is after 5 weeks, and compare plant lactobacillus NCU116 and ferment carrot juice group with diabetic groups more near normal group, prompting plant lactobacillus NCU116 and ferment carrot juice have impact on the metabolism process of diabetes rat to a certain extent.This result is consistent with early-stage Study (the hypoglycemic and reducing blood lipid that plant lactobacillus NCU116 and ferment carrot juice have diabetes rat).
(5) potential source biomolecule marker is analyzed.
The qualification of systematic search analysis to potential source biomolecule marker of database is significant.Different biomarkers is analyzed by non-paired t test.Utilize screening differentiation compound flow process, by AgilentMETLINPersonalMetaboliteDatabase, marker ID is identified, No. CAS, matched indicia thing, and utilize IDBrowser recognition function to determine possible biomarker (see table 7).
Table 7 serum potential source biomolecule marker and its variation tendency
Note: NDM: normal group, gavage physiological saline; DM: diabetic model group, gavage physiological saline; NCU: plant lactobacillus NCU116 group, gavage NCU116 plant lactobacillus NCU116 (10
9cFU/mL) physiological saline suspension; FCJ: ferment carrot juice group, gavage plant lactobacillus NCU116 ferment carrot juice (bacteria containing amount 10
9cFU/mL); NFCJ:(does not ferment) Radix Dauci Sativae juice group, the non-ferment carrot juice of gavage.* represent the variation tendency compared with normal group, # represents the variation tendency compared with model group.↓ represent that metabolite content is lowered, ↑ represent that metabolite content raises.n＝10。
Found by MPP software analysis, can obviously change some metabolic processes through plant lactobacillus NCU116 and the intervention of ferment carrot juice.Utilize the secondary ion fragment of UPLC-Q-TOF/MS/MS to differentiate potential biomarker.The information being the MS/MS collection of illustrative plates fragmention that 10-40eV obtains by collision voltage exports as CEF formatted file, by MSC software and Chemspider database analysis, and the biomarker in final this research of qualification.Analyze and find, positive ion feature metabolic markers mainly contains adenosine (adenosine, 1.36_268.1043), 5-oxindole acetaldehyde (5-hydroxyindoleacetaldehyde, 10.81_176.0707), proline(Pro) (proline, 1.40_116.0705), glycocholic acid (glycocholicacid, 12.30_466.3160), Taurochenodeoxycholic Acid (taurochenodeoxycholicacid, 13.68_500.3041), sphingosine (sphingosine, 17.59_300.2904); Negative ion characteristic metabolic marker mainly contains theophylline (theophylline, 1.70_179.0569) and taurocholate (taurocholicacid, 12.79_514.2847).
Claims (7)
1. a strain has plant lactobacillus (Lactobacillusplantarum) NCU116 of anti-diabetic function, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCC11885.
2. a composition, said composition is physiologically acceptable vehicle containing plant lactobacillus NCU116 described in claim 1 or thinner.
3. composition according to claim 2, is characterized in that, described plant lactobacillus NCU116 is the derivative of viable bacteria or inactivated bacteria or this bacterial strain, and described physiologically acceptable vehicle or thinner are a kind of food or medicine.
4. composition according to claim 3, is characterized in that, described food is fermentation fruits and vegetables, fermented-milk, cheese, milk-contained drink, milk powder or other contain any one of the food of this bacterial strain or this strain derivative.
5. composition according to claim 3, is characterized in that, described medicine is any one of capsule, pulvis, tablet or other pharmaceutically acceptable vehicle or thinner.
6. the composition according to any one of claim 2 to 5, is characterized in that, described composition is used for diabetes-alleviating symptom, is oral form.
7. composition according to claim 6, it is characterized in that described composition, for regulating body blood sugar, blood fat, hormonal readiness, raise short chain fatty acid levels, repair oxidativestress damage, pancreas and Pathological to damage, regulate the genetic expression of low density lipoprotein receptor, cholesterol 7α-hydroxylase, GLUT4, Peroxisome proliferator-activated receptorγ α and γ; Or, play antidiabetic effect for regulating glucose in body, lipid acid, cholic acid and amino acid metabolism approach.
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| CN116463264B (en) * | 2023-04-21 | 2024-02-06 | 四川大学 | Lactobacillus plantarum with colon cancer cell growth inhibition effect and application thereof |
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