CN117535174A - Enterococcus faecalis and application thereof in improving autism spectrum disorder - Google Patents
Enterococcus faecalis and application thereof in improving autism spectrum disorder Download PDFInfo
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- CN117535174A CN117535174A CN202311270388.2A CN202311270388A CN117535174A CN 117535174 A CN117535174 A CN 117535174A CN 202311270388 A CN202311270388 A CN 202311270388A CN 117535174 A CN117535174 A CN 117535174A
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- 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
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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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- 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
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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Abstract
The invention discloses enterococcus faecalis (Enterococcus faecalis), which is named as F1GAM5 and has a preservation number of CCTCC NO: m20231057, which was obtained from the first isolation of clinical FMT donor bacterial fluid. The enterococcus faecalis F1GAM5 belongs to enterococcus with dominant abundance, can improve the anxiety depression-like performance of an ASD mouse by promoting the exploring behavior and the spontaneous activity behavior of an ADS mouse model, can be used for improving the research and the application of autism spectrum disorder, and has important potential value in developing novel therapies of ASD probiotics.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to enterococcus faecalis (Enterococcus faecalis) and application thereof in improving autism spectrum disorders, wherein the strain is separated from clinical faecal fungus transplantation (Fecal microbiota transplantation, FMT) donor bacterial liquid.
Background
Autism spectrum disorder (autism spectrum disorder, ASD) is a type of developmental disorder characterized by severe autism, lack of emotional response, dysplasia in speech, repeated movement of the notch plate, and the like. Statistical data from the U.S. disease control center 2018 shows that autism incidence is as high as 1/59. The autism gradually appears in China with a high incidence, and the incidence rate is as high as 1%. The research finds that the dysbacteriosis of intestinal canal has a correlation with the occurrence and development of autism spectrum disorder.
Studies have shown that most patients with Autism Spectrum Disorder (ASD) suffer from varying degrees of gastrointestinal dysfunction, the most common gastrointestinal disorders being constipation, abdominal pain, diarrhea, abdominal distension, and gastrointestinal inflammation. It was found that the gastrointestinal disease co-occurrence rate of ASD children was 9% -91% and that the more severe the gastrointestinal symptoms, the more severe the ASD severity. Intestinal flora composition and proportion imbalance is also common in ASD infants. Crowd research shows that the ratio of bacteroidetes to thick-walled bacteria is obviously reduced in ASD infants and brothers and sisters thereof, the content of lactic acid bacteria and vibrio desulphurisation is increased in ASD infants, and the severity of repeated action of the notch board is proportional to the content of vibrio desulphurisation.
Since the probiotic intervention can effectively regulate the unbalance of intestinal microbiota, alleviate gastrointestinal tract disease symptoms, and also can influence brain functions through microorganism-intestinal-brain axes, promote active autonomous behaviors of animal models and ASD patients, the probiotic is expected to assist in treating ASD. After research on the combined preparation of bifidobacteria, lactic acid bacteria and streptococcus for ASD children patients, the intestinal flora of ASD patients is found to be normal, the symptoms of gastrointestinal diseases are relieved, and the autonomous behavior and emotion regulation are obviously improved. ASD mice immunized with maternal factors were treated with bacteroides fragilis, altering their microbial composition and improving ASD-related behavior, and ASD mice supplemented with lactobacillus reuteri also exhibited improvements in gastrointestinal tract and behavior. The application of probiotics can effectively regulate the cognitive function of a host and can also improve the problems of anxiety, depression and emotional dysfunction of the host. Results from a series of animal and clinical studies have shown that probiotics have become one of the adjunctive treatments for infants suffering from ASD as a non-drug and relatively risk-free option. However, because factors such as different species, strains, dosage size and duration have an influence on the therapeutic effect of probiotics, more intensive and standardized research is still required to provide a continuous and clinical basis. Enterococcus faecalis (Enterococcus faecalis) is one of the dominant flora in the human and animal intestinal tracts, can improve the intestinal microenvironment and is beneficial to the health of organisms, but its application in ASD is still less studied.
Disclosure of Invention
In order to overcome at least one problem in the prior art, the invention provides an enterococcus faecalis isolate isolated from clinical FMT donor bacterial liquid, which has remarkable promotion effect on exploring behavior and spontaneous activity behavior of an ASD model mouse and can be used for researching and applying to improving autism spectrum disorder.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides enterococcus faecalis F1GAM5, which has a preservation number of CCTCC NO: m20231057, which is classified and named as Enterococcus faecalis, the preservation date is 2023, 06 and 19 days, the preservation unit is China center for type culture collection (CCTCC for short), and the preservation unit address is university of Wuhan, china.
Further, the sequence of 16S rRNA of enterococcus faecalis F1GAM5 is shown in SEQ ID No. 1.
Further, the enterococcus faecalis F1GAM5 is characterized by the following morphological characteristics: culturing in Columbia blood plate to form medium-sized, round, smooth, milky white, and raised colony; the strain is in red color under a gram-dyed mirror and is in a typical enterococcus form.
In a second aspect, the present invention provides a microbial agent comprising at least one of a cell, a spore and a culture solution of enterococcus faecalis according to any one of the first aspect of the present invention.
Further, the microbial agent is freeze-dried bacterial powder of enterococcus faecalis, and the preparation of the freeze-dried bacterial powder comprises the following steps: and (3) centrifugally concentrating the bacterial liquid obtained by fermentation, mixing with an antifreeze agent and an excipient, pre-freezing, freeze-drying, grinding and uniformly mixing. Specifically, the bacterial liquid obtained by fermentation is centrifugally concentrated at 4 ℃, mixed with antifreeze agent (mannitol, 10-100 g/L) and excipient (skim milk powder, 50-200 g/L) and the like, pre-frozen for 1-2 hours at-40 ℃, placed into a vacuum freeze dryer for freeze drying for 15-25 hours, and ground and mixed uniformly. It will be appreciated that the cryoprotectants and excipients described above may also be other agents conventionally used in the art.
Further, the viable bacteria concentration of the freeze-dried bacteria powder is 1 multiplied by 10 7 ～1×10 9 CFU/g; specifically, it may be: 1X 10 7 ～5×10 7 CFU/g、5×10 7 ～1×10 8 CFU/g、1×10 8 ～5×10 8 CFU/g、5×10 8 ～1×10 9 CFU/g, etc.; more preferably, 1X 10 is selected 8 ～1×10 9 The CFU/g freeze-dried bacterial powder is used for preparing bacterial suspension.
Further, the microbial agent can be used for preparing a bacterial suspension of enterococcus faecalis, and the preparation of the bacterial suspension comprises the following steps: suspending lyophilized powder of enterococcus faecalis in PBS solution to obtain PBS bacterial suspension, wherein the viable bacteria concentration in the PBS bacterial suspension is 1×10 8 ～1×10 10 CFU/mL (e.g. 10 8 -10 9 CFU/mL、10 9 -10 10 CFU/mL, etc.).
In a third aspect, the invention provides the use of an enterococcus faecalis as described in any one of the first aspects of the invention, or a microbial agent as described in any one of the second aspects of the invention, in the manufacture of a product for ameliorating autism spectrum disorder. It is understood that the above-mentioned improvement of autism spectrum disorder includes the prevention and/or treatment of autism spectrum disorder diseases.
Further, the product may be used in a mammal having autism, the mammal being selected from the group consisting of rodents, artiodactyls, perissodactylas, lagomorphs, primates, and the like. Further, the rodent is preferably a mouse; the primate is preferably a monkey, ape or human.
Further, the application is for the preparation of a product for promoting exploratory and voluntary activity behavior in ASD patients.
Further, the use is for the preparation of a product for improving anxiety-depressive-like manifestations in ASD patients.
Further, the product comprises a food, a health product or a medicament.
Further, the dosage form of the product includes solid, liquid, gel, semi-fluid, aerosol or powder.
Further, the medicine also comprises a pharmaceutically acceptable carrier or auxiliary material.
By "pharmaceutically acceptable" is meant that the drug does not produce adverse, allergic or other untoward reactions when properly administered to an animal or human. The "pharmaceutically acceptable carrier or adjuvant" should be compatible with the active ingredient, i.e. it can be blended therewith without significantly reducing the efficacy of the drug in the usual manner, which are all substances conventionally used in the art, and suitable carriers or adjuvants are selected according to actual needs for helping stability of the formulation or helping to increase activity or bioavailability or to produce acceptable mouthfeel or odor in the case of oral administration.
Further, in one embodiment, in the above application, the ASD mice are given a gastric lavage treatment of enterococcus faecalis F1GAM5 strain; each time 10 8 -10 9 CFU (PBS suspension) was used, and the viable bacteria concentration in the PBS suspension was 10 9 -10 10 CFU/mL, the amount of the stomach irrigated each time is 0.1-0.2mL.
Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:
the probiotics strain used for ASD treatment research in the prior art is mainly bifidobacterium, and the invention separates and screens a enterococcus faecalis F1GAM5 from clinical faecal fungus transplantation (Fecal microbiota transplantation, FMT) healthy donor bacterial liquid for the first time, belongs to enterococcus with dominant abundance, has remarkable promotion effect on exploring behavior and spontaneous activity behavior of ASD, can be used for improving autism spectrum disorder, and has important potential value in developing novel therapies of ASD probiotics.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 is a 16S rRNA gene evolutionary tree of different species of enterococcus faecalis in an embodiment of the invention, wherein: the NCBI accession number of the sequence, the corresponding strain and the strain from which the sequence is derived are shown in the figure;
FIG. 2 is a graph showing the results of enterococcus faecalis F1GAM5 improving exploratory and spontaneous activity behavior in ASD mice in accordance with one embodiment of the present invention, wherein: (A) residence time of the mice in the central region, (B) movement distance of the mice in the central region, (C) representative movement track diagram of the mice, mean value +/-mean standard deviation;
the enterococcus faecalis F1GAM5 is preserved, and the preservation number is CCTCC NO: m20231057, which is classified and named as Enterococcus faecalis, the preservation date is 2023, 06 and 19 days, the preservation unit is China center for type culture collection (CCTCC for short), and the preservation unit address is university of Wuhan, china.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental procedures, which are not specified in the following examples, are generally determined according to national standards. The experimental materials not shown in the examples below are all commercially available. The equipment used in each step in the following examples is conventional equipment. If the corresponding national standard does not exist, the method is carried out according to the general international standard, the conventional condition or the condition recommended by the manufacturer. Unless otherwise indicated, all parts are parts by weight and all percentages are percentages by mass. Unless defined or otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any method and material similar or equivalent to those described may be used in the methods of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting.
EXAMPLE 1 isolation, screening and identification of enterococcus faecalis F1GAM5
The present example is a screening and identification process of enterococcus faecalis F1GAM5 (Enterococcus faecalis), which specifically comprises the steps of:
(1) Isolation of Strain F1GAM5
Strain F1GAM5 was isolated from clinical FMT donor stock and the isolation medium was modified anaerobic agar medium (Modified Gifu Anaerobic Medium agar) (from Haibo organism HB 8462). The specific method comprises the following steps: taking 100 mu L of bacterial liquid for clinical FMT, and carrying out gradient dilution for 10 by using a trypticase soybean peptone liquid culture medium (containing 15g/L of tryptone, 5g/L of soybean peptone, 2.5g/L of dipotassium hydrogen phosphate, 5g/L of sodium chloride, 5g/L of glucose and pH of 7.2-7.4) added with L-cysteine (0.05 percent) 4 -10 5 Doubling, coating 100 mu L of diluent on an improved anaerobic bacteria agar culture medium, and carrying out anaerobic culture for 48 hours at 37 ℃; single colony is picked from the plate, streaked and diluted on a Columbia blood plate (purchased from Ke Ma Jia), anaerobic culture is carried out for 48 hours at 37 ℃, the single colony is picked from the plate, the steps are repeated, and the purified strain is separated; spreading and culturing the obtained single colony on Columbia blood plate, anaerobic culturing at 37deg.C for 48 hr, scraping off thallus Porphyrae, re-suspending in brain heart infusion broth (beef powder 3g/L, sodium chloride 5g/L, peptone 12g/L, dipotassium hydrogen phosphate 2g/L, pH of about 7.5), and freezing at-80deg.C for seed preservation.
(2) Morphological characterization of strain F1GAM5
Strain F1GAM5 was medium-sized, round, smooth, milky white, raised colonies grown on golombia platelets; the product is red under the mirror after gram staining and is in the form of a typical enterococcus.
(3) Molecular characterization of Strain F1GAM5
And collecting thalli for carrying out 16S rRNA gene amplification sequencing, and identifying strains. The strain 16S rRNA gene is amplified by universal primers 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). A commercial kit, such as a Premix Taq (Ex Taq Version 2.0plus dye) kit, is adopted, a reaction system is given according to the instruction, the total amount of the DNA template is 10ng per reaction, and the reaction conditions are as follows; 98℃for 10 seconds, 55℃for 20 seconds, 72℃for 90 seconds, 30 cycles; the amplified product was detected by agarose gel electrophoresis. The sequence obtained after sequencing of the product was 99.93% identical to the 16S rRNA gene sequence of E.faecalis strain ATCC 19433 (NCBI accession number NR_ 115765.1), confirming that it belongs to the species enterococcus faecalis, i.e.the above strain identified as enterococcus faecalis.
The 16S rRNA gene sequence of the strain was determined as follows:
in addition, after the sequences are aligned with the 16S rRNA gene reference sequences of different strains of enterococcus (downloaded from NCBI database) by MUSCLE software, a maximum likelihood method is adopted to construct a evolutionary tree (bootstrap value is set to 1000), as shown in figure 1, the isolated strain and the enterococcus faecalis reference strain are gathered on the same branch, further shows that the strain F1GAM5 is enterococcus faecalis (Enterococcus faecalis), and the strains are preserved, wherein the preservation number is CCTCC NO: m20231057.
EXAMPLE 2 cultivation of enterococcus faecalis F1GAM5 and preparation of bacterial suspension
This example is a preferred method for culturing enterococcus faecalis F1GAM5 obtained by screening in example 1, and a method for preparing a microbial inoculum and a microbial suspension thereof, comprising the following steps:
in Columbia blood plates orCulturing the recovered enterococcus faecalis F1GAM5 by streaking with improved anaerobic agar medium (37 ℃), picking single colony, culturing at 37deg.C, scraping thallus, inoculating to agar-free thioglycolate liquid medium or other suitable liquid medium, culturing at 37deg.C, centrifuging and concentrating the fermented bacterial liquid at 4deg.C, mixing with antifreeze agent such as mannitol (10-100 g/L) and excipient such as skim milk powder (0-200 g/L), pre-freezing at-40deg.C for 1-2 hr, lyophilizing in vacuum freeze dryer for 15-25 hr, and grinding and mixing. The viable bacteria concentration of the obtained dry bacterial powder is 1×10 7 ～1×10 9 CFU/g; specifically, it may be: 1X 10 7 ～5×10 7 CFU/g、5×10 7 ～1×10 8 CFU/g、1×10 8 ～5×10 8 CFU/g、5×10 8 ～1×10 9 CFU/g, etc. Taking 1×10 8 ～1×10 9 Suspending CFU/g lyophilized powder in PBS solution to obtain viable bacteria concentration of 1×10 8 ～1×10 10 CFU/mL bacterial suspension.
EXAMPLE 3 use of enterococcus faecalis F1GAM5 in the amelioration of autism spectrum disorders
This example demonstrates the use of enterococcus faecalis F1GAM5 in the treatment of autism spectrum disorders (autism spectrum disorder, ASD) comprising the steps of:
(1) Constructing an ASD mouse model by a valproic acid induction method;
SPF (specific pathogen free) C57BL/6 Male young mice were intraperitoneally injected with 200mg/kg valproic acid (VPA) on day 7 post-natal to construct a VPA mouse autism model. Mice received drinking water treatment with tetrad antibiotics, including metronidazole 200mg/L, ampicillin/ampicillin 200mg/L, vancomycin hydrochloride 100mg/L, neomycin sulfate 200mg/L on day 14. Weaning the mice on the 21 st day, and continuously receiving the drinking water treatment of antibiotics; normal drinking water was resumed on day 28 to obtain an ASD mouse model.
(2) Performing gastric lavage treatment on enterococcus faecalis F1GAM 5;
mice on day 30, ASD mice were randomized into experimental and PBS control groups. The experimental group was subjected to gastric lavage treatment with enterococcus faecalis F1GAM5 strain; each time 10 8 -10 9 CFU (prepared using example 2)10 of (2) 9 -10 10 CFU/mL of PBS bacterial suspension, 0.1 mL), gastric lavage once every two days. PBS control was given an equal volume of PBS lavage. Gastric lavage treatment was performed for 4 weeks.
(3) Enterococcus faecalis F1GAM5 lavage improves the exploring and spontaneous activity behavior of ASD mice;
after the gastric lavage treatment is finished, the exploring behavior and the spontaneous activity behavior of each group of mice are detected by adopting an open field experiment and are carried out according to standard steps.
The experiment adopts the open field experimental equipment of the mice, the camera system is used for monitoring the activity condition of animals in the open field, and animal track tracking and data acquisition analysis are carried out through visual track video recording analysis software. The specific operation is as follows:
1) Taking out the mice from the rearing cage, and placing the mice back to the experimenter in the central area of the open field, wherein the experimenter rapidly leaves;
2) Rapidly starting video acquisition of software or video recording of a camera system, and recording the activity condition of animals in an open field for 10 min;
3) Taking out the mice after the experimental monitoring time is over, putting the mice back into a rearing cage, cleaning the whole open field box body by using 75% alcohol or experimental sterile water, and continuously repeating the experiment after waiting for the mice to be replaced after the mice are dried;
4) And (5) data analysis.
And (3) determining the residence time and the movement distance of each group of mice in the central area, wherein the related indexes can reflect the exploring behaviors and the spontaneous activity behaviors of experimental animals, so as to evaluate the autonomous behaviors, the exploring behaviors and the tension degree in a new different environment. Autism mice receiving enterococcus faecalis F1GAM5 lavage were found to have significantly improved retention time in the central region (part A of FIG. 2) and central region movement distance (part B of FIG. 2) compared to the PBS control group (both p <0.05, t-test), and representative movement trace patterns are shown in part C of FIG. 2. The results show that enterococcus faecalis F1GAM5 has remarkable promotion effect on exploring behavior and spontaneous activity behavior of ASD mice, and is suggested to have improvement effect on anxiety-depression-like manifestations in ASD.
From the above examples, the invention provides an enterococcus faecalis isolate F1GAM5 isolated from clinical FMT donor bacterial liquid, and the enterococcus faecalis isolate F1GAM5 can be proved to improve the anxiety depression-like expression in ASD by promoting the exploratory behavior and the spontaneous activity behavior of an autism mouse model, and the strain is helpful for the research and application of intestinal flora improvement of ASD.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, and it will be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the description and illustrations of the invention, and are intended to be included within the scope of the invention.
Claims (10)
1. Enterococcus faecalis (Enterococcus faecalis), which is characterized by being named as F1GAM5 and having a preservation number of CCTCC NO: m20231057.
2. A microbial agent, wherein the microbial agent comprises at least one of a bacterial cell, a spore and a culture solution of enterococcus faecalis according to claim 1.
3. The microbial agent according to claim 2, wherein the microbial agent is a lyophilized powder of enterococcus faecalis, and the preparation of the lyophilized powder comprises the steps of: and (3) centrifugally concentrating the bacterial liquid obtained by fermentation, mixing with an antifreeze agent and an excipient, pre-freezing, freeze-drying, grinding and uniformly mixing.
4. A microbial agent according to claim 3, wherein the viable bacteria concentration of the lyophilized powder is 1 x 10 7 ～1×10 9 CFU/g。
5. Use of the enterococcus faecalis of claim 1, or the microbial agent of any one of claims 2 to 4, in the manufacture of a product for ameliorating autism spectrum disorder.
6. The use according to claim 5, wherein the use is for the preparation of a product for promoting exploratory and voluntary activity of ASD patients.
7. The use according to claim 5, wherein the use is for the preparation of a product for improving anxiety-depression-like manifestations in ASD patients.
8. Use according to any one of claims 5 to 7, wherein the product comprises a food product, a health product or a medicament.
9. The use according to claim 8, wherein the dosage form of the product comprises a solid, a liquid, a gel, a semi-fluid, an aerosol or a powder.
10. The use according to claim 8, wherein the medicament further comprises a pharmaceutically acceptable carrier or adjuvant.
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