CN110396487B - Lactobacillus acidophilus capable of improving intestinal flora and regulating immunity and application thereof - Google Patents
Lactobacillus acidophilus capable of improving intestinal flora and regulating immunity and application thereof Download PDFInfo
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Abstract
The application discloses lactobacillus acidophilus LA-06 capable of improving intestinal flora and regulating immunity. The application takes Lactobacillus acidophilus LA-06 as a research object, 30 volunteers are selected, and the daily dose of the Lactobacillus acidophilus LA-06 for each person is 3.0 multiplied by 109CFU, before taking, 30d taking, 60d taking and 30d stopping taking are respectively used for measuring the change condition of the intestinal flora of the volunteers and blood immunity indexes CD4, CD8, CD4/CD8, IgA, IgG and IgM, and the effect of Lactobacillus acidophilus LA-06 on the structure and the immunity of the intestinal flora of the human body is proved.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to lactobacillus acidophilus capable of improving intestinal flora and regulating immunity and application thereof.
Background
The lactobacillus has close relationship with human life, and is one of beneficial microorganisms widely applied to the fields of food fermentation, industrial lactic acid fermentation and medical care. Many strains of lactobacillus and bifidobacterium have been recognized to have specific superior properties and genetic traits, exhibiting very strong probiotic properties. Such as resistance to acids, bile acids, colonizing growth and aerobic growth in the digestive tract and lactose intolerance, anti-mutagen, serum cholesterol lowering, anti-tumor, intestinal pathogenic bacteria inhibiting and anti-cancerHelicobacter pyloriAnd the like, the discovery and research application of the lactobacillus is still lacked.
Disclosure of Invention
One of the purposes of the application is to provide lactobacillus acidophilus capable of improving intestinal flora and regulating immunity, the other purpose of the application is to provide application of lactobacillus acidophilus in preparation of products influencing the immune system of a human body, and the other purpose of the application is to provide application of lactobacillus acidophilus in preparation of products influencing the intestinal flora structure and the blood immune index of the human body.
The purpose of the application is realized by the following technical scheme:
the lactobacillus acidophilus LA-06 is an acid-resistant, bile acid-resistant probiotic isolated from a naturally fermented dairy product, mare's milk. Specifically, the method is obtained by the following separation method:
step 1, taking the mare's milk as a lactobacillus separation sample, putting 2mL of mare's milk into a small sterilized test tube, and putting 1mL of mare's milk into a small sterilized test tube filled with CaCO3Placing the powder in a sampling bottle with the sterile volume of 2mL as a sample in a refrigerator for refrigeration at the temperature of 4 ℃; sucking 1mL of the mare's yogurt sample by using a sterilization straw and inoculating the mare's yogurt sample into 10mL of litmus milk culture medium;
step 3, putting the BL agar culture medium into a BBL gaspak anaerobic culture tank, and culturing for 48-72h at the temperature of 30-37 ℃ to form a bacterial colony; selecting colony with inoculating loop, inoculating in MRS culture solution, culturing at 30-37 deg.C for 24-48 hr to make strain grow;
step 4, after the bacterial strain grows well, inoculating the bacterial strain into a BL agar culture medium by using an inoculating loop scribing line again, culturing for 24-48h at the temperature of 30-37 ℃, observing and recording the morphological characteristics of bacterial colony and gram staining cell, performing gram positive and catalase tests, and separating out bacilli which are negative in the test, wherein the bacilli are lactobacillus;
step 6, determining the number of viable bacteria by using a BCP agar culture medium, and screening out lactobacillus strains with high acid resistance growth; and further determining the survival ability of the lactobacillus strain in different artificial gastric juice and intestinal juice, and obtaining lactobacillus acidophilus by an in vitro cholate resistance test and a cholesterol reduction test.
The application provides lactobacillus acidophilus LA-06 (L. acidophilus) capable of improving intestinal flora and regulating immunityLactobacillus acidophilusLA-06), the Lactobacillus acidophilus LA-06 is acid-resistant and bile acid-resistant probiotics separated from the fermented dairy product acid mare milk, the strain is preserved in China general microbiological culture Collection center with the preservation number: CGMCC NO. 17751; classified and named Lactobacillus acidophilus (Lactobacillus acidophilus) (ii) a Preservation time: year 2019, month 05, and day 10; and (4) storage address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the survival of the strain was detected.
The application takes Lactobacillus acidophilus LA-06 (Lactobacillus acidophilus LA-06) as a research object, 30 volunteers are selected, and the daily dose of the Lactobacillus acidophilus LA-06 taken by each person is 3.0 multiplied by 109CFU, before taking, taking 30 days, taking 60 days and stopping taking 30 days respectively, measuring the change condition of the intestinal flora of the volunteers and the blood immunity indexes CD4, CD8, CD4/CD8, IgA, IgG and IgM, and proving the influence of Lactobacillus acidophilus LA-06 on the structure and the immunity of the intestinal flora of the human body.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the effect of administration of Lactobacillus acidophilus LA-06 on the immune index;
FIG. 2 is a sparse curve;
fig. 3 is a shannon curve.
Detailed Description
The application provides lactobacillus acidophilus LA-06 (L. acidophilus) capable of improving intestinal flora and regulating immunityLactobacillus acidophilus LA-06), the Lactobacillus acidophilus LA-06 is acid-resistant and bile acid-resistant probiotics separated from the fermented dairy product acid mare milk, the strain is preserved in China general microbiological culture Collection center with the preservation number: CGMCC NO. 17751; classified and named Lactobacillus acidophilus (Lactobacillus acidophilus) (ii) a Preservation time: year 2019, month 05, and day 10; and (4) storage address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the survival of the strain was detected.
The lactobacillus acidophilus LA-06 is obtained by the following separation method:
step 1, taking the mare's milk as a lactobacillus separation sample, putting 2mL of mare's milk into a small sterilized test tube, and putting 1mL of mare's milk into a small sterilized test tube filled with CaCO3Placing the powder in a sampling bottle with the sterile volume of 2mL as a sample in a refrigerator for refrigeration at the temperature of 4 ℃; sucking 1mL of the mare's yogurt sample by using a sterilization straw and inoculating the mare's yogurt sample into 10mL of litmus milk culture medium;
step 3, putting the BL agar culture medium into a BBL gaspak anaerobic culture tank, and culturing for 48-72h at the temperature of 30-37 ℃ to form a bacterial colony; selecting colony with inoculating loop, inoculating in MRS culture solution, culturing at 30-37 deg.C for 24-48 hr to make strain grow;
step 4, after the bacterial strain grows well, inoculating the bacterial strain into a BL agar culture medium by using an inoculating loop scribing line again, culturing for 24-48h at the temperature of 30-37 ℃, observing and recording the morphological characteristics of bacterial colony and gram staining cell, performing gram positive and catalase tests, and separating out bacilli which are negative in the test, wherein the bacilli are lactobacillus;
step 6, determining the number of viable bacteria by using a BCP agar culture medium, and screening out lactobacillus strains with high acid resistance growth; and further determining the survival ability of the lactobacillus strain in different artificial gastric juice and intestinal juice, and obtaining lactobacillus acidophilus by an in vitro cholate resistance test and a cholesterol reduction test.
Specifically, the research design of lactobacillus acidophilus LA-06 includes:
a, aligning the sequences by using PyNAST software, and then carrying out UCLUST division on the sequences with 100% similarity to obtain a 16S rRNA gene sequence set without duplication;
establishing a classification operation unit (OTU), and further dividing the OTU under the similarity of 98.65% on the basis of dividing the similarity of 100%;
c, removing the OTU sequence containing the chimera sequence by using a Chimeraslayer;
d, comparing the OTU sequence with the chimera sequence removed with RDP (Ribosol Database project) and Greenenennes (Version-3.8) databases, and performing taxonomic identification;
step e, evaluating structural diversity and abundance of the flora of the sample, and simultaneously evaluating the sequencing sequence diversity and the sequencing quantity by using a sparse curve (Rarefection curve) and a Shannon curve (Shannon curve) to judge whether the sequencing sequence diversity can represent the species diversity of the sample;
the application uses acidophilus milkBacillus LA-06: (Lactobacillus acidophilusLA-06) was the subject, and 30 volunteers were selected from a group of persons of different sexes and ages (as shown in table 1), each of which was administered a dose of 3.0 × 10 per day9And (3) respectively measuring feces and blood samples of the CFU lactobacillus acidophilus LA-06 in four time periods (before taking, 30 days after taking, 60 days after taking and 30 days after stopping taking) of the volunteers to analyze the intestinal flora and the blood change condition. The change of the structure of intestinal flora of a human body can be influenced during taking of lactobacillus acidophilus LA-06, and the contents of CD4, CD8, CD4/CD8, IgG, IgA and IgM in blood are all improved to different degrees, so that the immunity of the human body can be enhanced by taking lactobacillus acidophilus LA-06.
TABLE 130 volunteer information
Experimental example 1: sample collection and preservation
The samples were: fecal and blood samples collected from 30 volunteers during the test period before taking, 30d, 60d and 30d discontinued.
The preliminary treatment process of the fecal sample and the blood sample in the sample is as follows:
collecting about 10 g of fresh excrement sample by using a sterile sampling spoon, putting the fresh excrement sample into a non-enzyme sterile 50 mL sampling centrifuge tube, adding 2-3 times of protective solution, fully mixing uniformly, and storing the sample in an ultra-low temperature freezer at minus 80 ℃ for later use.
The blood sample is collected by a hospital instead of fasting blood from morning and is used for measuring various clinical immunity indexes.
Experimental example 2: analysis of intestinal flora by Lactobacillus acidophilus
The experimental method comprises the following steps: the QIAGEN DNA Stool Mini Kit and the OMEGA DNA Kit are adopted to respectively extract the metagenomic DNA of the Stool sample. The 16S rRNA gene sequence in the macro gene DNA was PCR amplified using forward primer 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') containing Barcode and reverse primer 1492R (5'-ACCTTGTTACGACTT-3'). The purified DNA was subjected to a repair treatment using the SMRTbellTM Template Prep Kit 1.0 Kit, and the DNA concentration was measured again. Measuring the repaired DNA, carrying out inactivation enzyme treatment on the DNA, determining the DNA concentration of the library by using a LabchipGX instrument, calculating the addition amount of the reagent by using Calculater v2.3.3 software according to the concentration and the size of the library, adding the reagent into the library, uniformly mixing, and carrying out on-machine sequencing by using a PacBio RS II sequencer. According to SMRT Portal (V2.7), using RS _ ReadsOfinsert.1 to control the quality of the original sequence, extracting a high-quality sequence, dividing the high-quality sequence into each sample according to Barcode information, and then removing the Barcode. Bioinformatics analysis of the processed sequences was performed using the QIIME (v1.7.0) analysis platform.
Sparse curves and shannon curves are drawn as shown in fig. 2 and fig. 3 according to the results of collecting the fecal samples of volunteers at 4 time points (0 d, 30d, 60d, and stopping taking 30 d), the number of OTUs does not reach the equilibrium state, which indicates that new species will be found with the increase of sequencing amount. And the shannon curve reaches the horizontal state completely, which indicates that the bacterial diversity can not change with the increase of the sequencing quantity, so that the current sequencing quantity can meet the requirement of subsequent analysis.
At the genus level, the differences in intestinal flora among patients at four time points (0 d, 30d, 60d and 30d of withdrawal) were significant, but the main genera among the intestinal flora were bacteroides, prevotella and coprophilus as a whole. Compared analysis on relative content of the genus at four time points by using Kruskal-Wallis test shows that,Curvibactergenus Delftia (a)Delftia) Enterococcus genus (A)Enterococcus) Genus Germinium (Germinium) (II)Gemmiger) Lactobacillus (I) and (II)Lactobacillus) And rare species of Pediococcus: (Subdoligranulum) The relative content of the 6 genera varied significantly (table 2). Wherein Lactobacillus is the main genus of Lactobacillus acidophilus LA-06, and the relative content of Lactobacillus in the intestinal flora of volunteers is increased to different extent after taking Lactobacillus acidophilus LA-06, but is not detected in the intestinal tract after stopping taking, and is presumed to act as low-abundance passerby to volunteerThe influence of the intestinal flora structure is small.
TABLE 2 relative content of Difference genera
Note: calculation by Kruskal-Wallis detectionpValue of
At the species level, the predominant species in the patient's intestinal flora at four time points (0 d, 30d, 60d and 30d withheld) wereBacteroides doreiAndPrevotella copri. The comparative analysis of the strain content at four time points by Kruskal-Wallis test shows that the formic acid budding bacteria (A)Gemmiger formicilis) Lactobacillus crispatus: (Lactobacillus crispatus) Lactobacillus acidophilus (a)Lactobacillus acidophilus) Lactococcus lactis (A)Lactococcus lactis）、Blautia faecis、Delftia lacustris、Roseburia inulinivoransAndSubdoligranulum variabilethe relative content of the 8 species was significantly altered (table 3). The relative content of beneficial bacteria lactobacillus crispatus, lactobacillus acidophilus and lactococcus lactis is increased.
TABLE 3 relative content of different species
Differential species findings at four time points were analyzed by Mann-Whitney test comparison (table 4), with 26 species having significant differences: (table 4) (ii)p<0.05). Administering Lactobacillus acidophilus LA-06 at 60d with coprosterol-producing eubacterium: (Eubacterium coprostanoligenes) Bacteroides thetaiotaomicron: (A)Bacteroides thetaiotaomicron) Bacteroides ovatus (II)Bacteroides ovatus) Bacteroides fragilis: (B.fragilis) (B.fragilis)Bacteroides fragilis) The relative content of 14 beneficial strains is increased, and 6 strains such as bacteroides fragilis and bacteroides thetaiotaomicron are decreased after drinking is stopped. Wherein the Bacteroides thetaiotaomicron and the Bacteroides ovorans can be digested and utilizedOligosaccharides in the diet provide nutrients to the host and other bacteria in the host's intestinal tract.
Table 4 four time points significantly different relative strain contents
Note: the different letters represent significant differencesp<0.05, Mann-Whitney test calculate significant differences.
From the above results, it can be seen that the beneficial bacteria in the intestinal tract are increased and the conditioned pathogenic bacteria are decreased during the administration of lactobacillus acidophilus LA-06, and the relative contents of some strains tend to the level before the test after the administration is stopped, so that lactobacillus acidophilus LA-06 can regulate the change of the intestinal flora of human body.
Experimental example 3: analysis of lactobacillus acidophilus on human immunity
The T cells are mainly composed of a helper T cell (Th) of CD4 and a suppressor T cell (Ts)/killer T cell (Tc) of CD8, and the CD4 assists other cells to participate in immune response, secretes lymphokines, activates other cells to generate inflammatory response and mediates the effect of the inflammatory response. CD8 is an immunosuppressive cell that also secretes lymphokines, inhibiting the function of other immune cells. The two components are maintained in certain proportion in vivo, and participate in immune response process together, and the physiological balance disorder of the two components can cause immune dysfunction. The level of T lymphocyte subpopulation can be used as a marker for detecting the immune status of cells and the immune regulation level.
IgG is an immunoglobulin with the highest human content and the most predominant, and plays an anti-infection role through opsonization, toxin neutralization and virus neutralization. IgA plays a role in resisting infection by regulating saliva and dissolving, and can also inhibit pathogens and antigens from adhering to mucosa to prevent invasion into the body. IgM has the functions of lysing bacteria and blood cells, and can neutralize viruses.
The changes in CD4, CD8, CD4/CD8, IgG, IgA and IgM in the blood during administration of Lactobacillus acidophilus LA-06 are shown in Table 5 and FIG. 1.
TABLE 5 variation of the blood immune index at four time points
After taking lactobacillus acidophilus LA-06, the blood of the volunteers is improved in different degrees in CD4, CD8, CD4/CD8, IgG, IgA and IgM, which reflects that lactobacillus acidophilus LA-06 can enhance the immunity of human body.
Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.
It will be understood that the present application is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.
Claims (3)
1. Lactobacillus acidophilus LA-06 (for improving intestinal flora and regulating immunity)Lactobacillus acidophilus LA-06), characterized in that the Lactobacillus acidophilus LA-06 is acid-resistant and bile acid-resistant probiotics separated from the fermented dairy product sour mare milk, and the strain is preserved in China general microbiological culture Collection center with the preservation number: CGMCC NO. 17751.
2. Use of lactobacillus acidophilus LA-06 according to claim 1 for the preparation of a preparation that affects the human immune system.
3. Use of lactobacillus acidophilus LA-06 according to claim 1 for the preparation of a product that affects the structure of the intestinal flora and the immune index of the blood of humans.
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