CN110964653B - Lactobacillus paracasei ET-22 capable of adjusting intestinal flora balance - Google Patents
Lactobacillus paracasei ET-22 capable of adjusting intestinal flora balance Download PDFInfo
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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
- 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
- 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
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
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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
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Abstract
The invention relates to lactobacillus paracasei ET-22 capable of adjusting intestinal flora balance. The present invention provides a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain and its use for promoting the growth of bifido-and/or lactic-bacteria and/or for modulating the flora balance in a subject and related methods of use. The invention also provides a method for preparing a composition containing the strain for promoting the growth of bifidobacteria and/or lactic acid bacteria and/or regulating the flora balance of a subject. The strain can promote the growth of bifidobacteria and/or lactic acid bacteria and/or regulate the flora balance of a subject, and has good effect.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus paracasei capable of remarkably promoting the growth of bifidobacteria and lactic acid bacteria and application thereof.
Background
1. Health hazard due to intestinal dysbacteriosis
The balance of intestinal flora is closely related to human health. The intestinal flora comprises beneficial bacteria, neutral bacteria and harmful bacteria. Beneficial bacteria in healthy human intestinal tracts occupy dominant positions and continuously interact with harmful bacteria to maintain human health. If beneficial bacteria in the intestinal tract are reduced due to various factors, harmful bacteria can be propagated in a large quantity, the intestinal microecological balance is broken, and various clinical symptoms such as enteritis, diarrhea and the like are caused. Meanwhile, a large amount of antibiotics are generally used clinically, and although harmful bacteria can be effectively killed, beneficial bacteria can be killed at the same time, so that intestinal flora imbalance is caused, and diseases are caused.
For people with dysbacteriosis of intestinal canal, oral probiotic supplement is a direct effective method for regulating intestinal canal flora. The oral probiotic preparation or the product containing the probiotic can directly or indirectly regulate the composition of intestinal flora and activate the endogenous microbiota or the activity of an immune system of a host to realize the probiotic effect.
2. Safety of probiotics and application thereof
The definition of probiotic products by the world health organization is that foods contain sufficient numbers of living microorganisms, and the proper number and activity of living bacteria can be maintained after various processes of food processing and entering human intestinal tracts. Therefore, after the strain is used for preparing the bacterial powder, producing and processing products and being stressed by gastric acid and bile salt in the gastrointestinal tract of a human body, the strain can keep a stable viable count. In addition, even though lactic acid bacteria are well-known safe strains, recent research shows that many lactic acid bacteria, especially food-borne lactic acid bacteria and intestinal lactic acid bacteria, have toxic factor and antibiotic resistance, and pose potential risks to human health. Therefore, the probiotic properties of the strain should be evaluated and the safety during the eating process and the stability during the production process should be considered comprehensively.
Disclosure of Invention
In view of the above problems in the prior art, the present application provides a lactobacillus paracasei and its use. The strain has the advantages that a single bacterium has the capacity of remarkably promoting the growth of intestinal bifidobacteria and lactic acid bacteria, can tolerate the environment in vitro simulated gastrointestinal fluid stress, has no oral acute toxicity and no antibiotic tolerance, and can be safely used for food processing.
In some embodiments, the invention provides Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain, which is deposited in China general microbiological culture Collection center (accession No. CGMCC No. 15077).
In some embodiments, the present invention provides a culture comprising said Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain. In some embodiments, the culture may also include a suitable lactic acid bacteria medium. In some embodiments, the lactic acid bacteria culture medium comprises a solid medium and a liquid medium, such as MRS medium. In some embodiments, the culture comprises a freeze-dried culture of Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain.
In some embodiments, the present invention provides the use of a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain for modulating the microflora balance in a subject.
In some embodiments, the present invention provides the use of a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain for promoting the growth of bifidobacteria and/or lactic acid bacteria.
In some embodiments, the present invention provides a method for modulating the microflora balance in a subject, the method comprising administering a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain to the subject to modulate the microflora balance thereof.
In some embodiments, the strain is an active strain. In some embodiments, the strain may be administered to the subject by a gastrointestinal route. In some embodiments, the strain may be administered to the subject orally. In some embodiments, the strain is administered to the subject by other means suitable for intestinal absorption, for example by means other than through the mouth, throat and esophagus, such as a feeding tube.
In some embodiments, the subject may be a mammal. In some embodiments, the subject may be a mammal, e.g., human, monkey, horse, cow, dog, cat, mouse, rat, pig, and the like.
In some embodiments, the methods and compositions of the present invention promote the growth of bifidobacteria and lactic acid bacteria.
In some embodiments, the methods and compositions of the present invention are not affected by the growth of enteric bacteria such as enterobacteria after use.
In some embodiments, the present invention provides a method of promoting the growth of bifido-and/or lactic-bacteria, the method comprising administering Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain to the subject to promote the growth of bifido-and/or lactic-bacteria.
In some embodiments, the methods and compositions of the present invention are not affected by the growth of enteric bacteria such as enterobacteria after use.
In some embodiments, the present invention provides the use of a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain for promoting the growth of bifidobacteria and/or lactic acid bacteria and/or for modulating the flora balance of a subject.
In some embodiments, the present invention provides the use of a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain for the manufacture of a composition for promoting the growth of bifidobacteria and/or lactic acid bacteria and/or for modulating the microbiota balance in a subject. In some embodiments, the composition comprises a composition suitable for intestinal absorption by a subject. In some embodiments, the composition comprises a food composition. In some embodiments, the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain may be formulated into suitable food compositions, including health foods, nutritional supplements, functional foods, and the like. In some embodiments, buffers, excipients, diluents, carriers, stabilizers, and/or preservatives, for example, may be included in the compositions. In some embodiments, the food product may include a beverage product such as a food product, oil, meat product, dairy product, seafood, can, sugar-containing food, cold food, wine, pet food, and the like. In some embodiments, the food product may include beverages such as dairy drinks, tea, coffee, chewing gum and dentrifice, jerky for pets, or combinations thereof. In some embodiments, the strain of the invention may be prepared as a food composition, e.g. a milk drink, tea, coffee, wherein the milk drink may comprise fermented milk, yoghurt, cheese or milk powder. In the composition, the strain may be present in an effective amount. For example, in a food composition or a pharmaceutical composition, the number of lactic acid bacteria strains is 106CFU or more, e.g. 107CFU above, 108CFU above, 109CFU above, 1010CFU above, 1011Above CFU; preferably, the number of lactic acid bacteria strains is 109Above CFU.
In this context, probiotics may refer to living microorganisms that can be added to compositions such as food products and the like, maintain activity and exert their physiological effects on the subject ingesting the composition containing the probiotics. Prebiotics may refer to substances added to compositions such as food products that act on components of the microbiota, facilitating the establishment of bacteria beneficial to health and/or hindering the establishment of pathogenic bacteria. In some embodiments, the strains of the invention may be prepared as probiotic or prebiotic compositions.
In some embodiments, it has been found that the effect of probiotics on promoting the growth of bifidobacteria and/or lactic acid bacteria and/or on regulating the flora balance is strain specific. It has been found that the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain has an excellent effect of promoting the growth of bifido-and/or lactic-bacteria and/or regulating the flora balance compared to a control or control strain, such as other Lactobacillus strains, e.g.Lactobacillus paracasei strain, not comprising said strain. In some embodiments, the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain has an increased effect of promoting the growth of bifido-and/or lactic-bacteria and/or regulating the flora balance compared to a control or control strain, such as other Lactobacillus strains (e.g., Lactobacillus paracasei strain), not comprising said strain. In some embodiments, the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain is capable of promoting the growth of bifido-and/or lactic-bacteria and/or increasing the flora balance (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) of a subject as compared to a control or control strain, such as other Lactobacillus strains (e.g., Lactobacillus paracasei strains), that does not contain the strain. In some embodiments, promoting the growth of bifidobacteria and/or lactic acid bacteria and/or modulating the flora balance of a subject may be measured by methods known in the art. In some embodiments, promoting the growth of bifidobacteria and/or lactic acid bacteria and/or modulating the population balance in a subject may be performed, for example, by comparing the changes in bifidobacteria, lactic acid bacteria, enterococci, enterobacteria, both before and after the experiment, per se and between groups. In some embodiments, a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain may prepare a composition and/or kit for promoting the growth of bifido-and/or lactic-bacteria and/or modulating microbiota balance in a subject. In some embodiments, the composition and/or kit for promoting the growth of bifidobacteria and/or lactic acid bacteria and/or for modulating the microbiota balance in a subject may further comprise other suitable substances for promoting the growth of bifidobacteria and/or lactic acid bacteria and/or for modulating the microbiota balance in a subject.
In some embodiments, the present invention provides a method for producing a composition for promoting the growth of bifidobacteria and/or lactic acid bacteria and/or modulating the microbiota balance in a subject, the method comprising adding Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain to the composition.
Drawings
FIG. 1 shows the results of evaluation of acid-and bile salt-resistant properties of Lactobacillus paracasei ET-22.
FIG. 2. results of intestinal adhesion evaluation of Lactobacillus paracasei ET-22.
FIG. 3. Lactobacillus paracasei ET-22 regulates intestinal flora.
FIG. 4 comparison of Lactobacillus paracasei ET-22 with Lactobacillus casei LC-01 for modulating intestinal colony changes.
[ biological Material Collection ]
The Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain of the present invention is deposited in:
china center for type culture Collection/China Committee for culture Collection of microorganisms common microbiological center (CGMCC), wherein the preservation date is 18 days 12 months in 2017, and the preservation number is CGMCC No. 15077; the address of the depository is: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
Detailed Description
Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art.
The freeze-dried culture of the lactobacillus strain is preserved in China center for type culture Collection and China Committee for culture Collection of microorganisms. The details of the deposit are shown in the following table:
deposited data of lactic acid bacteria strains
Morphological and general Properties of Lactobacillus strains
The taxonomical characteristics of the strain were confirmed based on the results of 16S rDNA sequence analysis and analysis by the API bacterial identification system. The morphological and general characteristics of the above strains are detailed in the following table:
morphological and general Properties of the Lactobacillus Strain
The fermentation conditions of the strain are as follows:
MRS liquid medium: peptone, 10.0 g; 10.0g of beef extract; 5.0g of yeast extract powder; glucose, 20.0 g; dipotassium hydrogen phosphate, 5.0 g; diammonium hydrogen citrate, 2.0 g; sodium acetate, 5.0 g; magnesium sulfate heptahydrate, 0.5 g; 0.2g of manganese sulfate tetrahydrate; tween 80, 1.0 g; 15.0g of agar; 1000mL of distilled water. Adjusting the pH value to 6.2-6.4, and sterilizing for 15 minutes at 121 ℃.
L. paracasei ET-22 is a microaerophilic bacterium, grows well in a facultative anaerobic environment, produces lactic acid, has acid resistance, can resist an acid environment with a pH value of 2.5 and a 0.4% bile salt environment for 4 hours, is mesophilic, has a growth temperature range of 15-45 ℃, and has an optimal growth temperature of about 37 ℃.
Example 1: tolerance of artificial gastric juice, intestinal juice and bile
Activating L.paracasei ET-22 for three times, subpackaging 8, taking one, counting bacterial colonies, and calculating the initial viable count. And (3) centrifuging the rest seven cells at 4000rpm for 10min, removing the supernatant, culturing in a culture medium with the pH value of 2.5 at 37 ℃ for 1-3 h, taking one tube of mixed solution per hour, centrifuging at 4000rpm for 10min, removing the supernatant, washing twice by using 5mL of RO water, and calculating the number of viable bacteria. And centrifuging the other 4 tubes at 4000rpm for 10min when the number of the 4 tubes is 3 hours, removing supernatant (pH 2.5MRS broth), fully mixing the 4 tubes with 1.5% oxgall MRS broth, evenly distributing the mixture to the 4 tubes, and culturing the mixture for 1-4 hours at 37 ℃. The number of viable bacteria was counted after taking 1 tube of the mixture per hour, centrifuging at 4000rpm for 10min, removing the supernatant, washing twice with 5mL of RO Water. Counting the change of the number of the live bacteria measured at different time, and comparing and analyzing, wherein the survival rate of the strain is shown in figure 1.
The results show that the total bacterial count is continuously treated by an acid culture medium and a bile salt environment, and the bacterial count of 5 th power of L.paracasei ET-22 is maintained after continuous treatment of gastric acid and choline for 7 hours in a simulated digestion environment, thereby proving that the L.paracasei ET-22 can pass the strict test of the human digestive system environment.
Example 2 intestinal cell adsorption Effect
And clamping the glass slide with tweezers to obtain a cover glass, sterilizing with an alcohol lamp, and placing into a 6-hole tray for later use. The Caco-2 cells were removed from the storage tube, conditioned, transferred to a 6-well plate for culture, and experiments were performed after the plate was filled. For the assay, the tray was drained and washed twice with PBS buffer, and 1.5mL of bacterial suspension (1X 10/well) was added9CFU/mL) and 1.5mL of a cell culture medium (containing 10% PBS and 1% penicilin-streptomycin) were mixed, and cultured in a constant temperature incubator (37 ℃ C., 5% CO)2) After 4 hours, the cells were washed twice with sterile PBS, fixed with methanol, gram stained, and counted for viable count under a microscope.
The adhesion of the strain to Caco-2 cells was detected by adhesion experiments, and the results are shown in FIG. 2, wherein L.paracasei ET-22 has strong adhesion to Caco-2 cells.
Example 3: intestinal flora regulating effect
This example is intended to demonstrate the effect of lactobacillus paracasei according to the invention on intestinal regulation.
36 healthy SPF-grade BABL/c mice were taken, weighing 18-22g (supplied by Beijing Huafukang Biotech, Inc.). After 3 days of acclimatization, they were randomly divided into 3 groups of 12 individuals, i.e., placebo group, sample group. And (3) separately intragastrically irrigating sterile water (intragastrically volume is 0.2mL/10g) dissolved with ET-22 bacteria powder for each group of animals, and intragastrically irrigating sterile water with the same volume for a blank control group. The preparation is administered 1 time per day by continuous feeding or intragastric administration for 14 days.And (3) gavage metering: 1.3X 107CFU/ml (2X 10 according to human body demand)9CFU/day, conversion of human and mouse conversion factor 0.0026). Taking sterilized centrifuge tubes, numbering, adaptively feeding, collecting 2-3 mice feces of about 100mg per mouse under sterile condition, and transferring to a sterile operating room under low temperature condition for detecting flora. At the end of the experiment, the mouse feces were collected again. The mice are numbered in groups by picric acid, weighed respectively on the 8 th day and the 14 th day of the administration of the test substance, the gavage amount of the mice is calculated, and the mice are weighed 1 time at the end of the experiment. And (3) counting colonies: preparing selective culture medium according to the strain to be identified, sterilizing the strain to be detected and corresponding culture medium as shown in Table 3, shaking up, cooling to 45-50 ℃, and pouring into a flat plate for later use.
TABLE 1 strains to be tested and corresponding selective media
The collected mouse feces are placed in a sterilization tube filled with 0.5mL of physiological saline to prepare bacterial suspension, and the suspension is shaken for 1min before use. Sucking 0.1mL of the bacterial suspension by using a 0.1mL micropipettor, slowly injecting the bacterial suspension into 0.9mL of sterilized physiological saline, and shaking or repeatedly blowing to uniformly mix the bacterial suspension to prepare a mixture with the weight ratio of 1: 10. Another 0.1mL micropipette tip is taken, and according to the method, the 10-fold gradient dilution is carried out until the 10-fold gradient dilution is reached-7g/ml. Two consecutive suitable dilutions were selected according to the viable count of the species to be identified, 10 μ L of bacterial suspension was aspirated using a 10 μ L micropipette for each dilution, surface coated on selective agar plates, and cultured under the culture conditions shown in table 2. The colony counting method is carried out by referring to GB 4789.2-2010 food safety national standard food microbiology test colony total number determination.
TABLE 2 culture medium for testing intestinal flora and identification method
Data statistics were performed using SPSS 17.0. The changes of bifidobacteria, lactic acid bacteria and enterobacteria before and after the experiment were compared.
The results of the weight change of the animals during the experiment are shown in table 3. In the experimental period, the animal characteristics are normal, no adverse reaction occurs after the test substance is given, and the body weights of two groups of animals do not have obvious difference in the experimental period. As can be seen from tables 4-6, Lactobacillus paracasei ET-22 can significantly promote the growth of bifidobacteria and Lactobacillus, and has no significant effect on enterobacteria. Compared with the strains before and after self-group intervention, the ET-22 has obviously higher amplification than the known strain Lactobacillus casei LC-01(DSM 19465) and has excellent effect.
Thus, lactobacillus paracasei ET-22 has efficacy in modulating intestinal flora in this study (fig. 3).
TABLE 3 weight changes in animals
TABLE 4 Change of Bifidobacterium longum in intestinal tract of animals before and after the test (LgCFU/g)
The results show that the amplification of the ET-22 group self-intervention prognosis is obviously improved compared with the amplification of the known strain LC-01: ET-22 increased by 1.24 orders of magnitude and LC-01 increased only by 0.76 orders of magnitude (FIG. 4). ET-22 is lactobacillus, but can increase the bifidobacterium by more than an order of magnitude remarkably, and the increase amount is higher than LC-01, which shows that the adjusting ability of the ET-22 for increasing intestinal tract bacteria is extremely strong.
TABLE 5 Change of intestinal Lactobacillus in animals before and after the test (LgCFU/g)
The results show that the increase of the ET-22 group self-intervention prognosis is higher than that of the known strain LC-01: ET-22 increased by 0.77 orders of magnitude and LC-01 increased by 0.61 orders of magnitude (FIG. 4).
TABLE 6 Change of Enterobacter enterobacter in animal intestinal before and after the test (LgCFU/g)
Example 4: intestinal flora regulating effect (sequencing results)
This example is intended to demonstrate the efficacy of the Lactobacillus paracasei of the present invention in terms of gut modulation, the principle and procedure of which are described in "test and evaluation of health food-criteria for functional assessment of gut flora modulation". The difference from example 3 is that the 16S rDNA sequencing results were used in this experiment.
Experimental samples: the bacterial powder sample (the number of viable bacteria is shown in the packaging specification) is provided by the company of the limited responsibility of the technical research institute of the inner Mongolia dairy industry.
Experimental materials: 182 healthy adult BABL/c mice, 6 weeks old, weigh about 18-20 g.
Experimental procedure
1. Sample preparation
Live bacteria sample (ET-22): according to the specification of the sample, 1g of viable bacteria sample is weighed respectively, and the viable bacteria sample is suspended to 40ml by using PBS solution, namely the viable bacteria concentration is 2.5x109CFU/ml。
High dose group: calculated according to the gavage amount of 0.2ml/10g of the mice, the gavage amount of 20g of the mice is 0.4ml, and the gavage dose of 10 high-dose group mice is9CFU/20g。
The medium dose group: respectively adding 5ml of the high-dose suspension into PBS to reach 50ml of constant volume, calculating according to the gavage amount of 0.2ml/10g of mice, the gavage amount of 20g of mice is 0.4ml, and the gavage amount of 10 medium-dose group mice is 108CFU/20g。
Low dose group: adding PBS into 5ml of the medium dose group suspension respectively to reach 50ml, and keeping the volume according to 0.2ml/1 of the mouseCalculated by the gavage amount of 0g, the gavage amount of 20g of mice is 0.4ml, and the gavage dose of the low-dose group mice is 107CFU/20g。
2. Experiment for regulating intestinal flora
(1) The BABL/c mice of 6 weeks old are raised in a clean animal room with temperature of 22 ℃, humidity of 10-60%, 12-hour illumination of alternating light and shade, and fed with standard feed and freely drinking water.
(2) After 5 days of acclimatization, 182 mice were randomly assigned to 13 groups of 14 mice each, and the groups are shown in Table 7.
TABLE 7 Experimental groups for regulating intestinal flora
(3) Before beginning the gavage, the feces of each mouse were collected under sterile conditions, labeled, stored at-20 ℃ and tested for intestinal flora.
(4) Experimental groups were each administered with a intragastric dose of 0.2ml/10g, control groups were administered with PBS for 1-14 days, and experimental groups were each administered with a corresponding dose of test substance intragavage according to table 7. Mice were weighed once a week and gavage was adjusted according to body weight.
(5) Collecting feces of each mouse under sterile condition after 14 days, marking, storing at-20 deg.C, and detecting intestinal flora.
3 variation in intestinal microbial diversity in mice
The microbial diversity is based on an Illumina HiSeq sequencing platform, and a small fragment library is constructed for sequencing by using a double-ended sequencing (Paired-End) method. Species composition of the sample can be revealed by performing splicing filtration on Reads, OTUs (operational Taxonomic units) clustering, and performing species annotation and abundance analysis; and clustering the optimized sequences, dividing the OTUs, and obtaining species classification according to the sequence composition of the OTUs. Based on the results of the OTU analysis, the samples were subjected to taxonomic analysis at each taxonomic level to obtain the colony structure of each sample at the genus level.
The species diversity in a single sample is researched through Alpha diversity analysis, Ace, Chao1, Shannon and Simpson indexes of each sample under the 97% similarity level are counted, and a sample dilution curve and a grade abundance curve are drawn; further performing Alpha Diversity analysis (Alpha Diversity), Beta Diversity analysis (Beta Diversity), and significant species difference analysis, among others, can mine the differences between samples. And (3) comparing the intestinal flora alpha diversity indexes: chao1 and ACE refer to measures of species abundance, i.e., number of species; the Shannon and Simpson indices are used to measure species diversity, influenced by species abundance and species uniformity in a population of samples. The larger the homogeneity of each species in the population, the larger the diversity of the population, and the larger the Shannon index value and the smaller the Simpson index value, the higher the diversity of species in the sample. At the genus level, ET-22 dried prognosis significantly increased the relative abundance in the mouse gut compared to the control group. The indexes of OTU, ACE and Chao1 of the low-dose group are obviously increased, and the ET-22 low dose is shown to be capable of obviously increasing the intestinal microbial diversity.
Yellow (underlined) indicates: statistically significant differences (p < 0.05) were observed compared to the control group.
At the level of pathopoiesia, vibrio desulfovis (Desulfovibrio) in the intestinal tracts of both the ET-22 low dose and medium dose groups of mice was significantly reduced compared to the control group; the number of Helicobacter pylori (Helicobacter pylori) in the middle dose group and the high dose group is obviously reduced; the high Escherichia-Shigella (Escherichia-Shigella) was significantly reduced in both the low and medium dose groups. ET-22 can significantly increase the relative abundance of Lactobacillus in the intestinal tract of mice, and the ET-22 low dose group has the most significant increase degree on Lactobacillus.
Therefore, different doses of ET-22 are supplemented to regulate the balance of intestinal flora and inhibit the number of harmful bacteria and even pathogenic bacteria, thereby achieving the potential health effect.
Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture, processing conditions, and so forth used in the specification (including the claims) are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. The term "at least" preceding a series of elements is to be understood as referring to each element in the series, unless otherwise indicated. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The appended claims are intended to cover such equivalents.
It will be apparent to those skilled in the art that many modifications and variations of the present invention can be made without departing from its spirit and scope. The specific embodiments described herein are provided by way of example only and are not meant to be limiting in any way. The true scope and spirit of the invention is indicated by the appended claims, and the specification and examples are exemplary only.
Claims (10)
1. Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 12 months and 18 days in 2017, and the preservation number is CGMCC No. 15077.
2. Use of the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain of claim 1 for modulating the flora balance in a subject.
3. Use of the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain of claim 1 for promoting the growth of bifidobacteria and/or lactic acid bacteria.
4. A method for modulating the microflora balance of a subject, the method comprising administering the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain of claim 1 to the subject to modulate the microflora balance thereof.
5. The method of claim 4, wherein the strain is administered to the subject by a gastrointestinal route.
6. The method of claim 4 or 5, wherein the subject is a mammal.
7. A method of promoting the growth of bifido-and/or lactic-bacteria, comprising administering the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain of claim 1 to the subject to promote the growth of bifido-and/or lactic-bacteria.
8. The method of claim 7, wherein the growth of enterobacteria is not affected.
9. Use of the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain of claim 1 for the preparation of a composition for promoting the growth of bifido-and/or lactic-bacteria and/or for modulating the flora balance of a subject.
10. A method of preparing a composition for promoting the growth of bifido-and/or lactic-bacteria and/or modulating the microbiota balance in a subject, the method comprising adding the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain of claim 1 to the composition.
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| US17/259,137 US11274275B2 (en) | 2018-09-30 | 2019-09-23 | Lactobacillus paracasei ET-22 and use thereof |
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