Lactic acid bacteria, composition for improving the composition of intestinal microbiota, and products and uses of said lactic acid bacteria

Abstract

To provide a lactic acid bacterial strain and a composition for improving a gut microbiota composition, and a product and an application of the lactic acid bacterial strain.SOLUTION: The primary object of the present invention is to provide a lactic acid bacterial strain and a composition for improving a gut microbiota composition, where the composition comprises the lactic acid bacterial strain and / or extracellular vesicles secreted by the lactic acid bacterial strain. Another objective of the present invention is to provide an application of the lactic acid bacterial strain to prepare the composition for improving the gut microbiota composition, and a product of the lactic acid bacterial strain. The composition of the present invention has the capability to influence the growth of Firmicutes and Bacteroidetes, thereby improving the gut microbiota composition.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to the technical field of improving the composition of intestinal microbiota species, and particularly to the technical field of compositions for improving the composition of intestinal microbiota species including novel lactic acid bacteria and / or extracellular vesicles secreted therefrom.

Background Art

[0002] The composition and changes of intestinal microbiota are related to human health. A complex nervous system is distributed in the intestinal tract, and information is transmitted and influenced between the intestinal tract and the brain through the gut-brain axis. Intestinal microbiota produce metabolites and other neurotransmitters, which affect intestinal cells and immune cells, thus affecting the host's gastrointestinal function and ultimately behavior and emotional expression.

[0003] The gut microbiota encompasses various symbiotic microorganisms, which are mainly composed of bacteria, most of which are obligate anaerobes, and are mainly classified into four phyla: Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria. In addition, research has found that the composition ratio of Bacteroidetes and Firmicutes varies according to diseases. In the gut microbiota of healthy humans, a large amount of Bacteroidetes is contained, while in the gut microbiota of patients suffering from diseases, a large amount of Firmicutes is contained. In the intestinal tract of obese patients, the microbiota of Bacteroidetes has decreased significantly, including the decrease of various intestinal probiotics. In the intestinal tract of obese patients, Bacteroides thetaiotaomicron has a relatively low quantity, and the relevance between metabolites related to obesity and hormonal changes has been suggested. To identify key microorganisms related to diseases and use them as targeted therapies, a high degree of feasibility is expected. At the same time, gut microbiota plays an important role in maintaining the stable state of the intestinal tract and the integrity of the mucosal immune system, and it is considered to help maintain the health of the intestinal tract and regulate the barrier permeability of the intestinal tract. Moreover, evidence has been confirmed that the reduction and change of the diversity of the microbial flora are already related to many diseases and dysfunctions, such as obesity, inflammatory bowel disease (IBD), autism, and neurodegenerative diseases. Specific gut microorganisms may vary accordingly as the disease progresses to different degrees (see Non-Patent Documents 1 to 4).

[0004] These days, the regulation and control of the gut microbiota are generally achieved by directly taking probiotics, which can actively increase beneficial bacteria and suppress harmful bacteria through the formation of probiotic colonies. Even now, in some cases, normal microbiota are transplanted into the host's digestive system by means of faecal microbiota transplant (FMT).

[0005] However, there is a common problem with probiotics that they cannot form colonies, which limits their impact on the microbiota. In addition, FMT treatment still has many drawbacks, and there is still room for discussion regarding its safety. At present, efforts to transplant faecal microbiota mainly include those via the upper digestive tract, the middle digestive tract, and the lower digestive tract. Among them, the transplantation of faecal microbiota via the upper digestive tract is carried out by oral administration of faecal microbiota. However, its drawback is that microorganisms are easily affected by bile salts. Among them, in the transplantation of faecal microbiota via the middle digestive tract, it can be operated by means such as nasal tubes or percutaneous endoscopic gastrostomy tubes. However, its drawback is that in case of inappropriate operation, there is a risk of aspiration and reflux, and microorganisms are also affected by bile salts. Among them, in the transplantation of faecal microbiota via the lower digestive tract, the operation is carried out by means such as colonoscopy, enema, or endoscopic transintestinal tube placement. Its drawback is that the faecal microbiota only covers the sigmoid colon area and it is necessary to perform faecal microbiota transplantation multiple times. In the past, FMT was recognized as one of the safe treatment methods. However, according to the latest clinical reports and cases from the well-informed US Food and Drug Administration, although a patient received faecal microbiota from a healthy provider, the Escherichia coli in the faecal microbiota already had multiple drug resistance genes, and thus the risk of causing death to the FMT recipient was reported. In addition, because the current technology still cannot effectively evaluate and determine the drug resistance genes in the microbiota of healthy bacterial donors for FMT, FMT still has potential risks.

[0006] In view of the limitations and drawbacks of the current methods for regulating and controlling the gastrointestinal microbiota, and considering that the composition and changes of the gut microbiota are closely related to human health and numerous diseases, the inventors of the present application earnestly hope that the use of spherical lipid bilayers on the nanoscale and extracellular vesicles (EVs) that encapsulate component structures such as nucleic acids and proteins can be employed. By interacting alternately with neighboring bacteria, it becomes possible to safely and effectively regulate and control the composition and changes of the gut microbiota. In view of this, as a result of intensive research and invention by the inventors of the present application, they have finally completed the research and development of a novel lactic acid bacterium of the present invention, a composition for improving the composition of gut microbiota species, and products and uses of the above lactic acid bacterium, and can achieve the efficacy of effectively improving intestinal health by extracellular vesicles secreted from trace amounts of microorganisms.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0008] The main object of the present invention is to provide a lactic acid bacterium and a composition for improving the composition of intestinal microbiota, wherein the composition includes the lactic acid bacterium and / or extracellular vesicles secreted therefrom. In addition, the composition for improving the composition of intestinal microbiota can be provided for oral ingestion and use.

Means for Solving the Problems

[0009] To achieve the above object, the present invention proposes a novel lactic acid bacterium, which is Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under the accession number NCIMB 44102.

[0010] Furthermore, the present invention provides a composition for improving the composition of intestinal microbiota, which includes an effective amount of extracellular vesicles, wherein the extracellular vesicles can be secreted from lactic acid bacteria, and the lactic acid bacteria are Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under the accession number NCIMB 44102.

[0011] The above effective amount in the composition of the present invention is the extracellular vesicles with a particle number concentration of at least 10 8 particles / ml, and such extracellular vesicles are any one selected from the group consisting of exosomes, microvesicles, ectosomes and apoptotic bodies.

[0012] In addition, the above composition of the present invention has the ability to improve the composition of intestinal microbiota that can affect the clinical course of Alzheimer's disease (AD).

[0013] Furthermore, such extracellular vesicles in the composition of the present invention as described above can affect the growth of the phylum Firmicutes and / or the phylum Bacteroidetes.

[0014] At the same time, such extracellular vesicles can also affect the growth of at least one bacterial class, and the bacterial class is at least one selected from the group consisting of the class Bacteroidia, the class Clostridia, and the class Bacilli.

[0015] In addition, such extracellular vesicles can affect the growth of at least one bacterial family, and the bacterial family is at least one selected from the group consisting of the family Lactobacillaceae, the family Muribaculaceae, the family Lachnospiraceae, the family Clostridiaceae, the family Desulfovibrionaceae, the family Erysipelotrichaceae, the family Eggerthellaceae, the family Akkermansiaceae, the family Ruminococcaceae, and the family Eubacteriaceae. In addition, such extracellular vesicles can also affect the growth of the genus Muribaculum and / or the genus Lachnospira.

[0016] Furthermore, in the composition of the present invention as described above, such extracellular vesicles can promote the growth of Lactobacillus acidophilus.

[0017] In one embodiment, the present invention provides a nutritional supplement, and the nutritional supplement includes Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under the accession number NCIMB 44102.

[0018] In one embodiment, the present invention provides a food product, and the food product includes Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under the accession number NCIMB 44102.

[0019] In one embodiment, the present invention provides a dietary supplement, and the dietary supplement includes Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under the accession number NCIMB 44102.

[0020] In one embodiment, the present invention provides a food additive, and the food additive includes Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under the accession number NCIMB 44102.

[0021] In one embodiment, the present invention provides a pharmaceutical composition, and the pharmaceutical composition includes Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under the accession number NCIMB 44102.

[0022] In one embodiment, the present invention provides a feed, which includes Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK under the accession number NCIMB 44102.

[0023] Another object of the present invention is to provide the use of lactic acid bacteria for preparing a composition for improving the intestinal microbial flora composition, wherein the lactic acid bacteria is Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK under the accession number NCIMB 44102. In addition, the present invention also provides a novel lactic acid bacteria and / or its secreted extracellular vesicles for use in improving the intestinal microbial flora composition, wherein the lactic acid bacteria is Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK under the accession number NCIMB 44102.

Advantages of the Invention

[0024] Yet another object of the present invention is to provide the application of a composition for improving the intestinal microbial flora composition. Such an application includes product forms that are easy to use in daily life, such as foods, beverages, health foods, additives, and medical compositions. It is provided to the general public for taking, so as to maintain intestinal health on a daily basis.

Brief Description of the Drawings

[0025]

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Mode for Carrying Out the Invention

[0026] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention pertains. Hereinafter, the details of the present invention will be explicitly described using examples, but these examples are merely illustrative and not restrictive, and the present invention is not limited to these examples. Unless otherwise specified, as materials used in the present invention, all commercially available materials that are easily obtainable are preferred, and the available routes shown below are merely examples.

[0027] The present invention provides a novel lactic acid bacterium and a composition for improving the composition of intestinal microbiota, wherein the composition includes the lactic acid bacterium and / or extracellular vesicles secreted therefrom, and the lactic acid bacterium is Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK under the accession number NCIMB 44102. The inventor of the present application conducted experiments using a transgenic mouse animal model of Alzheimer's disease (knock-in of the Amyloid beta precursor protein gene: APP gene), and found that the extracellular vesicles secreted from the Pediococcus acidilactici have novel uses and functions for improving the composition of intestinal microbiota.

[0028] In addition, the Pediococcus acidilactici of the present invention is a novel lactic acid bacterium isolate obtained by the inventor of the present application through screening from the intestinal tract of chickens in the laboratory.

[0029] The extracellular vesicles secreted from Pediococcus acidilactici of the present invention were found by analyzing a mouse animal model to be able to effectively improve the richness of the intestinal microbiota in mice, that is, the extracellular vesicles can effectively improve the intestinal microbiota species composition of transgenic mice with Alzheimer's disease, and can restore the intestinal microbiota species composition of the transgenic mice to a microbiota species composition similar to that under healthy conditions.

[0030] The aforementioned lactic acid bacteria isolates include progeny or mutant strains of their subcultures, but still have such bacterial species characteristics, genomic or uses (used to improve the intestinal microbiota species composition) similar to those of the present invention.

[0031] The compositions according to the present text may include, but are not limited to, application forms of products applicable to the present invention such as nutritional supplements, food products, dietary supplements, food additives, veterinary pharmaceutical compositions and human pharmaceutical compositions, feeds, beverages, health foods, animal drinking water additives, animal feed additives, beverage additives, etc.

[0032] The term "improve" means that a composition containing Pediococcus acidilactici and / or its extracellular vesicles can effectively improve the intestinal microbiota species composition compared to a composition without the use of Pediococcus acidilactici and / or its extracellular vesicles of the present invention.

[0033] The term "effective amount" means an effective amount of the active ingredient of one or more intestinal microbiota species that can effectively improve, treat or restore a disease, and is also referred to as a "therapeutically effective amount" or an "improvement effective amount". Thus, the term "pharmaceutically acceptable" means that the substance or composition needs to be compatible with the other components of the formulation and is harmless to the patient.

[0034] The composition of the present invention refers to those in which the above-mentioned Pediococcus acidilactici and / or its extracellular vesicles can be prepared into a dosage form applicable to the composition of the present invention together with a pharmaceutically or food-acceptable carrier by using techniques well-known to those skilled in this art. Among them, the dosage form includes, but is not limited to, solution, emulsion, suspension, powder, tablet, pill, lozenge, troche, capsule and other dosage forms similar or applicable to the present invention.

[0035] One or more solubilizers, buffers, preservatives, colorants, fragrances, flavoring agents, excipients, etc. commonly used in the pharmaceutical field can be appropriately added to the above composition as needed.

[0036] In another preferred embodiment, the composition provided by the present invention can be further added to an edible material and can be prepared as a food product or a health product. Among them, the edible material includes, but is not limited to, water, fluid milk product, milk, concentrated milk, fermented milk such as yogurt, frozen yogurt, sour milk, lactic fermenting beverage, milk powder, ice cream, cream cheese, dry cheese, soybean milk, fermented soybean milk, fruit and vegetable juice, juice, sports drink, confectionery, jelly, baby food, health food, animal feed, herbal medicine, dietary supplement, etc.

[0037] Furthermore, the present invention also provides a method for improving the intestinal microbiota composition, which involves subjecting an effective amount of the aforementioned composition to patients suffering from intestinal diseases or patients with Alzheimer's disease for use in improving their intestinal microbiota composition.

[0038] In addition, the present invention also provides a method or use for preparing a composition for improving the intestinal microbiota composition using the aforementioned Pediococcus acidilactici and / or its secreted extracellular vesicles.

[0039] As a composition provided by the present invention and a method used for improving its intestinal microbial flora composition, the administration route is not particularly limited as long as it can be appropriately adjusted according to requirements, and suitable administration routes include oral administration suitable for appropriate dosage forms and the like.

[0040] <Origin of the strain> The Lactobacillus acidophilus used in the following examples of the present invention is the standard strain Lactobacillus acidophilus BCRC 14079 purchased from the Food Industry Research and Development Institute (Bioresource Collection and Research Center: BCRC) in Hsinchu, Taiwan. The Lachnospiraceae sp. and Ruminococcaceae sp. used in the present invention are the standard strains Lachnospiraceae sp. TSD-26 and Ruminococcaceae sp. TSD-27 purchased from the American Type Culture Collection (ATCC), respectively. The novel lactic acid bacteria of the present invention are Pediococcus acidilactici selected from the intestinal tract of chickens in the laboratory, and have been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK under the accession number NCIMB 44102.

[0041] <Animal breed and strain> The animal breed and strain used in the following examples of the present invention is APP of the AD model using knock-in of the amyloid-β precursor protein gene. NL-G-F / NL-G-FA transgenic mouse (TG) into which the Swedish-type mutant gene (KM670 / 671NL) and the Iberian-type mutant gene (I716F) have been introduced into its Aβ sequence to increase the amount of Aβ produced and improve the Aβ42 / 40 ratio. In the said transgenic mouse, memory impairment first appears at 6 months of age. In the control group, C57BL / 6 (non-transgenic mouse: non-TG) mice were used as a control (Reference 1: Nilsson et al. ACS Chemical Neuroscience 2014, 5, 499 - 502).

[0042] <Preparation of extracellular vesicles> First, Pediococcus acidilactici of the present invention was cultured in MRS medium at 37°C for 24 hours, and the cells were removed by centrifugation at 10,000×g for 30 minutes. After filtering the supernatant, the obtained filtrate was subjected to ultra-high speed centrifugation at 247,537×g at 4°C. After removing the supernatant, the precipitate was then washed with Dulbecco's phosphate buffered saline (DPBS). After filtering the obtained wash solution through a 0.22 μm sterilizing membrane, it was again subjected to ultra-high speed centrifugation at 247,537×g at 4°C. After removing the supernatant, the precipitate was redissolved in DPBS, and then extracellular vesicles were obtained and stored at -80°C (Reference 2: Choi et al. Experimental Neurobiology 2019, 28, 158 - 171).

Example

[0043] <Example 1: Morphological characteristics of extracellular vesicles secreted from Pediococcus acidilactici of the present invention> In this example, the extracellular vesicles of Pediococcus acidilactici of the present invention are purified by centrifugation using an ultra-high-speed centrifugation method, and the morphological characteristics of the extracellular vesicles (extracellular vesicles derived from lactic acid bacteria, hereinafter abbreviated as LAB-EV) obtained by purification are observed through a transmission microscope. As shown in Fig. 1A, the extracellular vesicles have a spherical lipid bilayer structure on the order of nanometers. In addition, after appropriate dilution of the LAB-EV with Dulbecco's phosphate buffered saline (DPBS), further analysis can be performed using a nanoparticle tracking analyzer. When the diluted liquid is irradiated using a laser light source and the Brownian motion of scattered light nanoparticles is observed through a microscope, the average particle diameter and the number of particles (quantification) of the LAB-EV sample are calculated. Fig. 1B shows the average particle size distribution of the LAB-EV, and the average particle diameter of the LAB-EV is about 125 nanometers (nm).

Example

[0044] <Example 2: Relative abundance of intestinal microbial species in transgenic mice after oral administration of extracellular vesicles of Pediococcus acidilactici of the present invention> In this example, the daily dose was administered to mouse extracellular vesicles (LAB-EV) by oral administration once a day. After administering to mouse LAB-EV for 4 weeks (weeks), the mice were sacrificed, and then the relative abundance of intestinal microbial species in the mice was analyzed.

[0045] Figure 2 shows the relative abundances of intestinal microbial species in mice after oral administration of extracellular vesicles (LAB-EV) of Pediococcus acidilactici of the present invention. Among them, non-TG Veh represents oral administration of a solvent to non-transgenic mice (n = 6), TG Veh represents oral administration of a solvent to transgenic mice (knock-in of the amyloid beta precursor protein gene (APP gene)) (n = 6), and TG LAB-EV represents oral administration of LAB-EV to transgenic mice (knock-in of the APP gene) (n = 6). Here, Veh (Vehicle) is DPBS, which is the solvent for LAB-EV.

[0046] Figure 2 shows the results of the top 10 bacterial families in terms of relative abundance in the intestinal microbiota of mice. Those other than the top 10 in the ranking are classified as others. Among them, the top 10 bacterial families in the ranking include Lactobacillaceae, Muribaculaceae, Lachnospiraceae, Clostridiaceae, Desulfovibrionaceae, Erysipelotrichaceae, Eggerthellaceae, Akkermansiaceae, Ruminococcaceae, and Eubacteriaceae. In addition, as can be seen from the results of Figure 2, there are differences among the microorganisms at the family level in the intestinal tracts of non-TG Veh mice and TG Veh mice, and the intestinal microbiota composition of TG LAB-EV mice is relatively similar to that of non-TG Veh mice. That is, it can be seen that the intestinal microbiota species composition of transgenic mice is improved to a certain extent after the administration of LAB-EV. This also indicates that the intestinal microbiota species composition of transgenic mice can be restored to be similar to that of the intestinal bacteria of normal mice. Therefore, LAB-EV can effectively improve the intestinal microbiota species of transgenic mice and restore the intestinal microbiota species composition of mice to a composition similar to that under healthy conditions.

Example

[0047] <Example 3: Co-culture of extracellular vesicles of Pediococcus acidilactici of the present invention and Lactobacillus acidophilus> In this example, in order to conduct in vitro tests, LAB-EV was co-cultured with Lactobacillus acidophilus to analyze whether the LAB-EV of the present invention has the potential ability to promote the growth of Lactobacillus acidophilus. After co-culturing LAB-EV and Lactobacillus acidophilus, the absorbance values at a wavelength of 600 nm were measured at different time points, and finally, line graphs and bar graphs as shown in FIGS. 3A and 3B were obtained respectively. Among them, the data repeated three times were represented by the mean value (Mean) ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance, and then multiple group comparison tests using Tukey's test were performed. Significant differences are marked with different English alphabets.

[0048] As can be seen from the results shown in FIGS. 3A and 3B, it was found that LAB-EV promotes the growth of Lactobacillus acidophilus, and at relatively high concentrations of LAB-EV, the effect of promoting the growth of Lactobacillus acidophilus can be more preferably exerted. That is, the effect of promoting the growth of Lactobacillus acidophilus by the LAB-EV was confirmed to be an effect dependent on the dosage. For example, when co-culturing LAB-EV with a particle number concentration of 10 10 particles / mL and Lactobacillus acidophilus, the growth of Lactobacillus acidophilus can be significantly increased, and when co-culturing LAB-EV with a particle number concentration of 10 8 particles / mL and Lactobacillus acidophilus, the effect of promoting the growth of Lactobacillus acidophilus is second only to that.

Example

[0049] <Example 4: Co-culture of extracellular vesicles of Pediococcus acidilactici of the present invention and intestinal bacteria> In this example, LAB-EV was co-cultured with Lachnospiraceae sp. and Ruminococcaceae sp. of intestinal bacteria respectively, and then the absorbance values at a wavelength of 600 nm were measured at different time points. Finally, line graphs as shown in FIGS. 4A and 4C were obtained respectively. The number of bacteria (Log CFU / mL) was determined using the plate counting method at different time points, and finally bar graphs as shown in FIGS. 4B and 4D were obtained respectively. Among them, the data repeated three times were expressed as the mean value (Mean) ± standard error (SEM), and significant differences (p<0.05) were denoted by different English alphabets.

[0050] As can be seen from the results shown in FIGS. 4A, 4B, 4C, and 4D, LAB-EV has no effect on the growth of both Lachnospira and Ruminococcus of intestinal bacteria, and it was found that neither relatively high-concentration LAB-EV nor relatively low-concentration LAB-EV has any effect on the growth of both Lachnospira and Ruminococcus of intestinal bacteria.

Example

[0051] <Example 5: Fluorescent Staining by the Reaction between LAB-EV and Mouse Fecal Microorganisms> To clarify whether intestinal microorganisms ingest LAB-EV and to examine the types of intestinal bacteria that can ingest LAB-EV, in this example, while LAB-EV at 10 8 particles / ml was stained (green), mouse fecal bacteria were stained with DRAQ5 (red), and then added to a microplate. Among them, CFSE has an excitation wavelength of 492 nm and an emission wavelength of 517 nm, and DRAQ5 has an excitation wavelength of 647 nm and an emission wavelength of 665 nm. And CFSE is a carboxyfluorescein succinimidyl ester staining reagent, and DRAQ5 is purchased from Abcam (Bristol, UK).

[0052] Referring to the results shown in FIG. 5, among them, the red fluorescence labels the bacterial cells in the feces of the DRAQ5-stained mice, and the cells have a rod-shaped distribution or a spherical distribution, and the green fluorescence labels the CFSE-stained LAB-EV, and the overlapping region of the two is labeled in yellow. As shown by the test results, LAB-EV is ingested into the bacterial cells by the intestinal microorganisms, and the intestinal microorganisms are composed of various bacteria. There may be cases where the green fluorescence does not overlap at some cell positions, indicating that not all types of bacteria necessarily ingest LAB-EV, and only specific groups of microorganisms ingest extracellular vesicles.

Example

[0053] <Example 6: Possibility of LAB-EV being ingested by intestinal bacteria in mice> In this example, the intestinal microorganisms of mice without LAB-EV addition (FIG. 6A) and the intestinal microorganisms of mice with LAB-EV addition (FIG. 6B) were respectively fed into a flow cell sorter for analysis. Among them, the intestinal microorganisms of mice with LAB-EV addition refer to staining 10 10 particles of LAB-EV with CFSE and reacting with the intestinal bacteria (n = 6) of mice in an anaerobic environment at 37°C. CFSE is the Carboxyfluorescein succinimidyl ester staining reagent.

[0054] Referring to the results shown in FIG. 6A, after feeding the intestinal microorganisms of mice without LAB-EV addition into a flow cell sorter for analysis, the bacterial groups were selected and analyzed for this specific region. The selection range includes cells with sizes of 1μm, 2μm, and 10μm, and it was found that the majority of the intestinal microorganisms in the quadrant diagram entered the R8 quadrant, and still accounted for about 98.58% of the total bacterial group.

[0055] Referring to the results shown in Fig. 6B, among them, after sending the intestinal microorganisms of the mice supplemented with LAB-EV into a flow cell sorter for analysis, the range of existence of the microbial community was selected, which also included cells of 1 μm, 2 μm and 10 μm in size. And in the quadrant diagram, most of the plurality of cells fall into R8 quadrant. In addition, it was found that 32.31% of the cells fell into the R9 quadrant.

[0056] From the above results, it was found that the cell sizes in the selected range were 1 μm, 2 μm and 10 μm, and these lengths corresponded to the sizes of bacteria. By analyzing the intestinal microorganisms of the mice without supplementation of LAB-EV as a control group, it became possible to know the intervening range of the intestinal microbial community of the mice. Furthermore, a fluorescence reaction was observed in the intestinal microorganisms supplemented with fluorescently stained LAB-EV, indicating that some microorganisms in the mouse intestinal tract ingested LAB-EV, and among the intestinal microorganisms of the mice, about 32.31% of the bacteria ingested LAB-EV.

Example

[0057] <Example 7: Elucidation of the types of intestinal microorganisms in mice that ingest LAB-EV> In this example, the gut microbiota that ingested LAB-EV in Example 6 was extracted, fractionated, and subjected to nucleotide sequencing. Referring to the results shown in FIG. 7, the gut microbiota that reacted with LAB-EV, after nucleotide sequencing, was found to include a total of 1 kingdom, 9 phyla, 14 classes, 19 orders, 37 families, 74 genera, and 87 species among the multiple gut microbiota. Shown in FIG. 7, which is a bar graph showing relative abundance, is the proportion of bacteria in different taxonomic groups, all of which are eubacteria, and most of which are the phyla Firmicutes and Bacteroidetes, accounting for approximately 49% and 47% respectively. For the bacterial classes, the breakdown is 47% for the class Bacteroidia, 44% for the class Clostridia, and 4% for the class Bacilli. For the bacterial families, the most abundant is the family Lachnospiraceae, which accounts for 33%. For the bacterial genera and species, both 22% of the genus / species Muribaculum / Muribaculum sp. and 19% of the genus / species Lachnospira / Lachnospira sp. occupy the top two positions.

[0058] In FIG. 7, which is a bar graph showing relative abundance for easy comparison checking, it sequentially includes various microorganisms of each of the following classifications, that is, the kingdom Bacteria, the phyla Proteobacteria, Bacteroidetes, and Firmicutes, the classes Bacilli, Clostridia, and Bacteroidia, the orders Lactobacillales, Clostridiales, and Bacteroidales, the families Lactobacillaceae, Bacteroidaceae, Rikenellaceae, Ruminococcaceae, Prevotellaceae, and Lachnospiraceae, the genera Alloprevotella, Rikenella, Butyricicoccus, (ASF356), Ruminiclostridium, Alistipes, Lachnoclostridium, Roseburia, Lactobacillus, Bacteroides, Prevotella, Lachnospira, and Muribaculaceae, and the species alloprevotella sp., Rikenellaceae sp., Butyricicoccus sp., spore-forming Clostridium (ASF356 sp.), Ruminiclostridium sp., Lactobacillus sp., Alistipes sp., Lachnoclostridium sp., Roseburia sp., Bacteroides sp.) including Prevotella sp., Lachnospiraceae sp., and Muribaculaceae sp.

[0059] In addition, in the co-culture of LAB-EV and Lactobacillus acidophilus and the fluorescence staining test of LAB-EV and intestinal microorganisms in the above-mentioned multiple examples, LAB-EV was shown to be ingested by specific flora microorganisms. Also, in the flow cell sorter experiment, it was found that about 32.31% of intestinal microorganisms utilize LAB-EV. At the same time, in further sequencing, it was also found that the two genera of bacteria, Muribaculaceae and Lachnospiraceae, are the most abundant among the microorganisms that ingest LAB-EV, accounting for 22% and 19% respectively.

Example

[0060] <Example 8: Identification and discrimination of a novel Pediococcus acidilactici strain of the present invention> In this example, the complete genomic sequence of Pediococcus acidilactici of the present invention was analyzed using the platforms of the next-generation MiSeq (Illumina, Inc., USA) and the next-next-generation MinION (Oxford Nanopore Technologies, Inc., UK) sequencers, and differences were found by comparison with genomic databases (such as GenBank, Ensembl, BioCyc, and RefSeq). Twenty-seven genomes that completely assembled those of the same species as the Pediococcus acidilactici species registered in GenBank were analyzed for identity with the Pediococcus acidilactici of the present invention. As shown in Table 1 below, the average nucleotide identity (ANI) with the Pediococcus acidilactici of the present invention was calculated using FastANI v1.33. Thus, Figure 8 shows a phylogenetic tree calculated using Roary v 3.11.2 and FastTree 2.1 and drawn, which represents the genetic relationship among different strains of Pediococcus acidilactici. Pediococcus acidilactici PB22 is the strain most similar to the Pediococcus acidilactici of the present invention (ANI: 99.16%), and no Pediococcus acidilactici strain having a genome completely homologous to that of the Pediococcus acidilactici of the present invention was found.

[0061]

Table 1

[0062] Subsequently, in order to effectively identify and distinguish the DNA sequence of Pediococcus acidilactici of the present invention, this example designed primers that can identify genomic mutation regions to distinguish different strains of Pediococcus acidilactici. The multiple primers are summarized in Table 2 below. Table 3 shows the primer combination method and the amplified sequences of the target strains. After comparison with the RefSeq database (the database includes complete genome, scaffold sequences, and contig sequences), the amplified sequence of the primer combination CGPA01_1590-F / R for identifying Pediococcus acidilactici of the present invention has the same fragment only in the MA18 / 5M genome of Pediococcus acidilactici, while the amplified sequence of the primer combination MA18 / 5M_850-F / R has this fragment only in Pediococcus acidilactici MA18 / 5M, and moreover, this fragment does not exist in the genome of Pediococcus acidilactici of the present invention.

[0063]

Table 2

[0064]

Table 3

[0065] Continuously, polymerase chain reaction (PCR) is performed on the primer combinations in Table 2 above. Among them, the total reaction volume of PCR is 20 μl, containing 0.1 ng of strain DNA, 500 nM of primer, and 10 μl of Taq DNA Polymerase Master Mix RED (Ampliqon A / S, DK). As the reaction conditions for PCR, after heating at 95°C for 2 minutes (2 min), it is heated again at 95°C for 20 seconds (20 sec), combined with 30 seconds (30 sec) at 60°C, and then extended at 72°C for 30 seconds (30 sec), and this condition is repeated 30 times. Finally, it is extended at 72°C for 5 minutes (5 min). After the above PCR reaction is completed, the PCR product can be taken out, and using a 1.8% agarose gel, electrophoresis is performed in a 1XTAE buffer solution at a voltage of 100 V for 40 minutes. Subsequently, the electrophoresis gel sheet is stained with SYBR Safe staining reagent (Thermo Fisher Scientific, USA), and the stained electrophoresis gel sheet is placed in a light box with blue light (470 nm) for observation, and an image is taken and saved as a file.

[0066] Table 4 shows the strains for confirming the unique sequences of the Pediococcus acidilactici strains of the present invention. Except for the Pediococcus acidilactici and MA18 / 5M of the present invention, the strains listed in Table 4 were all purchased from the Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, and moreover, the plurality of strains all belong to the genus Pediococcus, including Pediococcus pentosaceus, Pediococcus parvulus, and different strains of Pediococcus acidilactici. MA18 / 5M (also known as R1001) of Pediococcus acidilactici was obtained by separating from the product of Jarro-Dophilus EPS (Jarrow Formulas, Inc.).

[0067]

Table 4

[0068] Referring to FIG. 9, it is an electrophoresis diagram after the PCR reaction of the CGPA01_1590-F / R primer and the DNA of the Pediococcus genus strain. As shown in FIG. 9, only the Pediococcus acidilactici and MA18 / 5M of the present invention show a clear band at 115 bp. Referring to FIG. 10, it is an electrophoresis diagram after the PCR reaction of the DNA of the Pediococcus acidilactici strain and MA18 / 5M of the present invention obtained by amplification using the MA18 / 5M_850-F / R primer. As shown in FIG. 10, only MA18 / 5M of the Pediococcus acidilactici strain shows a clear band at 505 bp.

[0069] In summary, genomic analysis has revealed the differences between Pediococcus acidilactici of the present invention and Pediococcus acidilactici strains. Among them, Pediococcus acidilactici of the present invention has the highest identity with the PB22 genome. PCR and specific primers are used to distinguish Pediococcus acidilactici of the present invention from other publicly available Pediococcus strains. The primer combinations are CGPA01_1590-F / R and MA18 / 5M_850-F / R, respectively. CGPA01_1590-F / R amplifies only Pediococcus acidilactici of the present invention and MA18 / 5M, and does not amplify other Pediococcus strains. Therefore, it is necessary to use it in combination with the MA18 / 5M_850-F / R primer, which further distinguishes Pediococcus acidilactici of the present invention from MA18 / 5M. Only the amplification clear band of the CGPA01_1590-F / R primer appears in Pediococcus acidilactici of the present invention, and the amplification clear bands of the CGPA01_1590-F / R primer and the MA18 / 5M_850-F / R primer appear in MA18 / 5M of Pediococcus acidilactici. In addition, as can be seen from the above genomic analysis and PCR results, Pediococcus acidilactici (Pediococcus acidilactici) SWP-CGPA01 provided by the present invention was found to be a novel lactic acid bacteria isolate.

[0070] As described above, the novel lactic acid bacteria of the present invention, the composition for improving the intestinal microbiota composition, and the products and uses of the lactic acid bacteria have already been sufficiently and clearly described. It should be emphasized that the above detailed description specifically describes the feasible embodiments of the present invention, and the patent scope of the present invention is not limited to these embodiments. As long as it does not deviate from the technical spirit of the present invention, its equivalent implementations or modifications are still included within the scope of the claims of this application.

[0071] The Pediococcus acidilactici of the present invention was deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom on January 12, 2023 (date of deposit), and its deposit number is NCIMB 44102.

Claims

**Claim 1** A lactic acid bacterium, characterized in that it is Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK under the accession number NCIMB 44102. **Claim 2** A composition for improving the intestinal microbiota composition, comprising an effective amount of extracellular vesicles, wherein the extracellular vesicles can be secreted from lactic acid bacteria, and the lactic acid bacteria are Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK under the accession number NCIMB 44102. **Claim 3** The effective amount is extracellular vesicles having a particle number concentration of at least 10 ８ per milliliter, and the composition according to claim 2, characterized in that it is the extracellular vesicles. **Claim 4** The composition according to claim 2, characterized in that the extracellular vesicle is any one selected from the group consisting of exosome, microvesicle, ectosome and apoptotic body. **Claim 5** The composition according to claim 2, characterized in that the extracellular vesicle is capable of affecting the growth of the phylum Firmicutes and / or the phylum Bacteroidetes. **Claim 6** The composition according to claim 2, characterized in that the extracellular vesicle is capable of affecting the growth of at least one bacterial class, and the bacterial class is at least one selected from the group consisting of the class Bacteroidia, the class Clostridia and the class Bacilli. **Claim 7** The extracellular vesicles are capable of affecting the growth of at least one bacterial family, and the bacterial family is at least one selected from the group consisting of Lactobacillaceae, Muribaculaceae, Lachnospiraceae, Clostridiaceae, Desulfovibrionaceae, Erysipelotrichaceae, Eggerthellaceae, Akkermansiaceae, Ruminococcaceae, and Eubacteriaceae. The composition according to claim 2, characterized in that it is one.

8. The composition according to claim 2, characterized in that the extracellular vesicles are capable of affecting the growth of the genus Muribaculum and / or the genus Lachnospira.

9. The composition according to claim 2, characterized in that the extracellular vesicles are capable of promoting the growth of Lactobacillus acidophilus.

10. A nutritional supplement, characterized by comprising Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK under the accession number NCIMB 44102.

11. A food product, characterized by comprising Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK under the accession number NCIMB 44102.

12. A dietary supplement, characterized by comprising Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK under the accession number NCIMB 44102.

13. A food additive, characterized by comprising Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd), UK, under the accession number NCIMB 44102.

14. A pharmaceutical composition, characterized by comprising Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd), UK, under the accession number NCIMB 44102.

15. A feed, characterized by comprising Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd), UK, under the accession number NCIMB 44102.

16. Use of lactic acid bacteria for preparing a composition for improving the composition of intestinal microbiota, characterized in that the lactic acid bacteria are Pediococcus acidilactici deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd), UK, under the accession number NCIMB 44102.

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