WO2025206195A1 - Composition for controlling uptake of microparticles - Google Patents

Abstract

Provided is a composition for controlling uptake of microparticles into a body, the composition containing a lactic acid bacterium. The lactic acid bacterium is preferably any one selected from bacteria belonging to the genus Lactobacillus and bacteria belonging to the genus Streptococcus. For example, the lactic acid bacterium is a bacterium belonging to Lactobacillus delbrueckii, Lactobacillus paracasei, Lactobacillus gasseri, or Streptococcus thermophilus.

Description

Composition for controlling uptake of microparticles

The present invention relates to a composition for controlling the uptake of microparticles.

It is estimated that 150 million tons of plastic waste is discarded in the world's oceans, with 8 million tons being newly discharged each year. Plastic that has been broken down into tiny particles by the effects of waves, ultraviolet rays, etc. is called microplastics, with those 5 mm or smaller being called microplastics and those 100 nm or smaller being called nanoplastics (NP). Microplastics do not decompose naturally and therefore remain in the natural environment. Humans ingest microplastics on a daily basis when they ingest marine organisms that have ingested them. Microplastics are also found in the contents of plastic bottles and toothpaste, and it has been reported that we ingest as much as 5 g of microplastics per week.

Microplastics have not only been detected in feces, but also in various organs, and research is ongoing into the harmful effects of long-term exposure. In vitro tests using cells suggest that NPs are taken up into cells, inducing oxidative stress, DNA damage, apoptosis, etc. (Non-patent literature 1-3). There is also concern that oxidative stress is involved in the development of inflammatory bowel disease, colon cancer, and small intestine cancer.

It is known that when nanoparticles come close to cells, they interact with the cell membrane, resulting in the nanoparticles being engulfed in the cell membrane and taken up into the cell by endocytosis (Non-Patent Document 4). Furthermore, it has been reported that polyethylene oxide microplastics have an enhanced toxicity effect in the mouse intestine and Caco-2 cells (Non-Patent Document 5).

Furthermore, the acute effects of 48-hour exposure to different concentrations of polystyrene nanoplastics (0, 5, 10, and 20 mg/L) on the intestinal microbiota and oxidative stress parameters of freshwater crayfish (Procambarus clarkii) were examined. It was reported that exposure to polystyrene nanoplastics may lead to a weakening of the immune system of P. clarkii, and that increasing nanoplastic concentrations significantly altered the levels of glutathione (GSH), superoxide dismutase (SOD), acid phosphatase (ACP), lysozyme (LZM), alkaline phosphatase (AKP), peroxidase (POD), glutathione peroxidase (GPX), and protein carbonylation, suggesting that such lactic acid bacteria may play an auxiliary role in the treatment of oxidative stress (Non-Patent Document 6). Furthermore, it has been argued that lactic acid bacteria collected from infant feces efficiently adsorbed three types of nanoplastics: polypropylene, polyethylene, and polyvinyl chloride, suggesting that this could be a new strategy for removing nanoplastics from the intestinal environment (Non-Patent Document 7).

Biomolecules 2021, 11(10), 1442; https://doi.org/10.3390/biom11101442 Int. J. Mol. Sci. 2021, 22(4), 2094; https://doi.org/10.3390/ijms22042094 Nanoscale, First published 08 Mar 2024, Advance Article. https://doi.org/10.1039/D3NR06638J ACS Nano. 2015 September 22; 9(9): 8655.8671. doi:10.1021/acsnano.5b03184. Sci Total Environ. 2023 Dec 10:903:166057. doi: 10.1016/j.scitotenv.2023.166057. Sci Total Environ. 2022 Aug 10:833:155722. doi: 10.1016/j.scitotenv.2022.155722. Chemosphere. 2023 Apr:320:138038. doi: 10.1016/j.chemosphere.2023.138038.

No food-derived materials have yet been reported that can control the uptake of microparticles such as nanoplastics into the body. It would be desirable if the uptake of microparticles could be controlled by ingesting ingredients that are commonly eaten.

The present invention provides the following:
[1] A composition for controlling the uptake of microparticles into the body, comprising lactic acid bacteria.
[2] The composition according to 1, wherein the lactic acid bacteria are any one selected from the group consisting of bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Streptococcus, and bacteria belonging to the genus Bifidobacterium.
[3] The composition according to 1 or 2, wherein the lactic acid bacteria are bacteria belonging to Lactobacillus delbrueckii, Lactobacillus paracasei, Lactobacillus gasseri, or Streptococcus thermophilus, Lactobacillus paragasseri, Lactobacillus lactis, or Bifidobacterium longum.
[4] The composition according to any one of [1] to [3], wherein controlling uptake means inhibiting uptake.
[5] The composition according to any one of [1] to [4], wherein the uptake into the body is uptake into intestinal epithelial cells.
[6] The composition according to any one of 1 to 5, wherein the microparticles are nanoplastics or microplastics.
[7] The composition according to any one of 1 to 6, for treating any one selected from oxidative stress, DNA damage, and apoptosis caused by the incorporation of microparticles into the body.
[8] The composition according to any one of [1] to [7], for use as a probiotic or synbiotic.
[9] The composition according to any one of items 1 to 8, wherein the uptake is controlled by an action on cells.
[10] The composition according to 9, wherein the effect on cells is due to the inhibition, prevention or restriction of the uptake of microparticles into cells.
[11] The composition according to any one of items 1 to 10, wherein the uptake is controlled by controlling endocytosis by cells.
[12] The composition described in any one of 1 to 11, wherein the control of the uptake is not due to adsorption of the microparticles by the lactic acid bacteria.
[13] The composition described in any one of 1 to 12, wherein the lactic acid bacteria are live or killed cells.
[14] The composition described in any one of 1 to 12, wherein the lactic acid bacteria are heat-killed bacteria.
[15] A method for reducing the risk of developing any one selected from inflammatory bowel disease, colon cancer, and small intestine cancer, comprising the step of administering a composition containing lactic acid bacteria to a subject, thereby controlling the uptake of microparticles into the body. [16] The method according to 15, wherein the control of the uptake is due to an action on cells.
[17] The method according to 15 or 16, wherein the effect on cells is due to the blocking, prevention, or restriction of the uptake of microparticles into cells.
[18] The method according to any one of 15 to 17, wherein the uptake is controlled by controlling endocytosis by cells.
[19] The method according to any one of 15 to 18, wherein the control of the uptake is not due to adsorption of the microparticles by the lactic acid bacteria.
[20] The method according to any one of 15 to 19, wherein the lactic acid bacteria are live or killed cells.
[21] The method according to any one of 15 to 19, wherein the lactic acid bacteria are heat-killed bacteria.

[22] A composition comprising lactic acid bacteria for use in a method for controlling the uptake of microparticles into the body. Use of lactic acid bacteria in the manufacture of a composition for controlling the uptake of microparticles into the body. A method or non-therapeutic method for controlling the uptake of microparticles into the body, comprising a step of having a subject ingest a composition comprising lactic acid bacteria. Use or non-therapeutic use of lactic acid bacteria for controlling the uptake of microparticles into the body.
[23] The composition, use in production, method or non-therapeutic method, use or non-therapeutic use according to 1, wherein the lactic acid bacteria are any one selected from bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Streptococcus, and bacteria belonging to the genus Bifidobacterium.
[24] The composition, use in production, method or non-therapeutic method, use or non-therapeutic use according to 1 or 2, wherein the lactic acid bacterium is a bacterium belonging to Lactobacillus delbrueckii, Lactobacillus paracasei, Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus paragasseri, Lactobacillus lactis, or Bifidobacterium longum.
[25] The composition, use in manufacturing, method, or non-therapeutic method, use, or non-therapeutic use according to any one of 1 to 3, wherein controlling uptake means inhibiting uptake.
[26] The composition, use in manufacturing, method, or non-therapeutic method, use, or non-therapeutic use according to any one of 1 to 4, wherein the uptake into the body is uptake into intestinal epithelial cells.
[27] The composition, use in manufacture, method or non-therapeutic method, use or non-therapeutic use according to any one of 1 to 5, wherein the microparticles are nanoplastics or microplastics.
[28] The composition, use in manufacturing, method, or non-therapeutic method, use, or non-therapeutic use according to any one of 1 to 6, wherein the composition is for treating any one selected from oxidative stress, DNA damage, and apoptosis caused by the uptake of microparticles into the body.
[29] The composition, use in manufacture, method or non-therapeutic method, use or non-therapeutic use according to any one of claims 1 to 7, wherein the composition is for use as a probiotic or synbiotic.
[30] A composition comprising lactic acid bacteria for reducing the risk of developing any one selected from inflammatory bowel disease, colon cancer, and small intestine cancer by controlling the uptake of microparticles into the body. A composition comprising lactic acid bacteria for use in a method for controlling the uptake of microparticles into the body, thereby reducing the risk of developing any one selected from inflammatory bowel disease, colon cancer, and small intestine cancer. Use of lactic acid bacteria in the manufacture of a composition for controlling the uptake of microparticles into the body, thereby reducing the risk of developing any one selected from inflammatory bowel disease, colon cancer, and small intestine cancer. A non-therapeutic method for reducing the risk of developing any one selected from inflammatory bowel disease, colon cancer, and small intestine cancer, comprising the step of administering a composition comprising lactic acid bacteria to a subject, thereby controlling the uptake of microparticles into the body. Use of a composition comprising lactic acid bacteria for reducing the risk of developing any one selected from inflammatory bowel disease, colon cancer, and small intestine cancer by controlling the uptake of microparticles into the body.

In one embodiment, the composition controls the uptake of microparticles into the body.

One embodiment of the composition makes it possible to control the uptake of microparticles into the body using lactic acid bacteria, which are commonly consumed as food.

L. bulgaricus 2038 and S. thermophilus 1131 inhibit PSNP uptake. Mean ± standard error (n = 5-6/group). The fluorescence intensity of PSNP taken up by Caco-2 is expressed as median fluorescence intensity. The values for each group were normalized by dividing by the median fluorescence intensity of the control group so that the value for the control group was 1.000. *p < 0.05 (Tukey-Kramer test). 16S rRNA gene, Lactobacillus delbrueckii ssp. bulgaricus 2038 (SEQ ID NO:1) 16S rRNA gene, Streptococcus thermophilus 1131 (SEQ ID NO:2) Live L. bulgaricus 2038 and live S. thermophilus 1131 bacteria inhibit PSMP uptake. Mean ± standard error (n = 7/group). The fluorescence intensity of PSNP taken up by Caco-2 is expressed as median fluorescence intensity. The values for each group were normalized by dividing by the median fluorescence intensity of the control group so that the value for the control group was 1.000. *: p<0.05 (Tukey-Kramer test) Both live and heat-treated L. bulgaricus 2038 cells and live and heat-treated S. thermophilus 1131 cells inhibit PSNP uptake. Mean ± standard error (n = 6/group). The fluorescence intensity of PSNP taken up by Caco-2 is expressed as the median fluorescence intensity. The values for each group were normalized by dividing by the median fluorescence intensity of the control group so that the value for the control group was 1.000. *: p<0.05 (Tukey-Kramer test) Heat-treated cells of various strains of L. bulgaricus all suppress PSNP uptake. Mean value (n = 3/group). The fluorescence intensity of PSNP taken up into Caco-2 is expressed as the median fluorescence intensity. The values for each group were normalized by dividing by the median fluorescence intensity of the control group so that the value for the control group was 1.000. Heat-treated cells from various strains of S. thermophilus all inhibit PSNP uptake. Mean values (n = 3/group). The fluorescence intensity of PSNP taken up into Caco-2 is expressed as the median fluorescence intensity. The values for each group were normalized by dividing by the median fluorescence intensity of the control group so that the value for the control group was 1.000. Heat-treated L. lactis P2306614, LG21, and R-1 all inhibit PSNP uptake. Mean values (n = 3/group). The fluorescence intensity of PSNP taken up by Caco-2 is expressed as the median fluorescence intensity. The values for each group were normalized by dividing by the median fluorescence intensity of the control group so that the value for the control group was 1.000. Heat-treated L. paracasei YIT9029 and B. longum BB536 cells both inhibit PSNP uptake. Mean values (n = 3/group). The fluorescence intensity of PSNP taken up by Caco-2 is expressed as the median fluorescence intensity. The values for each group were normalized by dividing by the median fluorescence intensity of the control group so that the value for the control group was 1.000.

This embodiment relates to a composition containing a specific bacterium. More specifically, it relates to a composition for controlling the uptake of microparticles into the body.

[Active ingredient]
The composition of the present invention contains any of the lactic acid bacteria as an active ingredient. "Any" means that the type and number are optional. "Containing as an active ingredient" means that the composition uses an effective amount to exert the intended function, and that the ingredient is specified in the label as contributing to the intended purpose. In functional food products, the active ingredient is sometimes referred to as a functional ingredient (an ingredient that contributes to a specific health purpose (excluding those related to reducing the risk of disease)).
Regardless of the material used, the ingredients contained in the composition of the present invention are used at an intake level that is guaranteed to be safe or below the acceptable daily intake (ADI), giving priority to the food safety laws of each country.

(lactic acid bacteria)
In the present invention, unless otherwise specified, the term "lactic acid bacteria" refers to a general term for microorganisms that utilize glucose to produce lactic acid, including bifidobacteria and microorganisms that produce lactic acid at a sugar yield of 50% or more (lactic acid bacteria in the narrow sense). Lactic acid bacteria in the narrow sense are those that produce lactic acid at a sugar yield of 50% or more and that, in addition to being Gram-positive cocci or bacilli, are non-motile, often non-spore-forming (although some lactic acid bacteria, such as Bacillus coagulans, can form spores), and have catalase-negative properties. Bifidobacteria are Gram-positive, anaerobic bacilli that belong to the genus Bifidobacterium and produce acetic acid in addition to lactic acid.

(bacteria belonging to the genus Lactobacillus)
In one embodiment, bacteria belonging to the genus Lactobacillus are used as active ingredients. In the present invention, when bacteria belonging to the genus Lactobacillus are described, unless otherwise specified, they are classified according to the following reclassification: Zheng J, Wittouck S, Salvetti E, Franz CMAP, Harris HMB, Mattarelli P, O'Toole PW, Pot B, Vandamme P, Walter J, Watanabe K, Wuyts S, Felis GE, Ganzle MG, Lebeer S.: A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol. 2020 Apr; 70(4): 2782-2858.

In one embodiment, the active ingredient is a bacterium belonging to the genus Lactobacillus, such as Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus paragasseri, or the like. Bacteria belonging to the species Lactobacillus paragasseri, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, or Lactobacillus johnsonii are used.

In another embodiment, the active ingredient is a bacterium belonging to the genus Lactobacillus, such as Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus gasseri, or Lactobacillus paragasseri. This is because all of these bacteria have been widely consumed. Furthermore, when any of these bacteria is used, other effects can be expected in addition to the intended effect.

(Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus)
In a preferred embodiment, the active ingredient used is 2038, OLL1255 (Accession No.: NITE BP-76), OLL1171 (Accession No.: NITE BP-01569), OLL1247 (Accession No.: NITE BP-01814), OLL205013 (Accession No.: NITE BP-02411), OLL1073R-1 (Accession No.: FERM BP-10741), or a taxonomically equivalent strain of bacteria belonging to L. delbrueckii subsp. bulgaricus.
OLL1073R-1 is also available from Meiji Co., Ltd. under the trade name Meiji Probio Yogurt R-1.

L. delbrueckii ssp. bulgaricus 2038 can be isolated from Meiji Bulgaria Yogurt LB81 (Meiji Co., Ltd.) and is commercially available. It is also stored at Meiji Co., Ltd. (Meiji Innovation Center, 1-29-1 Shichikuni, Hachioji, Tokyo).

The mycological properties of 2038 are shown below.
Morphology: Bacillus, does not produce gas from glucose, GC content 49.7%, Gram positive, catalase negative, lactic acid optical rotation: D type, does not grow at 15°C, produces acid by utilizing glucose, lactose, etc., does not form spores, non-motile.

The sequence of the 16S rRNA gene of 2038 is shown in SEQ ID NO: 1 in the sequence listing and Figure 2.

(Lactobacillus paragasseri, Lactobacillus gasseri)
In one embodiment, Lactobacillus paragasseri or a bacterium belonging to Lactobacillus gasseri is used as the active ingredient. Lactobacillus paragasseri is a Gram-positive bacterium that performs homolactic fermentation, and JCM5343 ^T is the type strain. Lactobacillus paragasseri can be distinguished from Lactobacillus gasseri by average nucleotide identity (ANI) or DNA-DNA hybridization. Examples of lactic acid bacteria strains belonging to Lactobacillus paragasseri include JCM 5343 ^T , Lactobacillus paragasseri OLL2809 (NITE BP-72), FERM BP-6999 (LG21), and NITE BP-224.

Strains with numbers beginning with JCM can be purchased from the Microbial Materials Development Division, BioResource Research Center, RIKEN (3-1-1 Takanodai, Tsukuba, Ibaraki 305-0074).
Lactobacillus paragasseri FERM BP-6999 can also be isolated from "Meiji Probio Yogurt LG21" sold by Meiji Co., Ltd.

(Lactobacillus paracasei)
In one embodiment, the active ingredient is a bacterium belonging to the genus Lactobacillus paracasei. Lactobacillus paracasei is a Gram-positive bacterium that performs heterolactic fermentation, and the type strain is mNCDO 151. Examples of lactic acid bacteria strains belonging to Lactobacillus paracasei include YIT9029 (FERM BP-1366) and YIT9018 (FERM BP-665).

(Bacteria belonging to the genus Streptococcus)
In one embodiment, a bacterium belonging to the genus Streptococcus is used as the active ingredient.

In a preferred embodiment, the active ingredient is Streptococcus thermophilus (also known as Streptococcus salivarius subsp. thermophilus), a bacterium belonging to the genus Streptococcus. More specifically, Streptococcus thermophilus 1131 or a strain taxonomically identical to it is used.

S. thermophilus 1131 can be isolated from Meiji Bulgaria Yogurt LB81 (Meiji Co., Ltd.).

The mycological properties of 1131 are shown below.
Morphology: Streptococcus, does not produce gas from glucose, gram-positive, catalase-negative, lactic acid rotatory L-form, grows at 45°C, produces acid by utilizing glucose, lactose, fructose, etc., does not form spores, is non-motile.

The partial sequence (489 bases long) of the 16S rRNA gene of 1131 is shown in sequence number 2 in the sequence listing and in Figure 2.

(Bifidobacteria)
In one embodiment, bifidobacteria are used as the active ingredient.
Examples of bifidobacteria include, but are not limited to, bacteria of the genus Bifidobacterium.
Further examples of bifidobacteria include, but are not limited to, Bifidobacterium bifidum, Bifidobacterium longum, and Bifidobacterium lactis.
In one embodiment, bacteria belonging to Bifidobacterium longum are used as the active ingredient. Examples of bifidobacterial strains belonging to Bifidobacterium longum include B. longum BB536.
B. longum BB536 can also be isolated from "Bifidus BB536 Plain Yogurt Fat-Free" sold by Morinaga Milk Industry Co., Ltd.

(taxonomically equivalent strains)
A strain taxonomically equivalent to a certain strain (hereinafter referred to as strain S) refers to, for example, any of the following:
A strain belonging to the same species as strain S, whose 16S rRNA gene (hereinafter sometimes referred to as "16S" or "16S rRNA gene") sequence, or a characteristic part thereof (such as the V1 region, the V2 region, or all or part of the V1 and V2 regions, or a part including the V1 and V2 regions), has 90% or more, preferably 95% or more, more preferably 98% or more, even more preferably 98.5% or more, even more preferably 98.7% or more, even more preferably 99% or more, and even more preferably 100% sequence identity with the sequence of strain S. A strain having the same mycological properties as strain S.

In the context of the present invention, unless otherwise specified, sequence identity means the percentage of matching bases shared between two sequences when the sequences are optimally aligned. Analysis of base sequence identity can be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When using a program, parameters can be set appropriately by those skilled in the art, or the default parameters of each program may be used. The specific techniques for these analysis methods are also well known to those skilled in the art. Commercially available genetic information processing software may be used to calculate identity.

With regard to criteria for determining species similarity based on 16S rRNA gene sequences, those skilled in the art can refer to Stackebrandt E, Ebers J. Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 2006;33:152-155.

The composition may contain microorganisms other than lactic acid bacteria, such as yeast, as long as the intended function is not impaired. In one preferred embodiment, the composition contains one or more types of lactic acid bacteria but does not contain other microorganisms, such as yeast.

(Production, acquisition, and form of lactic acid bacteria)
The lactic acid bacteria used as an active ingredient can be produced by culturing. The culturing conditions are not particularly limited as long as the desired effect is achieved.

The lactic acid bacteria may be included in any state as long as they can exert the desired effect. For example, the lactic acid bacteria may be in the form of the bacteria themselves, or in the form of a culture of effective bacteria (consisting of bacteria and culture supernatant). The bacteria may be in a live state (viable bacteria) or a dead state (dead bacteria) as long as they can exert the desired effect.

Killed bacteria can be obtained by sterilizing effective bacteria. There are no particular limitations on the sterilization method, as long as it can produce the desired effect, and it can be achieved by heat, a germicidal lamp (UV), ozone, chemicals, high osmotic pressure, etc.

The killed bacteria are preferably heat-killed bacteria obtained by heat-treating live bacteria. The heat treatment to obtain heat-killed bacteria is not particularly limited as long as the desired effect is achieved, and is carried out at a temperature and for a time sufficient to kill the effective bacteria used. While these conditions vary depending on the effective bacteria used, the heat treatment temperature is, for example, 55°C or higher, preferably 60°C or higher, more preferably 65°C or higher, and even more preferably 70°C or higher, and may be 80°C or higher, or even 90°C or higher. The upper limit of the heat treatment temperature can be set appropriately, for example, 121°C or lower, 100°C or lower, 90°C or lower, or 80°C or lower. Depending on the heat treatment temperature, the heat treatment time can be 1 minute or more, 3 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, or 45 minutes or more. The upper limit of the heat treatment time can be, for example, 120 minutes or less, 100 minutes or less, 90 minutes or less, or 80 minutes or less.

The lactic acid bacteria to be contained in the composition, whether live or dead, can be prepared in the form of a dried substance, suspension, paste, gel, etc.

[Application]
(Action/Function)
The composition of this embodiment can be used to control the uptake of microparticles into the body. Control includes upward regulation (promotion, increase, suppression of decline) and downward regulation (suppression, decrease, suppression of rise). In a preferred embodiment, the uptake of microparticles into the body is suppressed.
In a preferred embodiment, the control (preferably inhibition) of the uptake is preferably achieved by an action on cells, more preferably by controlling endocytosis, blocking, preventing, or restricting the uptake of microparticles into cells, or controlling the permeation of tight junctions. That is, in this embodiment, the composition of the present embodiment is preferably one in which the lactic acid bacteria act on cells to control endocytosis by the cells, inhibiting, preventing, or restricting the uptake of microparticles by the cells, or controlling the permeation of tight junctions, thereby inhibiting the uptake of microparticles.
In another preferred embodiment, the control of endocytosis is preferably inhibition of endocytosis.
In another preferred embodiment, the control of uptake is preferably achieved by controlling the permeability of the microparticles to the cell membrane. In the above embodiment, the control of uptake is more preferably achieved by inhibiting the uptake, and more preferably by inhibiting the permeability of the microparticles to the cell membrane.
In another preferred embodiment, the control of tight junction permeability is preferably inhibition of tight junction permeability.
Furthermore, as will be shown in the examples below, it is preferable that the uptake is not controlled by adsorption of microparticles by lactic acid bacteria.

In vitro tests using intestinal epithelial cells have reported that NPs are taken up into the cells, inducing oxidative stress, DNA damage, apoptosis, etc. (Non-Patent Documents 1-3). Oxidative stress is also feared to be involved in the development of inflammatory bowel disease, colon cancer, and small intestine cancer. Therefore, the composition of this embodiment is suitable for treating any of oxidative stress, DNA damage, and apoptosis caused by the ingestion of microparticles into the body, and is also suitable for reducing the risk of developing any of inflammatory bowel disease, colon cancer, and small intestine cancer.

Inhibition of uptake into the body means that the use of a composition or active ingredient reduces the amount taken up into the body compared to when the composition or active ingredient is not used, or when the composition or active ingredient is used in a smaller amount. Inhibiting uptake into the body includes inhibiting an increase (enhancement) in the amount taken up into the body. For example, in a situation where the uptake of microparticles into cells is enhanced, using the composition or active ingredient of this embodiment to inhibit the enhanced uptake of microparticles is also an example of inhibiting the uptake of microparticles into the body.

In the context of this invention, "in the body" can be interpreted as "inside a living organism" or "inside a living organism." Controlling uptake into the body includes controlling uptake into cells.

This embodiment is expected to control the uptake of microparticles in various cells, tissues, and organs. There are no particular limitations on the cells, tissues, and organs for which uptake of microparticles can be inhibited. Examples of organs include the esophagus, stomach, small intestine, large intestine, rectum, appendix, trachea, lungs, eyes, nasal cavity, pharynx, thyroid gland, spleen, liver, gallbladder, kidneys, bladder, ureter, urethra, heart, arteries, and veins. Examples of cells include epithelial cells. Epithelial cells are present on the surfaces of the body (skin), body cavities (intestinal tract), organs, etc., and also constitute exocrine and endocrine glands.

In a preferred embodiment, the composition is used to control the uptake of microparticles in intestinal epithelial cells or alveolar epithelial cells, more particularly in intestinal epithelial cells. The intestinal tract refers to the digestive organs in humans and animals that digest and absorb ingested food. The intestinal tract includes the small intestine and large intestine, and is preferably the small intestine.

Whether a certain component can control the uptake of microparticles into the body can be evaluated, for example, using appropriate cultured cells. More specifically, cultured cells are cultured on a Transwell plate insert to form a monolayer, and a labeled standard nanoplastic is added to the apical side of the Transwell, with the test component added and cultured for a while. The cells are then detached, and the amount of nanoplastic taken up into the cells is analyzed using the labeled substance as an indicator. If necessary, this can be compared with the amount taken up in a system without the test component added. An example of cultured cells that can be used is Caco-2 cells.

In the context of this invention, microparticles refer to particles that are insoluble in water and have a size (particle diameter or length) of 1 mm or less. Microparticles can also be referred to as micromaterials or microsubstances. Particles that are insoluble in water can also be referred to as solid particles. Microparticles can be tiny substances that have adverse effects on the human body.

The size of the microparticles can be 100 μm or less, 10 μm or less, or 1 μm or less. The microparticles can also be, for example, 1 nm or more, 2 nm or more, 3 nm or more, 4 nm or more, 5 nm or more, or 10 nm or more.

Regarding microparticles, there are no particular limitations on the material (also referred to as components, ingredients, or raw materials). Examples of materials include plastics, metals (titanium dioxide, aluminum oxide, zinc oxide, cerium oxide, magnesium oxide, iron oxide, titanium, silver, gold, iron, etc.), carbons (fullerenes, carbon tubes, etc.), and ceramics (silicon dioxide, nanoclay, silicic acid, etc.).

Examples of plastic materials include:
Polystyrene (PS), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyethylene terephthalate (PET), polycarbonate (PC), ethylene vinyl acetate (EVA), acrylonitrile styrene (SAN), acrylonitrile butadiene styrene (ABS), acrylate styrene acrylonitrile (ASA), polyacetal (POM), alkyd (Alkyd), unsaturated polyester (UPR), polyvinyl acetate (PVAc), polyacrylonitrile (PAN), polymethyl methacrylate (PMMA), polyallylamine-acrylonitrile butadiene styrene-polymethacrylimide (PAA-MABS-PMI), polyamide (PA), urea resin (UF), polyurethane (PUR), epoxy resin (EP), melamine resin (MF), phenolic resin (PF), silicone resin (SI).

An example of a microparticle whose uptake can be suitably controlled by this embodiment is a microplastic, and its size may be 1000 nm or less, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, or 1 nm or more, 2 nm or more, 3 nm or more, 4 nm or more, 5 nm or more, or 10 nm or more. In one embodiment, the uptake of nanoplastics can be suitably controlled. In the present invention, the term nanoplastics refers to plastics with sizes between 1 and 100 nm, unless otherwise specified.

Other examples of fine particles for which intake control can be suitably performed using this embodiment are suspended particulate matter (SPM, particles of 10 μm or less suspended in the air) and fine particulate matter (PM2.5, small particles of 2.5 μm or less suspended in the air). The material of suspended particulate matter and fine particulate matter varies depending on the source. When the source is yellow sand, volcanic smoke, etc., the material is inorganic elements, etc. When the source is a factory, automobile, etc., the material is elemental carbon, organic carbon, inorganic elements, etc. When the source is secondary particles (produced by the reaction of chemicals in the air), the material is sulfate ions, nitrate ions, ammonium ions, organic carbon, etc. The size can be 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2.5 μm or less, 2 μm or less, 1 μm or less, 0.5 μm or less, or 0.01 μm or more, 0.05 μm or more, 0.1 μm or more.

The specific gravity of microparticles that can be suitably controlled for uptake by this embodiment can be, for example, 1.6 or less, 1.5 or less, or 1.4 or less.

(subject)
The composition of this embodiment is suitable for administration to healthy subjects in whom it is desirable to control the uptake of microparticles into the body, healthy subjects in whom it is desirable to control the uptake of microparticles into intestinal epithelial cells, for example, healthy subjects in whom it is desired to control the uptake of microparticles into the body, healthy subjects who are thought to be ingesting a large amount of microparticles, and healthy subjects who are concerned about the ingestion of microparticles. A healthy subject refers to a human who has not been diagnosed with any disease (pre-disease) or a companion animal, as described below. In other words, when the composition of this embodiment is used on these healthy subjects, the composition of this embodiment is not provided for therapeutic purposes.

The composition of this embodiment is also suitable for administration to healthy subjects who have increased uptake of microparticles into their bodies.

The subject is preferably healthy (e.g., free of intestinal disease, infection, and cancer).

The composition of this embodiment can be used non-therapeutically. Non-therapeutic means not intended to treat a disease. In one embodiment, the composition of this embodiment can be used in the form of a food or the like (for example, as a food composition). In the above aspect, the composition is not a medicine, but is provided in the form of, for example, a supplementary food, health food, or dietary supplement. Non-therapeutic use means maintaining the current state, temporary improvement or relief, support, etc.

The composition of this embodiment is also suitable for administration to subjects with a disease in whom it is desirable to control the uptake of microparticles into the body, subjects with a disease in whom it is desirable to control the uptake of microparticles into intestinal epithelial cells, for example, subjects with a disease in whom it is desired to control the uptake of microparticles into the body, subjects with a disease who are thought to be ingesting large amounts of microparticles, and subjects with a disease who are concerned about the ingestion of microparticles.

The composition of this embodiment is also suitable for administration to subjects with a disease in which the body's uptake of microparticles is enhanced.

The composition of this embodiment can also be used for non-therapeutic purposes in subjects with a disease. In one embodiment, the composition of this embodiment can be used as a composition to support treatment in the form of a food or the like (e.g., as a food composition). Here, "supporting treatment" refers to non-therapeutic uses for treating a disease or illness. Examples of support for treatment include use in subjects undergoing treatment for a disease or illness to enhance the therapeutic effect or provide nutritional support during treatment; use in subjects treated for a disease or illness to improve the prognosis, maintain a good prognosis, or provide nutritional support after treatment; and use in subjects scheduled to be treated for a disease or illness to enhance the therapeutic effect after treatment or provide nutritional support before treatment. In these embodiments, the composition is not a pharmaceutical, but is provided in the form of, for example, a supplementary food, health food, liquid food, highly functional liquid food, supplement, etc.
The composition of the present embodiment can also be used for the purpose of treating a subject with a disease. In one embodiment, the composition of the present embodiment can be used as a composition for treatment in the form of a medicine or the like (for example, as a pharmaceutical composition).

When using the composition of this embodiment for non-therapeutic purposes, judgments as to whether it is desirable or necessary may include judgments as non-therapeutic procedures, such as advice other than diagnosis, by medical professionals such as doctors, nurses, pharmacists, midwives, and clinical laboratory technicians; judgments by those involved in non-therapeutic procedures, such as nutritionists (including registered dietitians and sports nutritionists), public health nurses, sports instructors, pharmaceutical manufacturers, pharmaceutical distributors, food manufacturers, and food distributors; and judgments by the subject themselves or their family. Furthermore, the above judgments include judgments based on the output results of questionnaires on lifestyle habits, eating habits, and subjective symptoms, and judgments based on subjective symptoms (concerns about obesity or lifestyle-related diseases, etc.).

When using the composition of this embodiment for therapeutic purposes, the necessity of such use can be determined by a medical professional such as a doctor, nurse, pharmacist, midwife, or clinical laboratory technician as a therapeutic procedure.

The subject may be a human or a non-human animal. Non-human animals include mammals, birds, reptiles, amphibians, fish, etc. Non-human animals may be commercial animals, research animals, or companion animals. The term "companion animal" refers to a domestic or domestic animal whose physical, emotional, behavioral, and social needs can be readily met as a domestic companion or through close daily association with one or more humans. In one embodiment, species included within the definition of companion animal include dogs, canines, cats, felines, cows, horses, goats, sheep, pigs, primates (such as monkeys), rabbits, ferrets, rodents (such as guinea pigs, hamsters, mice, and rats), and other small mammals. In another embodiment, species included within the definition of companion animals are dogs, cats, horses, rabbits, ferrets, guinea pigs, and other small mammals, birds, small reptiles, fish, and livestock animals.

There are no particular limitations on the age of the subject to which the composition of this embodiment is administered, and when the subject is human, examples of such subjects include newborns (within 28 days of birth), infants (less than 1 year of age), toddlers (1-6 years of age), children (7 years of age or older, under 15 years of age), adults (15 years of age or older), middle-aged and elderly people, people 60 years of age or older, elderly people (65 years of age or older), those who are ill or recovering from an illness, pregnant women, and women who have just given birth.

[Composition]
(Food composition, etc.)
The composition of this embodiment may be a food composition, pharmaceutical composition, or cosmetic composition. Foods, pharmaceuticals, and cosmetics, unless otherwise specified, include not only those intended for humans but also those intended for non-human animals. Foods, unless otherwise specified, include general foods, functional foods, and nutritional compositions, as well as therapeutic foods (those intended for therapeutic purposes, prepared based on a menu prepared by a nutritionist or other professional prescribed by a doctor), dietary therapy foods, ingredient-adjusted foods, nursing care foods, and therapeutic support foods. Unless otherwise specified, foods include not only solids but also liquids, such as beverages, energy drinks, liquid diets, and soups. Functional foods are foods that can impart specific functionality to living organisms, and include a wide range of health foods, including foods with specified health uses (including conditional FOSHUs [specified health foods]), foods with functional claims, foods with health claims, foods with nutrient function claims, foods for special dietary uses, foods with special dietary uses, dietary supplements, health supplements, supplements (e.g., tablets, coated tablets, sugar-coated tablets, capsules, liquids, etc.), and beauty foods (e.g., diet foods). In addition, in the present invention, "functional foods" encompass health foods to which health claims based on the Codex Alimentarius (Joint FAO/WHO Food Standards Commission) are applied. In the present invention, pharmaceuticals include topical skin preparations. In the present invention, cosmetics include quasi-drugs, and also include cosmetics that do not contain active ingredients and medicated cosmetics that contain active ingredients.

(Route of administration)
The composition of this embodiment may be administered orally, parenterally, for example, via a tube (gastrostomy, enterostomy), or transnasally. It may also be administered transdermally. In the context of the present invention, the term "administer" refers not only to administering a pharmaceutical to a subject, but also to ingesting food or applying cosmetics to the skin. "Administer" can sometimes be read as "intake," and "intake" can also be read as "administer."

(Active ingredient content and dosage)
The content of the active ingredient in the composition of this embodiment may be any amount sufficient to achieve the desired effect. The content of lactic acid bacteria in the composition can be appropriately determined taking into account various factors such as the age, weight, and symptoms of the subject to be ingested or administered. The content of lactic acid bacteria per unit of the composition (when multiple types of bacteria are active ingredients, this may be the content of each active ingredient or the total content; the same applies hereinafter when referring to the content) can be, for example, 1 x ¹⁰ or more, 1 x ¹⁰ or more, 1 x 10 or more, ⁵ x ¹⁰ or more, 1 x ¹⁰ or more, 5 x ¹⁰ or more, preferably 1 x ¹⁰ or more, more preferably 1 x ¹⁰ or more, and even more preferably 2 x ¹⁰ or more. The upper limit can be set as appropriate, and whatever the lower limit, it may be, for example, 1 x ¹⁰ or less, 1 x ¹⁰ or less, or 1 x ¹⁰ or less. One unit of the composition can be ingested by a subject once a day, or may be ingested by a subject multiple times a day, for example, three times a day.

When the active ingredient is a dried bacterial cell, the content of the active ingredient in the composition of this embodiment can be, for example, 1 mg or more, or may be 2 mg or more, 5 mg or more, 10 mg or more, 15 mg or more, or 20 mg or more, preferably 30 mg or more, more preferably 50 mg or more, and even more preferably 100 mg or more. The upper limit can be set as appropriate, and whatever the lower limit, it may be, for example, 1000 mg or less, 750 mg or less, or 500 mg or less.

The content of lactic acid bacteria per 1 g of the composition may be, for example, 1 x ¹⁰ or more, 5 x ¹⁰ or more, 1 x ¹⁰ or more, 5 x ¹⁰ or more, 1 x ¹⁰ or more, 5 x ¹⁰ or more, 1 x ¹⁰ or more, 5 x ¹⁰ or more, 1 x ¹⁰ or more, 5 x ¹⁰ or more, 1 x 10 or more, 5 x ¹⁰ or more, 1 x ¹⁰ or more, 5 x ¹⁰ or more, and preferably 5 x 10 or more. The upper limit can be set appropriately, and whatever the lower limit, the content may be, for example, 1 x ¹⁰ or less, 1 x ¹⁰ or less, 1 x ¹⁰ or less, 1 x ¹⁰ or less, or 1 x ¹⁰ or less.

Furthermore, when the lactic acid bacteria content is as described above, in either case, one unit of the composition can be, for example, 0.5g or more, 1g or more, 5g or more, 10g or more, 20g or more, or 30g or more. When the composition is in the form of a food product such as fermented milk, one unit can be an amount that is easy to ingest in one sitting as a food product, for example, 50g or more, 60g or more, 70g or more, 80g or more, 90g or more, or 100g or more. The upper limit can be set as appropriate, and in either case, the lower limit can be 500g or less, 400g or less, 300g or less, 200g or less, 150g or less, or 125g or less.

The composition of this embodiment contains lactic acid bacteria with a long history of consumption as its active ingredient. Therefore, the composition of this embodiment may be administered repeatedly or over a long period of time, for example, continuously for three days or more, preferably one week or more, more preferably four weeks or more, and particularly preferably one month or more.

(Other ingredients, additives)
The composition of this embodiment may contain ingredients acceptable for use as food, medicine, or cosmetics. Examples of ingredients acceptable for use as food or medicine include lipids (e.g., milk fat, vegetable oil, medium-chain fatty acid-containing oil), proteins (e.g., milk protein, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), heat-denatured whey protein, and enzyme-treated whey protein), amino acids (e.g., lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, valine), kojibiose, and kojibiose-containing sugars. carbohydrates other than oligosaccharides having the above-mentioned constituent sugars (glucose, sucrose, fructose, maltose, trehalose, erythritol, maltitol, palatinose, xylitol, dextrin), electrolytes (e.g., sodium, potassium, calcium, magnesium), vitamins (e.g., vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, biotin, folic acid, pantothenic acid, and nicotinic acids), minerals (e.g., copper, zinc, iron, cobalt, manganese), antibiotics, dietary fiber, etc. Examples of ingredients acceptable for cosmetics include aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizing ingredients, fragrances, pigments, etc.

The composition of this embodiment may further contain additives that are acceptable for use in foods, pharmaceuticals, or cosmetics. Examples of such additives include inert carriers (solid or liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, colorants, preservatives, buffers, pH adjusters, emulsifiers, stabilizers, sweeteners, antioxidants, flavorings, acidulants, and natural products. More specifically, these include water, other aqueous solvents, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, sucralose, stevia, aspartame, acesulfame potassium, citric acid, lactic acid, malic acid, tartaric acid, phosphoric acid, acetic acid, fruit juice, vegetable juice, etc.

(Dosage form/shape)
In one aspect, the food composition may be prepared in any form, such as a solid, liquid, mixture, suspension, powder, granules, paste, jelly, gel, or capsule. The food composition according to the present invention may also be prepared in any form, such as dairy products, supplements (e.g., tablets, coated tablets, sugar-coated tablets, enteric-treated agents such as enteric coating, capsules (enteric-coated soft capsules, enteric-coated hard capsules, large intestine delivery capsules, etc.), confectioneries, beverages, energy drinks, seasonings, processed foods, prepared dishes, soups, etc. More specifically, the composition of this embodiment may be prepared in any form, such as liquid foods (semi-liquid foods, concentrated liquid foods, etc.), jellies, gels, powders, infant formula, infant formula, powdered milk and liquid milk for pregnant and lactating women, fermented milk, bars, and milk products. cheese, chocolate, biscuits, ice cream, fermented milk, lactic acid bacteria drinks, dairy drinks, soft drinks, fruit juice drinks, tablets, cheese, bread, biscuits, crackers, pizza crust, whiskey, bourbon, spirits, liqueur, wine, fruit wine, sake, Chinese alcohol, shochu, beer, non-alcoholic beer with an alcohol content of 1% or less, happoshu, other miscellaneous alcohol, alcoholic beverages such as chuhai, mineral water; processed products using eggs, processed products (including delicacies) of seafood and meat (including liver and other offal) The formula can be in the form of miso paste, soy sauce, furikake (a seasoning), or other condiments, soups such as miso soup, foods for the sick, nutritional foods, frozen foods, or processed foods. It can also be in the form of granules, powders, pastes, thickened liquids, or other forms for mixing with beverages or foods. Granules and powders can be in cube or stick form (single-serving packets). Mineral water includes both sparkling and non-sparkling mineral water. For the purposes of this invention, "formulated milk powder" refers to a powdered product obtained by processing raw milk, cow's milk, special cow's milk, or raw buffalo milk, or a food made from these ingredients, or using them as the main ingredient, and adding nutrients necessary for infants. For the purposes of this invention, "formulated liquid milk" refers to a liquid product obtained by processing raw milk, cow's milk, special cow's milk, or raw buffalo milk, or a food made from these ingredients, or using them as the main ingredient, and adding nutrients necessary for infants.

In one aspect, the pharmaceutical composition can be in any dosage form suitable for oral administration, such as solid preparations such as tablets, granules, powders, pills, and capsules (enteric-coated soft capsules, enteric-coated hard capsules, colon delivery capsules, etc.); liquid preparations such as solutions, suspensions, and syrups; gels, aerosols, and live bacterial preparations. Solid preparations can be coated or sugar-coated, and can be enterically treated with enteric coating or the like. They can also be in the form of ointments, creams, topical liquid preparations, eye drops, nasal drops, suppositories, patches, and inhalants suitable for topical administration.

In one embodiment, the cosmetic composition may be in the form of a solution, emulsion, suspension, gel, cream, mask pack, sheet, foam, or aerosol.

(others)
The composition of this embodiment is suitable for administration to a subject at the same time as or before ingesting a food that contains a large amount of microparticles or a food that is suspected to contain a large amount of microparticles.

In the production of the composition of this embodiment, the stage of blending the active ingredient can be selected as appropriate. The stage of blending is not particularly limited as long as it does not significantly impair the properties of the active ingredient. For example, the ingredients (acidulant, sweetener, stabilizer, fruit juice, flavoring, lactic acid bacteria cells, water) can be mixed with a heat-treated lactic acid bacteria (approximately ¹⁰ cells are suspended in ion-exchanged water and heat-treated at 95°C for 3 minutes) and bottled to prepare a composition in the form of a 100 mL fruit juice-flavored soft drink.

In one aspect, the composition can be labeled with its intended use (application), and in another aspect, the function of the composition or active ingredient or the usage based on that function is labeled. Examples of usage based on that function are as explained above regarding functions, actions, and effects. Furthermore, the composition of this embodiment can be labeled with the fact that it can be used as a prebiotic or as a synbiotic (a combination of probiotics and prebiotics).

Specific examples of labeling include "controlling the intake of fine particles into the body," "suppressing the intake of fine particles into the body," "controlling the intake of microplastics into the body," "suppressing the intake of microplastics into the body," "controlling the intake of nanoplastics into the body," "suppressing the intake of nanoplastics into the body," "controlling the intake of PM2.5 into the body," and "suppressing the intake of PM2.5 into the body." Control or suppression encompasses assisting in control, assisting in suppression, temporarily alleviating, etc.

In one embodiment, the composition is labeled to recommend administration to a specific subject. Examples of subjects for which the labeling is provided are as described above.

Labeling can be explicit or implicit. Examples of explicit labeling are direct inscriptions on tangible objects such as the product itself, packaging, containers, labels, and tags. Examples of implicit labeling (which can also be called implied) include advertising and promotional activities in various places and by various means, such as websites, in-store, pamphlets, exhibitions, media seminars, books, newspapers, magazines, television, radio, video streaming sites, social media, influencer marketing, mail, email, and audio.

The present invention will be explained in more detail below using examples.

[Example 1: Inhibition of nanoplastic uptake]
L. bulgaricus ssp. bulgaricus 2038 was cultured in MRS medium for 18 hours, washed with PBS, and then suspended in PBS to an OD ₆₀₀ of 10. Caco-2 cells were cultured on inserts of a 0.4 μm pore size Transwell plate (12 wells, 0.4 μm, cell culture surface treated, sterilized, product number: 3401) for 2 weeks to form a monolayer.

Fluorescently labeled polystyrene nanoplastics (PSNPs) with a particle size of 20 nm were added to the apical side of the Transwell at a concentration of 200 μg/mL.
Fluorescently labeled PSNP
Manufacturer: Thermo Fisher Scientific
Product name: FluoSpheres ^™ Carboxylate-Modified Microspheres
Model number: F8787
To prevent aggregation, Tween 20 was added to PSNP at a final concentration of 0.005%, and the mixture was treated with an ultrasonic cleaner for 30 seconds twice.

Live L. bulgaricus 2038 cells were added to the apical side of the medium at 1%. The bacterial concentration in the medium was calculated to be approximately 5 x ¹⁰ CFU/mL. After 18 hours of culture, the cells were washed three times with PBS, and the Caco-2 cells were detached using Accumax. The amount of PSNP incorporated into Caco-2 cells was measured by flow cytometry analysis using FACSVerse.

Live S. thermophilus 1131 cells were cultured in M17 medium containing 1% lactose for 18 hours, washed with PBS, and then suspended in PBS to an OD ₆₀₀ of 10. Subsequent experiments were carried out in the same manner as for L. bulgaricus 2038. The bacterial concentration in the medium was calculated to be approximately 1 x 10 ⁷ CFU/mL.

The measurement results are shown in FIG.
The measurement results were normalized by dividing the numerical value of each group by the median fluorescence intensity of the control group so that the value of the control group was 1.000. In other words, the vertical axis of the graph is the relative value of the median fluorescence intensity (MFI). This is also true for Figures 3 to 8 below.
As a result, in the group to which L. bulgaricus 2038 was added (2038) or the group to which S. thermophilus 1131 was added (1131), PSNP uptake into Caco-2 was significantly suppressed compared to the group to which PBS was added (Control) (Figure 1, n = 5-6, *: p < 0.05 (Tukey-Kramer test)).

[Example 2: Confirmation of nanoplastic adsorption by lactic acid bacteria]
L. bulgaricus 2038 and S. thermophilus 1131 were prepared as described in Example 1. PSNP was added to Caco-2 culture medium at 200 μg/mL. To prevent aggregation, Tween 20 was added to a final concentration of 0.005% and the medium was ultrasonically cleaned for 30 seconds twice. L. bulgaricus 2038 or S. thermophilus 1131 was added to the medium at 1%. A control group was also prepared without lactic acid bacteria. After incubation in a _CO2 incubator at 37°C for 18 hours, the medium was passed through a 0.22 μm filter. The filter was washed with PBS using a syringe, and the trapped lactic acid bacteria were recovered. The fluorescence intensity was measured using a fluorescent plate reader.

As a result, the fluorescence was comparable to the background in all groups: the control group, the L. bulgaricus 2038-added group, and the S. thermophilus 1131-added group; no fluorescence was detected. This suggests that the suppression of PSNP uptake is not due to adsorption of PSNP to the two bacterial strains.

[Example 3: Inhibition of microplastic uptake]
Instead of the fluorescently labeled PSNP with a particle size of 20 nm used in Example 1, fluorescently labeled polyethylene microplastics (PSMP) with a particle size of 200 nm were added.
Other than that, the culture system, amount added, addition method, etc., and measurement method used were the same as in Example 1.

The measurement results are shown in FIG.
As shown in Figure 3, the uptake of microplastics was significantly suppressed in the group to which live L. bulgaricus 2038 bacteria were added (2038) or the group to which live S. thermophilus 1131 bacteria were added (1131) compared to the group to which PBS was added (Control) (n = 7, *: p < 0.05 (Tukey-Kramer test)).

[Example 4: Inhibition of nanoplastic uptake by heat-treated bacterial cells]
The cells in Example 1 were incubated at 75°C for 1 hour to prepare heat-treated cells, and the same experiment was carried out.
Specifically, the above-mentioned nanoplastic (PSNP) was added to the apical side of a monolayer of Caco-2 cells cultured on a Transwell insert at 200 μg/mL. Simultaneously with the addition of PSNP, live or heat-treated L. bulgaricus 2038 cells, live or heat-treated S. thermophilus 1131 cells, or heat-treated S. thermophilus 1131 cells, which had been adjusted to an OD600nm of 10 and incubated at 75°C for 1 hour, were added to the medium at 1%. After 18 hours of culture, the amount of PSNP taken up by Caco-2 cells was measured using a FACSVerse.

The measurement results are shown in FIG.
As shown in Figure 4, nanoplastic uptake was significantly suppressed in the group to which heat-treated L. bulgaricus 2038 cells were added (heat-treated 2038) or the group to which heat-treated S. thermophilus 1131 cells were added (heat-treated 1131) compared to the group to which PBS was added (Control) (n = 6, *: p < 0.05 (Tukey-Kramer test)).

[Example 5: Inhibition of nanoplastic uptake by heat-treated L. bulgaricus cells]
Various strains of L. bulgaricus (P2306601, JCM 1002T, P2306602, P2306603, P2306604, P2306605, P2306606, P2306607, P2306608, 2038) were cultured in the same manner as in Example 1, and heat-treated to produce cells in the same manner as in Example 4. The inhibition of nanoplastic uptake in each strain was evaluated in the same manner as in Example 4.

The measurement results are shown in FIG.
Figure 5 suggests that for all strains of L. bulgaricus, nanoplastic uptake was suppressed compared to the PBS-added group (control) (n=3).

[Example 6: Inhibition of nanoplastic uptake by heat-treated S. thermophilus cells]
Various strains of S. thermophilus (P2306609, P2306610, P2306611, P2306612, P2306613, JCM 17834T, 1131) were cultured in the same manner as in Example 1, and heat-treated in the same manner as in Example 4. The inhibition of nanoplastic uptake in each strain was evaluated in the same manner as in Example 4.

The measurement results are shown in FIG.
Figure 6 suggests that nanoplastic uptake was suppressed in all strains of S. thermophilus compared to the PBS-added group (control) (n=3).

[Example 7: Inhibition of nanoplastic uptake by heat-treated cells of other bacterial species and strains (1)]
Various lactic acid bacteria (L. lactis P2306614, LG21, R-1, S. thermophilus 1131) were cultured, and the heat-treated cells were evaluated for their ability to inhibit the uptake of nanoplastics (PSNP) in the same manner as in Example 4.
[Culture method]
L. lactis P2306614: Cultured in the same manner as S. thermophilus 1131 in Example 1, except that it was cultured in MRS medium.
LG21 (FERM BP-6999): This strain was cultured in the same manner as S. thermophilus 1131 in Example 1, except that it was cultured in MRS medium.
R-1 (FERM BP-10741): This strain was cultured in the same manner as S. thermophilus 1131 in Example 1, except that it was cultured in MRS medium.
1131 (S. thermophilus 1131): Cultured in the same manner as S. thermophilus 1131 in Example 1.
2038 (L. bulgaricus 2038): Cultured in the same manner as in Example 1 for L. bulgaricus 2038.

The measurement results are shown in FIG.
Figure 7 suggests that the uptake of nanoplastics was suppressed in all bacterial species and strains compared to the PBS-added group (control) (n=3).

[Example 8: Inhibition of nanoplastic uptake by heat-treated cells of other bacterial species and strains (2)]
Various lactic acid bacteria (L. paracasei YIT9029, B. longum BB536) were cultured, and the bacteria of Example 4 were cultured and the inhibition of nanoplastic uptake was evaluated as heat-treated bacterial cells in the same manner as in Example 4.
[Culture method]
L. paracasei YIT9029: Cultured in the same manner as S. thermophilus 1131 in Example 1, except that it was cultured in MRS medium.
B. longum BB536: Cultured in the same manner as S. thermophilus 1131 in Example 1, except that it was cultured in GAM medium.

The measurement results are shown in FIG.
Figure 8 suggests that the uptake of nanoplastics was suppressed in all bacterial species and strains compared to the control group where no addition was made (n=3).

[Summary]
From the above experimental results, it can be said that lactic acid bacteria, particularly bacteria belonging to the genus Lactobacillus such as Lactobacillus delbrueckii ssp. bulgaricus, and bacteria belonging to the genus Streptococcus such as Streptococcus thermophilus, control the uptake of microparticles into the body (including intracellularly).

The present invention provides a method for inhibiting the uptake of microparticles into the body and a method for producing food suitable for inhibiting the uptake of microparticles into the body, which prevents or improves diseases or conditions that can be improved by inhibiting the uptake of microparticles, and supports the maintenance and improvement of health in people who are not yet ill. The present invention can provide a food composition for maintaining and improving health, and a method for producing food. The present invention can also improve the nutrition of a variety of people, ensure healthy lifestyles, and promote welfare.

[Sequences listed in the sequence listing]
SEQ ID NO:1 16S rRNA gene, Lactobacillus delbrueckii ssp. bulgaricus 2038
SEQ ID NO:2 16S rRNA gene, Streptococcus thermophilus 1131

Claims (9)

A composition containing lactic acid bacteria for controlling the uptake of microparticles into the body. The composition according to claim 1, wherein the lactic acid bacteria are selected from the group consisting of bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Streptococcus, and bacteria belonging to the genus Bifidobacterium. The composition according to claim 1, wherein the lactic acid bacteria are bacteria belonging to Lactobacillus delbrueckii, Lactobacillus paracasei, Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus paragasseri, Lactobacillus lactis, or Bifidobacterium longum. The composition of claim 1, wherein controlling uptake means inhibiting uptake. The composition according to claim 1, wherein the uptake into the body is into intestinal epithelial cells. The composition of claim 1, wherein the microparticles are nanoplastics or microplastics. The composition described in claim 1 is for treating any of oxidative stress, DNA damage, and apoptosis caused by the ingestion of microparticles into the body. A composition according to any one of claims 1 to 7 for use as a probiotic or synbiotic. A method for reducing the risk of developing any of inflammatory bowel disease, colon cancer, and small intestine cancer, comprising the step of administering a composition containing lactic acid bacteria to a subject, thereby controlling the uptake of microparticles into the body.