CN109966321B

CN109966321B - Composition comprising a lactobacillus casei strain for protecting cells and tissues from particulate matter toxicity

Info

Publication number: CN109966321B
Application number: CN201811557788.0A
Authority: CN
Inventors: 姜京秀; 判哲镐; 车光贤; 洪性哲; 金周妍; 李昭暎; 崔一东; 李明熙
Original assignee: Korea Advanced Institute of Science and Technology KAIST
Current assignee: Korea Advanced Institute of Science and Technology KAIST
Priority date: 2017-12-21
Filing date: 2018-12-19
Publication date: 2022-12-27
Anticipated expiration: 2038-12-19
Also published as: KR101912258B1; CN109966321A

Abstract

The invention discloses a composition which contains a Lactobacillus casei strain as an effective ingredient and has a protective effect on the toxicity of particulate matters. The composition comprises a Lactobacillus casei strain, a disrupted product thereof, a culture thereof, or an extract of the strain, disrupted product or culture as an active ingredient, to inhibit cell damage caused by particulate matter toxicity, and to improve resistance to particulate matter toxicity.

Description

Composition comprising a strain of lactobacillus casei for protecting cells and tissues from particulate matter toxicity

Technical Field

The invention discloses a composition which contains a Lactobacillus casei strain as an effective ingredient and has a protective effect on the toxicity of particulate matters.

Background

Recently, yellow sand, which has appeared due to desertification in northern China and dry Mongolia areas, contains harmful heavy metals such as cadmium, copper and lead, and various harmful bacteria and molds, and has been recognized as a serious health hazard. In addition, recently, carbon compounds, sulfates, nitrates, and compounds of harmful heavy metals generated by combustion have also been rapidly increased due to the use of fossil fuels. It is known that such micro-combustion products (particulates, fine particulates) have many side effects on the human body.

Particulate Matter (PM) refers to dust floating in the air for a long time, and has a particle size of 10 μm or less, referred to as PM10, a particle size of 5 μm or less, referred to as PM5, a particle size of 2.5 μm or less, referred to as PM2.5, and a particle size of 1 μm or less, referred to as PM1. Among these particles, those having a particle size of 2.5 μm or less are referred to as fine particles.

These particles directly permeate into the human body through the respiratory system or the skin, directly damage the human body, and, when it rains, it is mixed into the rain water, and also affects crops and livestock. In particular, particulate matter having a particle size of 10 μm or less permeates into the bronchus through respiration, and causes various respiratory diseases such as asthma and chronic obstructive pulmonary disease, and various inflammations in body parts exposed to the air such as skin and eyeball. In addition, it is also a cause of cerebrovascular diseases such as cerebral hemorrhage, migraine and dementia, cardiovascular diseases, depression, intestinal dysfunction, reproductive dysfunction, hypokinesia, fetal growth disorder, and other diseases, and once absorbed into the body, it is accumulated without being easily excreted, and it is necessary to take measures as soon as possible to induce cancer or genetic diseases.

Prior art documents

Patent document

Patent document 1: KR 10-2017-0025450A

Disclosure of Invention

Technical problem

It is an object of the present invention to provide a composition comprising a lactobacillus casei strain as an active ingredient and having a protective effect against particulate matter toxicity.

Another object of the present invention is to provide a composition comprising a lactobacillus casei strain as an active ingredient and inhibiting cell damage caused by toxicity of particulate matter.

It is still another object of the present invention to provide a composition comprising lactobacillus casei strain as an active ingredient and having enhanced resistance to particulate matter toxicity.

Technical scheme

In one aspect, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by toxicity of particulate matter, comprising a lactobacillus casei strain, a disrupted product thereof, a culture thereof, or an extract of the strain, disrupted product or culture as an effective ingredient.

In the examples, the strain may be Lactobacillus casei HY2782 (Lactobacillus casei HY 2782), lactobacillus casei KCTC3109 (Lactobacillus casei KCTC 3109) or Lactobacillus casei KCTC13086 (Lactobacillus casei KCTC 13086).

In an embodiment, the pharmaceutical composition can prevent or treat growth inhibition caused by toxicity of the particulate matter.

In an embodiment, the pharmaceutical composition can prevent or treat reproductive dysfunction caused by particulate toxicity.

In embodiments, the pharmaceutical composition can prevent or treat a decrease in exercise performance caused by toxicity of the particulate matter.

In another aspect, the present invention provides a food composition for inhibiting cell damage caused by particulate toxicity, comprising a lactobacillus casei strain, a disrupted product thereof, a culture thereof, or an extract of the strain, disrupted product or culture as an active ingredient.

In an embodiment, the food composition can improve growth inhibition caused by toxicity of the particulate matter.

In an embodiment, the food composition can inhibit germ cell damage caused by particulate toxicity.

In an embodiment, the food composition can ameliorate reproductive dysfunction caused by particulate toxicity.

In an embodiment, the food composition can ameliorate the decline in exercise performance caused by particulate matter toxicity.

In another aspect, the present invention provides a cosmetic composition for inhibiting cell damage caused by particulate toxicity, comprising a lactobacillus casei strain, a disrupted product thereof, a culture thereof, or an extract of the strain, disrupted product or culture as an active ingredient.

In embodiments, the strain may be Lactobacillus casei HY2782 (Lactobacillus casei HY 2782), lactobacillus casei KCTC3109 (Lactobacillus casei KCTC 3109), or Lactobacillus casei KCTC13086 (Lactobacillus casei KCTC 13086).

In another aspect, the present invention provides a feed composition for inhibiting cytotoxicity caused by toxicity of particulate matter, comprising a lactobacillus casei strain, a disrupted product thereof, a culture thereof, or an extract of the strain, disrupted product or culture as an active ingredient.

In an embodiment, the feed composition can improve growth inhibition caused by toxicity of the particulate matter.

In embodiments, the feed composition can inhibit germ cell damage caused by particulate matter toxicity.

In embodiments, the feed composition can ameliorate reproductive dysfunction caused by particulate matter toxicity.

In embodiments, the feed composition can ameliorate the decline in exercise performance caused by particulate matter toxicity.

In an embodiment, the feed composition may have a feeding use selected from one or more of cattle, pigs, sheep, horses, rabbits, dogs, cats, chickens, turkeys, ducks, turtles, snakes, fish, earthworms, spiders, insects, and nematodes.

In an embodiment, the nematode can be caenorhabditis elegans.

Effects of the invention

The invention provides a new application of lactobacillus casei strain.

In one aspect, the present invention provides a composition comprising a lactobacillus casei strain as an active ingredient and having a protective or mitigating effect on particulate matter toxicity.

In another aspect, the present invention provides a composition comprising a lactobacillus casei strain as an active ingredient and inhibiting cell damage caused by toxicity of particulate matter.

In another aspect, the present invention provides a composition comprising a lactobacillus casei strain as an active ingredient and enhancing resistance to particulate matter toxicity.

Drawings

Fig. 1 is a graph confirming the protective efficacy of lactobacillus casei HY2782 strain used in the present invention against CCD-18Co human apoptosis caused by particulate matter 1 containing Polycyclic Aromatic Hydrocarbons (PAHs).

For CCD-18Co cells, granule 1 (500. Mu.g/mL) or Lactobacillus casei HY2782 strain (1X 10) was added separately ⁶ CFU/mL), or both of the pellets 1 and the lactobacillus casei HY2782 strain were added and cultured for 72 hours, and then the cell survival rate was measured. Statistical significance was shown together, compared to the group of particles dosed alone, as statistical significance<0.001. From this graph, it can be clearly confirmed that the lactobacillus casei strain has a protective effect against apoptosis of human cells caused by the particulate matter 1.

FIG. 2 is a graph confirming the protective efficacy of the Lactobacillus casei HY2782 strain used in the present invention against CCD-18Co human cells caused by the particulate matter 2 containing heavy metals.

For CCD-18Co cells, granular 2 (500. Mu.g/mL) or cheese was dosed aloneLactobacillus HY2782 strain (1X 10) ⁶ CFU/mL), or both of the granules 2 and the lactobacillus casei HY2782 strain were added and cultured for 72 hours, and then the cell survival rate was measured. Statistical significance is shown together, compared to the group of particles dosed alone, statistical significance is P<0.05，***P<0.001. From this graph, the protective efficacy of the lactobacillus casei strain against human apoptosis caused by particulate matter 2 can be clearly confirmed.

Fig. 3 is a photograph showing the protective effect of the lactobacillus casei HY2782 strain used in the present invention on the human apoptosis of CCD-18Co caused by the particulate matter 1 containing Polycyclic Aromatic Hydrocarbons (PAH), which is observed by a fluorescence microscope after being simultaneously stained with Hoechst33342 and propidium iodide (propidium iodide).

For CCD-18Co cells, granule 1 (500. Mu.g/mL) or Lactobacillus casei HY2782 strain (1X 10) was added ⁶ CFU/mL), or both of the pellet 1 and the lactobacillus casei HY2782 strain were added, and after 24 hours of culture, the cells were stained with Hoechst33342 (blue) and propidium iodide (red) fluorescent reagents, and then the fluorescence detected from CCD-18Co was observed by a fluorescence microscope. Hoechst33342 has the property of staining all cells, whereas propidium iodide has the property of staining only cells with disrupted cell membranes. Thus, when staining with two fluorescent reagents was performed simultaneously, the surviving normal cells were stained only with Hoechst33342 reagent and were blue, while the cells with disrupted cell membranes were stained with Hoechst33342 reagent and propidium iodide reagent simultaneously and showed pink fluorescence. As a result of the experiment, when the granules 1 were injected alone, propidium iodide-stained cells (PI-positive cells) were high, whereas when the lactobacillus casei HY2782 strain and the granules 1 were injected simultaneously, propidium iodide-stained cells were significantly reduced. From this, it was clearly confirmed that Lactobacillus casei has a protective effect against apoptosis of human cells caused by particulate matter.

FIG. 4 is a graph showing the results of fluorescence microscopic observation of FIG. 3 quantitatively by using the proportion of Propidium Iodide (PI) stained cells to all stained CCD-18Co cells. Will be statistically significantTogether, the results show that the statistical significance was P compared to the control group<0.05，***P<0.001, when the group to which the pellet 1 was fed alone was compared with the group to which the pellet 1 and the Lactobacillus casei HY2782 strain were fed simultaneously, ^### P<0.001. from this graph, it can be clearly confirmed that the lactobacillus casei strain has protective efficacy against apoptosis of human cells caused by particulate matter.

FIG. 5 is a graph confirming the protective efficacy of Lactobacillus casei HY2782 strain used in the present invention against particulates 1 and 2 through animal experiments for which C.elegans (Caenorhabditis elegans) was used as a model animal.

For caenorhabditis elegans, escherichia coli OP50 (e.coli OP 50) or lactobacillus casei HY2782 strain of the present invention was fed as a conventional bait for four days. At this time, in order to confirm the toxicity of pellets 1 and 2, pellets were further fed, respectively. To confirm the effect of the particulate matter on the growth of the model animals, the lengths of caenorhabditis elegans were measured four days later, and the lengths of 50 individuals per group were measured. Statistical significance is shown together, with statistical significance P <0.001 compared to control group fed with e. From this graph, it can be clearly confirmed that the lactobacillus casei strain has a protective efficacy against growth inhibition caused by the particulate matters 1 and 2.

Fig. 6 is a graph confirming the protective efficacy of lactobacillus casei HY2782 strain used in the present invention against the granules 1 and 2 through an animal experiment, and for the experiment, the reproductive ability of Caenorhabditis elegans (Caenorhabditis elegans) as a model animal was investigated.

For caenorhabditis elegans, escherichia coli OP50 (e.coli OP 50) or the lactobacillus casei HY2782 strain of the present invention was fed as a conventional bait for four days. At this time, in order to confirm the toxicity of pellets 1 and 2, pellets were further fed, respectively. To verify the effect of the particles on reproductive function of the model animals, the total number of eggs laid per group was observed. As a result of the experiment, it was confirmed that the toxicity of the particulate matter has an effect on the reproductive function, and that the strain of Lactobacillus casei has a protective effect on the toxicity of the particulate matter affecting the reproductive function. The total number of eggs laid was measured for 30 C.elegans per group. Statistical significance is shown together, compared to the control group fed with e.coli only, the statistical significance is P <0.01 compared to the control group fed with e.coli only. From this graph, it can be clearly confirmed that the lactobacillus casei strain has a protective effect against the inhibition of reproductive function caused by the particulate matters 1 and 2.

Fig. 7 is a graph in which the protective efficacy of lactobacillus casei HY2782 strain used in the present invention against the particulate matter 1 was confirmed by animal experiments, and for this experiment, the exercise capacity of c.elegans (Caenorhabditis elegans) as a model animal was investigated.

For caenorhabditis elegans, escherichia coli OP50 (e.coli OP 50) or lactobacillus casei HY2782 strain of the present invention was fed as a general bait. At this time, in order to confirm the toxicity of the pellets 1, the pellets 1 were further fed, respectively. To confirm the effect of the particulate matter on the locomotor ability of the model animals, 15 C.elegans per group were used as subjects, and the number of body bends in 20 seconds was measured. In the group fed with E.coli OP50 as a conventional bait, the granules significantly reduced the motility of C.elegans, whereas in the group fed with Lactobacillus casei strain HY2782, this reduction did not occur. Statistical significance was shown together, with statistical significance of P <0.001 compared to control groups fed with e. From this graph, the protective efficacy of the lactobacillus casei strain against the inhibition of the motor capacity by the particulate matter can be clearly confirmed.

Fig. 8 is a graph in which the protective efficacy against the reduction of reproductive function caused by particulate matter toxicity according to the kind of lactobacillus casei strain used in the present invention was confirmed, and the data of the following table 4 was graphed.

In caenorhabditis elegans fed with escherichia coli OP50 (e.coli OP 50), the total number of eggs laid was significantly reduced in the group to which both escherichia coli OP50 and granular material 2 were simultaneously fed, as compared to the group to which escherichia coli OP50 was fed alone, showing reproductive toxicity caused by granular material 2. Similarly, it was also shown in C.elegans fed with Lactobacillus gasseri KCTC3163 strainThe reproductive toxicity caused by the particles 2. In contrast, in caenorhabditis elegans fed with both the strain lactobacillus casei HY2782 and the strain lactobacillus casei KCTC3109, no reproductive toxicity occurred due to the granulation 2. Statistical significance is shown together, compared to control group fed with e.coli OP50 alone, statistical significance is P<0.01 or P<0.001. When various microorganisms were introduced, the difference between the group into which the microorganisms were introduced alone and the group into which the microorganisms and the particulate matter were simultaneously introduced was # P<0.05 or ^## P<0.01, and, when there is no statistical significance, is expressed in n.s. From the graph, it can be clearly confirmed that the lactobacillus casei strain used in the present invention exhibits a protective effect against the reduction of reproductive function caused by particulate matter toxicity, and that such an effect is different depending on the kind of the strain.

Detailed Description

Hereinafter, the present invention will be described in detail.

The present invention provides the use of a lactobacillus casei strain, a homogenate thereof, a culture thereof, an extract of said strain, an extract of said homogenate, or an extract of said culture, for the preparation of a composition for the mitigation of particulate matter toxicity.

In embodiments, the particulate matter may comprise polycyclic aromatic hydrocarbons (polycyclic hydrocarbons) or heavy metals.

In an embodiment, the particulate matter may be PM10. The PM10 refers to particulate matter having a particle diameter of 10 μm or less.

Lactobacillus casei strains are representative lactic acid bacteria used for producing fermented milk products, and it has been known so far that lactobacillus casei improves intestinal flora, increases intestinal beneficial bacteria, reduces intestinal harmful bacteria, improves defecation function, enhances immunity, reduces allergy and asthma symptoms, etc., but the efficacy of alleviating particulate toxicity of lactobacillus casei is not disclosed.

The "active ingredient" in the present specification means an ingredient which exhibits a desired activity alone or together with a carrier or the like which is not active itself.

In one aspect, the compositions disclosed herein have a particulate toxicity-mitigating effect, a particulate toxicity-protecting effect, an effect of inhibiting cell damage caused by particulate toxicity, and an effect of enhancing resistance to particulate toxicity.

In embodiments, the composition can prevent or treat growth inhibition caused by toxicity of the particulate matter.

In embodiments, the composition can inhibit germ cell damage caused by particulate matter toxicity, or prevent or treat reduced reproductive function caused by toxic harmful substances.

In embodiments, the compositions can prevent or treat hypokinesia caused by particulate matter toxicity. Exercise performance refers to the ability to utilize muscles to perform exercise.

The strain in the present specification is improved or modified to have the same activity or better activity than that by a conventional physical, chemical mutation method or the like well known in the art to which the present invention pertains.

In an example, the Lactobacillus casei strain may be a Lactobacillus casei HY2782 (Lactobacillus casei HY 2782) strain.

In examples, the Lactobacillus casei HY2782 can be obtained according to the method disclosed in Korean patent publication No. 1997-0009934B1 (publication No. 1997: 6/19/1997, application No. KR 10-1993-0026829). The above documents are incorporated by reference in their entirety in the present application.

In another example, lactobacillus casei HY2782, which is a strain widely used in the production of fermented milk products, can be easily separated from a commercially available fermented milk product beverage containing lactobacillus casei HY2782 and used. The fermented milk product beverage used contained no other strains than lactobacillus casei HY2782. One bottle of sartorius was purchased in the market (product name: sartorius, manufacturer: korean sartorius ltd.). The above Yanleduo (Yakult) contains 2 × 10 ⁸ Lactobacillus casei HY2782 strain above CFU. Diluting the YANGLUO beverage to 10% with sterilized saline water ⁵ ～10 ⁶ After doubling, 1mL of the dilution was spread on MRS agar (agar) plate medium. Then, at 37 deg.C,When cultured under aerobic conditions for 48 hours, a colony of Lactobacillus casei HY2782 strain was formed. One of the formed colonies was inoculated on an MRS liquid medium and cultured at 37 ℃ for 24 hours to obtain a culture solution containing Lactobacillus casei HY2782. In general, in the production of a beverage, a sterilization treatment step is performed so that the fermented milk product beverage containing lactobacillus casei HY2782 is free from other strains, and therefore, it is obvious to those skilled in the art that the colony formed by culturing the fermented milk product beverage containing lactobacillus casei HY2782 is lactobacillus casei HY2782. The following table 1 shows the composition of 1L of MRS solid medium for MRS agar, and table 2 shows the composition of 1L of MRS liquid medium for MRS broth.

TABLE 1

Peptone (Peptone)	10.0g
		Beef extract (Beef extract)	10.0g
Yeast extract (Yeast extract)	5.0g
		Glucose (Dextrose)	20.0g
Polysorbate 80 (Polysorbate 80)	1.0g
		Ammonium citrate (Ammonium citrate)	2.0g
Sodium acetate (Sodi)um acetate)	5.0g
		Magnesium sulfate (Magnesium sulfate)	0.1g
Manganese sulfate (Manganese sulfate)	0.05g
		Dipotassium hydrogen phosphate (dipotasssium phosphate)	2.0g
Agar (Agar)	15.0g

TABLE 2

Peptone (Peptone)	10.0g
		Beef extract (Beef extract)	10.0g
Yeast extract (Yeast extract)	5.0g
		Glucose (Dextrose)	20.0g
Polysorbate 80 (Polysorbate 80)	1.0g
		Ammonium citrate (Ammonium citrate)	2.0g
Sodium acetate (Sodium acetate)	5.0g
		Magnesium sulfate (Magnesium sulfate)	0.1g
Manganese sulfate (Manganese sulfate)	0.05g
		Dipotassium hydrogen phosphate (dipotasium phosphate)	2.0g

In an embodiment, the lactobacillus casei strain may be 1 or more selected from lactobacillus casei HY2782, lactobacillus casei KCTC3109, and lactobacillus casei KCTC 13086.

In the examples, the strain is prepared by culturing the strain and centrifuging the culture solution, washing the culture solution with sterilized physiological saline, dispersing the solution in a solvent such as sterilized milk, lyophilizing the solution, and producing and using the lyophilized powder.

The disrupted material may be a product obtained by disrupting the strain itself by chemical or physical force.

The culture may refer to a substance including part or all of substances contained in a medium in which the strain is cultured, whether in a liquid or solid form, and may refer to, for example, a substance including metabolites or secretions as a culture product of the strain or a disrupted product thereof, and the strain itself may also be included in the culture. In addition, the culture may be a culture solution.

The extract may refer to a product obtained by extracting the strain itself, a disrupted product of the strain, a culture of the strain, or a mixture thereof, regardless of the extraction method, the extraction solvent, the extracted component or the form of the extract, and is a broad concept including a substance which can be processed or treated by other methods such as fractionation, concentration, and the like after the extraction.

In an embodiment, the composition may be a pharmaceutical composition.

The pharmaceutical composition may further contain, in addition to the active ingredient, a preservative, a stabilizer, a wettable powder or an emulsion enhancer, a pharmaceutically acceptable adjuvant such as a salt for osmotic pressure regulation and/or a buffer, and other therapeutically useful substances, and may be formulated into various oral preparations or non-oral preparations according to conventional methods.

The oral preparations include, for example, tablets, pills, hard capsules, soft capsules, liquids, suspensions, emulsifiers, syrups, powders, fine granules, pellets and the like, and these preparations may contain, in addition to the active ingredient, surfactants, diluents (e.g., lactose, glucose, sucrose, mannitol, sorbitol, cellulose, glycine), lubricants (e.g., silicon dioxide, talc, stearic acid, magnesium stearate, calcium stearate and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinyl chloride pyrrolidone, and the like, and, according to circumstances, may contain pharmaceutical additives such as starch, agar, disintegrators of alginic acid or its sodium salt, absorbents, colorants, flavoring agents, and sweeteners. Such tablets may be prepared by conventional mixing, granulating or coating methods and the like.

In addition, the non-oral preparation may be in the form of a transdermal administration preparation, for example, injection, drops, ointment, emulsion, gel, cream (cream), spray, suspension, emulsion, suppository, patch, etc., but is not limited thereto.

The determination of the dose of the effective ingredient is within the level of those skilled in the art, and although the daily dose of the drug varies depending on various factors such as the degree of onset, the onset time, the age, the health condition, complications and the like of the subject, the dose is not limited in any way to the scope of the present invention, and may be administered in 1. Mu.g/kg to 2000mg/kg on the one hand and 50. Mu.g/kg to 500mg/kg on the other hand, in 1 to 3 divided doses per day, based on an adult.

The pharmaceutical composition can be a skin external preparation, which is a general name of any product applied to the skin, and can contain medicaments in various dosage forms.

In an embodiment, the composition may be a food composition.

The food composition may be in a liquid or solid form, and for example, may include various foods, beverages, chewing gums, teas, vitamin complexes, health supplementary foods, and the like, and may be used in the form of powders, granules, tablets, capsules, fermented milks, beverages, and the like. The food compositions of various dosage forms can be appropriately selected and mixed by those skilled in the art according to the dosage form or the purpose of use without difficulty, in addition to the effective ingredients, the ingredients generally used in the respective fields, and when used together with other raw materials, synergistic effects may occur.

Liquid components that may be contained in addition to the active ingredients disclosed in the present specification are not particularly limited, and various flavors or natural carbohydrates may be contained as additional components as in a usual beverage. As examples of the natural carbohydrates, monosaccharides, disaccharides such as glucose, fructose, etc., polysaccharides such as maltose, sucrose, etc., general saccharides such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc., are given. As the flavoring agent, natural flavoring agents (Thaumatin, stevia (stevia) extracts (e.g., steviolbioside a, glycyrrhizin, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) may be advantageously used, and the proportion of the natural carbohydrate may be generally about 1 to 20 g per 100 ml of the composition disclosed in the present specification, and on the other hand about 5 to 12 g.

In one aspect, the food composition may include a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colors and flavor enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents for carbonated beverages, and the like. In another aspect, pulp for making natural juices and vegetable beverages may be included. The above components may be used alone or in combination. The proportion of the above additives may vary, but is generally selected from the range of 0.001 to about 20 parts by weight per 100 parts by weight of the composition disclosed herein.

In an embodiment, the composition may be a cosmetic composition.

The cosmetic composition may further include a functional additive and components contained in general cosmetic compositions, in addition to the active ingredients. The functional additive may include a component selected from the group consisting of water-soluble vitamins, fat-soluble vitamins, high molecular peptides, high molecular polysaccharides, sphingolipids and seaweed extracts. Examples of the other ingredients to be contained include an oil and fat ingredient, a moisturizer, an emollient, a surfactant, an organic/inorganic pigment, an organic powder, a sunscreen agent, a preservative, a bactericide, an antioxidant, a plant extract, a pH adjuster, alcohol, a pigment, a perfume, a blood circulation promoter, a coolant, an antiperspirant, and purified water.

The formulation of the cosmetic composition is not particularly limited, and may be appropriately selected according to the purpose. For example, one or more formulations selected from powders, skin lotions, toilet lotions, skin lotions, milky lotions, milk lotions, moisturizing lotions, nourishing lotions, massage creams, nourishing creams, moisturizing creams, hand creams, foundation lotions, essence lotions, nourishing essences, facial masks, shampoos, soaps, face washes, facial cleansers, facial washes, body lotions, and body washes can be prepared, but not limited thereto.

When the formulation of the present invention is a paste, cream or gel, as a carrier component, animal fiber, vegetable fiber, wax, paraffin, starch, tragacanth, a cellulose derivative, polyvinyl alcohol, silicone, bentonite, silica, talc, zinc oxide, or the like can be used.

When the formulation of the present invention is a powder or a spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier ingredient, and particularly, when it is a spray, an accelerator such as chlorofluorocarbon, propane/butane or dimethyl ether may be further contained.

When the dosage form of the present invention is a solution or emulsion, as a carrier ingredient, a solvent, a solvating agent or an opacifying agent is used, and examples include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil (1, 3-butylglycol oil), glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitol.

When the dosage form of the present invention is a suspension, as a carrier ingredient, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as Ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and Polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, or the like can be used.

When the formulation of the present invention is a surfactant-containing detergent, as a carrier ingredient, fatty alcohol sulfate ether, sulfosuccinic acid monoester, isethionate, imidazoline derivative, methyltaurate, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or alkoxylated glycerin fatty acid ester, and the like can be included.

When the formulation of the present invention is a soap, as a carrier ingredient, alkali metal salts of fatty acids, fatty acid half esters, fatty acid protein hydrolysates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerin, sugars, and the like can be used.

In an embodiment, the composition may be a feed composition.

In an embodiment, the composition may be a livestock feed composition.

In examples, the composition may have a feeding use of 1 or more selected from the group consisting of fishes such as cattle, pigs, sheep, horses, rabbits, dogs, cats, chickens, turkeys, ducks, turtles, snakes, flounders, eels, earthworms, spiders, insects, and nematodes.

In an embodiment, the nematode can be caenorhabditis elegans.

Hereinafter, the present invention will be described in further detail by examples. It will be apparent to those skilled in the art that these examples are merely illustrative of the present invention, and the scope of the present invention is not limited to these examples.

Example 1: acquisition of particulate matter

There are two types of particles used in this specification, purchased and used from SIGMA-ALDRICH. Both of the particulate matters are PM10 (particulate matter 10 having a particle size of about 10 μm), the particulate matter 1 is characterized by containing a large amount of Polycyclic Aromatic Hydrocarbons (PAHs), and the particulate matter 2 is characterized by containing a large amount of heavy metals such as arsenic, cadmium, nickel, and lead.

Example 2: obtaining of Lactobacillus casei Strain

The strain used in this example was the well-known strain of Lactobacillus casei HY2782 (Lactobacillus casei HY 2782), which was obtained and used from the manufacturer (strain) korean playduo flat probiotic factory. The lactobacillus casei HY2782 lactobacillus powder (permit number: 20140017107, product manufacture number: 201400171077, approval date: 2016, 7 and 19) is a light yellow powder with no peculiar smell and odor and an inherent fragrance. The lactobacillus casei HY2782 strain is deposited at 19.12.2017 in the Korean type culture Collection (KCTC) of the depository and has the deposit number KCTC 13438BP.

In addition, as well-known strains that are easily obtained by those skilled in the art, commercially available strains of lactobacillus casei KCTC3109 (the same strain as lactobacillus casei ATCC393 that is easily obtained from the ATCC in the usa), lactobacillus casei KCTC13086, lactobacillus paracasei KCTC3189, lactobacillus gasseri KCTC3163 (the same strain as lactobacillus gasseri ATCC33323 that is easily obtained from the ATCC in the usa) were purchased from the korean typical strain collection (KCTC) and used in the following experiments.

In the following experimental examples, the efficacy of lactobacillus casei in the protection of particulate matter in the intestinal membrane cell (ex vivo) and caenorhabditis elegans (in vivo) models will be evaluated. Based on this, it is intended to provide a composition having a mitigating effect on the toxicity of particulate matter.

Experimental example 1: human apoptosis effect caused by particulate matter, and apoptosis protective effect based on Lactobacillus casei strain

In this experimental example, the apoptosis effect of human body caused by particulate matter and the apoptosis protective effect based on lactobacillus casei strain were confirmed. For this purpose, two kinds of particulate matter are used, the particulate matter 1 being a particulate matter containing a large amount of polycyclic aromatic hydrocarbons, and the particulate matter 2 being a particulate matter containing a large amount of heavy metals.

CCD-18Co cells, which are a human normal large intestine cell line, were purchased and used from Korean cell line Bank (Seal). CCD-18Co cells were cultured and maintained in 5% carbon dioxide at 37 ℃ using RPMI medium (Roswell Park Memori Institute medium) supplemented with 10% Fetal Bovine Serum (FBS), 10U/mL penicillin, 100. Mu.g/mL streptomycin.

CCD-18Co cells were dispensed into 48-well plates to achieve 2X 10 cells per well ⁴ The cells were cultured for 24 hours and allowed to attach. Then, pellet 1 (500. Mu.g/mL), pellet 2 (500. Mu.g/mL) or Lactobacillus casei HY2782 strain (1X 10) ⁶ CFU/mL), or both of the granules and lactobacillus casei HY2782 strain, and cultured for 72 hours. After the culture was completed, cell survival was measured using Ez-Cytox (Daeil Lab Science Co., ltd., korea) Kit (Kit). After adding the Ez-Cytox solution to the medium diluted 10 times, the culture was performed for 1 hour. Then, after completely removing the particulate matter by centrifugation, absorbance at 450nm was measured using only the supernatant. The cell survival rate is in direct proportion to the absorbance, and the cell survival rate of the control group is set as 100%, and the cell growth of the experimental group is calculatedAnd (4) storage rate.

As a result of the experiment, when a large amount of the particulate matter 1 (500. Mu.g/mL) containing polycyclic aromatic hydrocarbon was separately charged, the survival rate of CCD-18Co cells was significantly decreased. When granule 1 and Lactobacillus casei HY2782 strain (1 × 10) are added simultaneously ⁶ CFU/mL), it could be confirmed that the cell survival rate was significantly recovered compared to the experimental group treated with only the granule 1 alone (fig. 1). Similarly, a large amount of the particulate matter 2 containing heavy metals also decreased the cell survival rate, and apoptosis was inhibited by simultaneously administering the particulate matter 2 and the lactobacillus casei HY2782 strain (fig. 2). In conclusion, the lactobacillus casei strain has a alleviating effect on apoptosis of human cells caused by particles containing polycyclic aromatic hydrocarbons or heavy metals.

Experimental example 2: the human apoptosis effect caused by the particulate matter and the apoptosis protective effect based on the Lactobacillus casei strain were confirmed again by fluorescence microscopy

In this experimental example, the degree of damage of the cell membrane was observed by a fluorescence microscope, and the apoptosis effect of human body caused by the particulate matter and the apoptosis protective effect based on lactobacillus casei strain were confirmed again. For this purpose, cells were simultaneously stained with the DNA fluorescent stains Hoechst33342 (blue fluorescence) and propidium iodide (red fluorescence), and observed with a fluorescence microscope. Hoechst33342 reagent is cell membrane permeable while propidium iodide is not, so that living cells are stained only with Hoechst33342 reagent, and blue, whereas cells with damaged cell membranes are stained with Hoechst33342 and propidium iodide reagent at the same time, and show pink fluorescence.

CCD-18Co cells were dispensed into 6-well plates to reach 3X 10 per well ⁵ After the culture, the cells were cultured for 24 hours and allowed to attach.Then, granule 1 (500. Mu.g/mL) or Lactobacillus casei HY2782 strain (1X 10) ⁶ CFU/mL), or both of granule 1 and lactobacillus casei HY2782 strain, and cultured for 24 hours. Then, the cells were washed twice with a Dulbecco's phosphate-buffered saline solution, and stained for 15 minutes at 37 ℃ in the dark after being added to a medium containing Hoechst33342 (2 ng/mL) and propidium iodide (20. Mu.g/mL) reagents. The stained cells were washed with a Dulbecco's phosphate buffered saline solution 1 time and then dried in the dark at room temperature for two hours. Then, a fluorescence microscope (Nikon, TE-2000) was used to take a fluorescence microscope photograph. In order to quantitatively compare the degree of cell membrane damage, after three arbitrary shots of each experimental group, the proportion (%) of Propidium Iodide (PI) -stained cells to the total stained cells was calculated and compared.

As a result of the experiment, in the control group (control) and in the case of using only Lactobacillus casei HY2782 strain (1X 10) ⁶ CFU/mL) showed only blue fluorescence in all of the cells treated alone, and thus, no damage to cell membranes was observed at all. When a large amount of the pellet 1 (500. Mu.g/mL) containing the polycyclic aromatic hydrocarbon was separately injected, pink fluorescent cells were significantly increased, and thus it was confirmed that the cell membrane of the CCD-18Co cell was seriously damaged and the cell membrane was damaged by the pellet 1 (FIGS. 3 and 4). When the particulate matter 1 and Lactobacillus casei HY2782 strain (1X 10) are added simultaneously ⁶ CFU/mL), the proportion of pink cells was significantly reduced compared to the treatment group of granulate 1 alone. Therefore, it was confirmed again that the protective effect of the lactobacillus casei strain against apoptosis in human body caused by toxicity of particulate matter was extremely significant.

Experimental example 3: model animal caenorhabditis elegans growth inhibition effect caused by particulate matters and recovery efficiency of lactobacillus casei strain on particulate matter toxicity

To confirm the toxicity of the particulate matter and the recovery effect based on the lactobacillus casei strain, caenorhabditis elegans was used as an animal model. Caenorhabditis elegans used a wild-type N2 strain (strain) purchased from the american nematode Genetics Center (CGC). As bait, E.coli OP50 was fed, maintained at 20 ℃ and subjected to the experiment. Coli OP50 was also purchased and used from the american nematode genetics center.

Caenorhabditis elegans (Caenorhabditis elegans) is commonly used as a model animal in biological and chemical research. The caenorhabditis elegans is beneficial to animal research ethics by replacing rodent experiments. Pharmaceutical and functional food compositions have been further developed from existing ex vivo experiments (in vitro) to be able to evaluate individual level in vivo (in vivo) activity and efficacy. In addition, recently, such caenorhabditis elegans has been widely used in the research of functional discovery and action mechanism of lactic acid bacteria in a plurality of well-known research papers.

Eggs of only caenorhabditis elegans were collected and used in toxicity evaluation experiments. Escherichia coli OP50 and Lactobacillus casei HY2782 were spread on NGM (Medium) agar medium, and pellets 1 and 2 were added at a concentration of 1mg/mL for toxicity evaluation. Eggs of caenorhabditis elegans were scattered on the thus prepared medium coated with the granules and Escherichia coli or Lactobacillus casei HY2782 strain, and cultured for 3-4 days. Then, the morphology of caenorhabditis elegans was observed by a stereomicroscope, and a photomicrograph was taken, and the length of caenorhabditis elegans was measured by the Image J program (National Health Institute, USA). According to each experimental group, the length of 25-50 nematodes was measured.

As a result of the experiment, the Lactobacillus casei strain did not exert an influence on the growth of C.elegans as a model animal, as compared to E.coli OP50 as a conventional bait for C.elegans. That is, it was confirmed that the strain of Lactobacillus casei as a bait was not much different from that of Escherichia coli as a conventional bait. However, when Escherichia coli was charged with a large amount of the granules 1 containing polycyclic aromatic hydrocarbons or a large amount of the granules 2 containing heavy metals, the growth of C.elegans as a model animal was significantly reduced (FIG. 5). It can thus be confirmed that the particulate matter induces toxicity at an individual level. In contrast, when both the granules and the lactobacillus casei HY2782 strain were administered simultaneously, it was confirmed that there was no toxic effect at all on the growth of caenorhabditis elegans, and thus it was confirmed that the lactobacillus casei strain was able to alleviate the individual level of growth toxicity induced by the granules (fig. 5). This effect is independent of the kind of the particulate matter and can be confirmed in both the particulate matter 1 and the particulate matter 2.

Experimental example 4: inhibition effect of caenorhabditis elegans reproductive function of model animal caused by particulate matter and recovery effect of lactobacillus casei strain on particulate matter toxicity

In this experimental example, in order to verify again the toxicity of the granules and the recovery efficacy based on lactobacillus casei, not only the size of caenorhabditis elegans but also the total number of eggs laid were observed, thereby observing the influence on the reproductive ability.

Eggs of only caenorhabditis elegans were collected and used in toxicity evaluation experiments. Escherichia coli OP50 and Lactobacillus casei HY2782 were spread on NGM (Medium) agar medium, and pellets 1 and 2 were added at a concentration of 1mg/mL for toxicity evaluation. Eggs of caenorhabditis elegans were scattered on the thus prepared medium coated with the pellets and Escherichia coli or Lactobacillus casei HY2782 strain and cultured for 64 hours. Then, each group of caenorhabditis elegans was transferred to a medium coated with E.coli OP50 every day, and the number of eggs laid per day was observed. The number of eggs laid per day was divided by 30 to calculate the number of eggs laid per model animal, and the total number of eggs laid for 5 days was added as the total number of eggs laid per model animal.

As a result of the experiment, the Lactobacillus casei strain did not have a great influence on the reproduction of C.elegans as a model animal, compared to E.coli OP50 as a conventional feed for C.elegans. That is, it was confirmed that when feeding the lactobacillus casei strain as a bait, there was not much difference in reproductive ability compared to escherichia coli as a conventional bait. However, when a large amount of the particulate matter 1 (1 mg/mL) containing polycyclic aromatic hydrocarbon was administered to E.coli, the reproductive capacity of C.elegans as a model animal was decreased, and when a large amount of the particulate matter 2 (1 mg/mL) containing heavy metal was administered, the reproductive capacity of C.elegans as a model animal was significantly decreased (FIG. 6). It can thus be confirmed that the particulate matter induces toxicity at an individual level. Further, it was confirmed that when the granules and lactobacillus casei HY2782 strain were simultaneously administered, there was no toxic effect on the reproductive ability of c.elegans at all, and thus it was confirmed that the lactobacillus casei strain was able to alleviate the individual level reproductive toxicity induced by the granules (fig. 6). This effect is independent of the kind of particulate matter, and can be confirmed in both the particulate matter 1 and the particulate matter 2.

Experimental example 5: inhibition effect of caenorhabditis elegans movement ability of model animal caused by particulate matters and recovery effect of lactobacillus casei strain on particulate matter toxicity

In this experimental example, in order to verify again the toxicity caused by the particulate matter and the recovery efficacy based on lactobacillus casei, not only the size and total number of eggs laid by caenorhabditis elegans but also the movement of caenorhabditis elegans was observed, and the influence on the motor ability was verified.

Eggs of only caenorhabditis elegans were collected and used in toxicity evaluation experiments. Escherichia coli OP50 and Lactobacillus casei HY2782 were applied to NGM (growth medium) agar medium, and a large amount of pellet 1 containing polycyclic aromatic hydrocarbon was added at a concentration of 1mg/mL for toxicity evaluation. Eggs of caenorhabditis elegans were scattered on the thus prepared medium coated with the granules and the Escherichia coli or Lactobacillus casei HY2782 strain, and then cultured for 96 hours. Then, each group of caenorhabditis elegans was transplanted onto a glass slide with physiological buffered saline. The video was then taken with a stereomicroscope and a microscope camera (Jenopitk, progress griyphax, germany). The number of body bends (body bending) of C.elegans in 20 seconds was measured and investigated for 15 nematodes per group.

As a result of the experiment, the granule 1 significantly reduced the number of body-bending times of caenorhabditis elegans with the addition of E.coli OP50 as a conventional feed for caenorhabditis elegans. On the contrary, when lactobacillus casei HY2782 strain was administered alone or in combination with lactobacillus casei HY2782 strain and the granule 1, the number of times of body bending was not reduced at all as compared with the control group (fig. 7). It was thus confirmed that the decrease in exercise ability caused by the particulate matter could be significantly recovered by the ingestion of lactobacillus casei.

Experimental example 6: comparison of the efficacy of Lactobacillus casei strains in restoring particulate toxicity

In this experimental example, in order to compare the efficacy of different species of lactobacillus casei for reducing the toxicity of particulate matter, the effect of various microorganisms on the growth and reproductive ability of caenorhabditis elegans was tested according to the methods described in said experimental examples 3 and 4, respectively.

As a result of the experiment, as shown in table 3, when escherichia coli was fed, the growth inhibition rate by the particulate matter was 90.5%, while the growth inhibition rates of lactobacillus casei HY2782 and KCTC13086 were 100.3% and 96.7%, respectively, confirming that the lactobacillus casei strain effectively alleviated the growth inhibition toxicity by the particulate matter. In contrast, the growth inhibition rates of lactobacillus paracasei KCTC3189 and lactobacillus gasseri KCTC3163 were 82.3% and 89.1%, respectively, confirming that the strains had no efficacy of alleviating particulate toxicity. Therefore, it can be confirmed that even if the strains belong to the same genus lactobacillus (genus), when species (species) are different, the particulate matter toxicity-alleviating efficacy is different depending on the species of the strains. In view of the above, it could be confirmed that the lactobacillus casei strain effectively alleviates toxicity caused by particulate matter.

As shown in table 4 and fig. 8, when escherichia coli was administered, the reproduction inhibition rate by the particulate matter was 61.3%, while the reproduction inhibition rates of lactobacillus casei HY2782 and lactobacillus casei KCTC3109 were 82.2% and 82.7%, respectively, and it was confirmed that lactobacillus casei effectively reduced the reproduction inhibition toxicity by the particulate matter, whereas the reproduction inhibition rate of lactobacillus gasseri KCTC3163 was 66.7%, and it was confirmed that there was no efficacy of reducing the particulate matter toxicity. Therefore, it was confirmed that even if the strains belong to the same genus lactobacillus (genus), the efficacy of alleviating particulate toxicity differs depending on the species of the strain when the species (species) are different. In view of the above, it could be confirmed that the lactobacillus casei strain effectively alleviates toxicity caused by particulate matter.

TABLE 3

TABLE 4

The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the scope of the present invention.

Formulation example 1' tablet

150mg of Lactobacillus casei culture, 100mg of glucose, 50mg of red ginseng extract, 96mg of starch and 4mg of magnesium stearate were mixed, 40mg of 30% ethanol was added to form granules, and then the granules were dried at 60 ℃ and compressed into tablets by a tablet press.

Preparation example 2 granules

Mixing 150mg of Lactobacillus casei culture, 100mg of glucose, 50mg of Ginseng radix Rubri extract and 600mg of starch, adding 100mg of 30% ethanol, granulating, drying at 60 deg.C to obtain granule, and filling into bag. The final weight of the contents was adjusted to 1g.

Preparation example 3 drink

150mg of Lactobacillus casei culture, 10mg of glucose, 50mg of red ginseng extract, 2g of citric acid and 187.8g of refined water were mixed and bottled. The final volume of the contents was adjusted to 200mL.

Preparation example 4 preparation of health food

A.casei culture 1000mg

Vitamin mixture

70 mu g of vitamin A acetate

1.0mg of vitamin e

0.13mg

0.15mg of vitamin B2

0.5mg of vitamin B6

0.2 μ g

.

Inorganic mixture

1.75mg of ferrous sulfate

.

.55mg

.

The composition ratio of the vitamin and mineral mixture is a composition in which foods relatively suitable for health foods are mixed according to a preferred embodiment, but the mixing ratio may be changed arbitrarily, and the above components may be mixed and then granulated according to a conventional method for preparing health foods, and used in the preparation of health foods according to a conventional method.

Preparation example 5 preparation of health drink

.

A.once....2 g of the concentrated solution of Japanese apricot

.

The method is 900mL together with refined water

According to a conventional method for producing a health drink, the above components are mixed, stirred and heated at 85 ℃ for about 1 hour, and the resulting solution is filtered, filled in a sterilized 2-liter container, sealed and sterilized, and then stored under refrigeration. Thereafter, it is used in the preparation of the health drink composition of the present invention.

The composition ratio is a composition in which components suitable for favorite beverages are mixed according to a preferred embodiment, but the mixing ratio may be arbitrarily changed according to a preference degree of a region or a nation such as a demand hierarchy, a demand country, a use purpose, and the like. Those skilled in the art will be able to make numerous applications and variations within the scope of the present invention in light of the above teachings.

The foregoing has been a detailed description of certain portions of this disclosure. It will be apparent to those skilled in the art that this particular technique is only one preferred embodiment and the scope of the present invention is not limited thereto. Accordingly, the true scope of the invention is defined by the following claims and their equivalents.

Claims

1. Use of Lactobacillus casei HY2782 strain in the preparation of a composition for alleviating particulate toxicity,

wherein the particulate matter contains polycyclic aromatic hydrocarbons or heavy metals.

2. The use according to claim 1, wherein,

the particulate matter is PM10.

3. The use according to any one of claims 1 or 2, wherein,

the composition is a feed composition.