KR20230053941A - A Composition for improving function of sperm and its use - Google Patents

Abstract

The present application relates to a composition for improving sperm function and uses thereof. Specifically, the present application includes Lactobacillus rhamnosus as an essential component, which has the optimal effect in improving sperm function. In particular, the composition can increase sperm motility, viability, concentration, etc.

Description

A composition for improving sperm function and its use {A Composition for improving function of sperm and its use}

The present application relates to a composition for improving sperm function composed of Lactobacillus rhamnosus.

This application relates to a method for preparing the composition.

This application relates to various uses of the composition.

As the age of marriage and childbirth increases, male infertility with sperm deficiency and azoospermia is also increasing.

Male infertility is caused by various causes such as abnormal hormone secretion, exposure to environmental hormones, smoking, drinking, and stress.

In addition, as the size of the companion animal market grows, markets for efficient pregnancy of companion animals and artificial insemination for mating with specific species are also growing.

In order to solve male infertility and efficiently conceive companion animals, we are trying to freeze-preserve sperm in sperm banks.

However, when sperm are thawed at the necessary time after cryopreservation, there is a problem such as a decrease in sperm function (eg, sperm motility, viability, etc.).

That is, since sperm cryopreservation cannot increase sperm motility and simply stores sperm, it cannot be a fundamental solution to solve sperm deficiency and artificial insemination failure.

In order to solve this problem, it is required to develop a method capable of improving sperm function.

1. US Patent No. 7829079

1. Int J Reprod Biomed. 2021 Mar 21;19(3):235-244. 2. Randomized Controlled Trial Andrology. 2017 May;5(3):439-444.

An object of the present application is to provide a composition for improving sperm function composed of Lactobacillus rhamnosus.

Another object of the present application is to provide a method for preparing the composition.

Another object of the present application is to provide various uses of the composition.

In order to solve the above problems, the present application uses Lactobacillus rhamnosus, which is optimal for improving sperm function.

At this time, the sperm function is the number, concentration, motility, viability, straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), and linearity. , LIN), Straightness (STR), Amplitude of lateral head displacement (ALH), Beat-cross Frequency (BCF), Membrane Integrity, Acrosomal integrity, It may be any one or more selected from antioxidant activity and diversification of intestinal microorganisms.

To this end, in one aspect

A composition for improving sperm function containing Lactobacillus rhamnosus is provided.

In particular, a composition containing only Lactobacillus rhamnosus as an active ingredient as a strain is provided.

In this case, the Lactobacillus rhamnosus may be Lactobacillus rhamnosus NBRC 3425.

In addition, the composition may be a composition further comprising prebiotics. At this time, Lactobacillus rhamnosus and prebiotics may be included in a ratio of 1:1 to 10:1.

in another aspect,

prepared Lactobacillus rhamnosus NBRC 3425;

A method for preparing a composition for improving sperm function comprising a is provided.

At this time, the composition may be a culture medium, and the culture medium may be one or more selected from LB medium, MRS medium, and BL medium.

In the above method, the Lactobacillus rhamnosus may be included in a weight ratio of 5% to 10% in the culture medium.

In another aspect,

administering a composition for improving sperm function containing only Lactobacillus rhamnosus as an active ingredient as a strain to a subject;

A method for improving sperm function comprising a is provided.

In this case, the subject may be a dog, and the administration is oral administration.

In addition, in the method for improving sperm function, a method of administering 100 mg to 10000 mg of Lactobacillus rhamnosus at one time is provided.

1 is a view showing the results of confirming changes in sperm motility before and after refrigeration when culture solutions of various types of Lactobacillus species (L.plantarum, L.acidophilus, L.reuteri, and L.rhamnosus) were added to refrigerated semen medium of dogs.
Figure 2 is a diagram showing the antioxidant activity analysis results before and after refrigeration when various types of Lactobacillus species (L.plantarum, L.acidophilus, L.reuteri, L.rhamnosus) culture medium were added to dog refrigerated semen medium.
Figure 3 is a composition consisting of Lactobacillus rhamnosus NBRC 3425 and FOS (fructooligosaccharides) measured before and after feeding dogs exercise performance (Fig. 3 (a)), viability (Fig. 3 (b)), concentration (Fig. 3 (c)) A diagram showing the result.
Figure 4 is a view showing the results of measuring the diversity of intestinal microorganisms before and after feeding a composition consisting of Lactobacillus rhamnosus NBRC 3425 and fructooligosaccharides (FOS) to dogs.
Figure 5 is a view showing the results of analyzing the taxanomy abundance of intestinal microorganisms at the Phylum level before and after feeding a composition consisting of Lactobacillus rhamnosus NBRC 3425 and FOS (fructooligosaccharides) to dogs.
Figure 6 is a view showing the results of measuring the taxanomy abundance of intestinal microorganisms at the genus level before and after feeding a composition consisting of Lactobacillus rhamnosus NBRC 3425 and FOS (fructooligosaccharides) to dogs.
Figure 7 is a view showing the results of measuring the taxanomy abundance of intestinal microorganisms at the species level before and after feeding a composition consisting of Lactobacillus rhamnosus NBRC 3425 and fructooligosaccharides (FOS) to dogs.
8 shows exercise performance (FIG. 8 (a)), viability (FIG. 8(a)), and viability (FIG. b)), a diagram showing the results of measuring the concentration (FIG. 8(c)).
9 is a view showing the results of measuring the diversity of intestinal microorganisms before and after feeding a composition consisting of Lactobacillus acidophilus KACC 12419, Lactobacillus reuteri KACC 11452, Lactobacillus salivarius KACC 10006, FOS (fructooligosaccharides) and GOS (galactooligosaccharides) to dogs.
10 is a view showing the results of analyzing the taxanomy abundance of intestinal microorganisms at the phylum level before and after feeding a composition consisting of Lactobacillus acidophilus KACC 12419, Lactobacillus reuteri KACC 11452, Lactobacillus salivarius KACC 10006, FOS (fructooligosaccharides) and GOS (galactooligosaccharides) to dogs. am.
11 is a diagram showing sequencing results.

Unless defined otherwise, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or identical to those described in this application may be used in the practice or testing of this application, suitable methods and materials are described below. All publications, patent applications, articles and other references mentioned in this application are incorporated by reference in their entirety. Additionally, the materials, methods, and practices are illustrative only and are not intended to be limiting.

In this application, the term "probiotic" refers to beneficial bacteria that benefit the human body. For example, it may be a strain that has a beneficial effect on the intestinal environment when it is ingested and reaches the intestine. The terms probiotic and probiotics can be used interchangeably.

In this application, the term "prebiotic" refers to a substance that is a nutrient source of probiotics. For example, inulin, oligosaccharides, and the like may be included. The terms prebiotic and prebiotics can be used interchangeably.

To date, various methods have been tried to improve sperm function in infertile men. Recently, there has been a report that intake of probiotics may affect sperm count increase. In addition, as described below, it has been reported that sperm function of infertile men can be improved with a composition containing not only probiotics but also prebiotics, which are their food.

Int J Reprod Biomed. 2021 Mar 21;19(3):235-244. administered Familact containing Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus acidophilus, Bifidobacterium breve/longum, and Streptococcus thermophiles and FOS to men with idiopathic infertility to The quality improvement effect was confirmed.

Randomized Controlled Trial Andrology. In May;5(3):439-444, 2017, Lactobacillus paracasei B21060, Arabinogalattano, FOS, and Flortec containing L-glutammina were administered to men with idiopathic infertility and the sperm quality improvement effect was confirmed.

As such, the strains of probiotics used in the literature reported to date are not only different from each other in the type of strain, but also use a composition in which multiple strains are combined rather than one strain.

Therefore, it is necessary to find a specific strain excellent in improving sperm function among various probiotic strains.

● Technical features of this application

The present application is characterized by using a strain most effective in improving sperm function among various strains. In particular, it is characterized by using a specific strain that is most effective in improving sperm function among various Lactobacillus strains.

The specific Lactobacillus strain used in this application is Lactobacillus rhamnosus.

Accordingly, the present application relates to a composition for improving sperm function having Lactobacillus rhamnosus as an essential component. In addition, the composition is characterized in that it can optionally further include prebiotics that can act as nutrients for probiotics.

The composition diversifies the intestinal microflora; or/and an increase in antioxidant activity; and furthermore, it may show an effect of improving sperm motility, viability, and the like.

Therefore, this application has the following technical features.

First, strains optimized for sperm function improvement were experimentally identified and selected.

Second, a composition containing the Lactobacillus rhamnosus selected above as an essential component is used.

Third, when a composition containing Lactobacillus rhamnosus is used, not only the sperm function improvement effect is exhibited, but also the effect of diversifying the intestinal microflora and/or increasing antioxidant activity is exhibited.

Fourth, a composition further selectively containing prebiotics, a nutrient source of Lactobacillus rhamnosus, may be used.

Hereinafter, the composition for improving sperm function of the present application having the above technical characteristics and its use will be described in detail.

1. Composition for improving sperm function

One aspect of the present application relates to a composition for improving sperm function.

The sperm function is the number of sperm, concentration, motility, viability, straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), linearity (LIN) , Straightness (STR), Amplitude of lateral head displacement (ALH), Beat-cross Frequency (BCF), Membrane Integrity, Acrosomal integrity, Antioxidant activity, It may be one or more selected from the diversity of intestinal microorganisms.

The term “improvement” refers to fixing a deficiency in an existing state, function, effect, form, etc., or adding a specific element or newly transforming the result to make it better. The method for determining the improvement is not limited to those that include both objective methods such as experiments and surveys or subjective methods. For example, in the present application, the improvement means an increase in the number, concentration, motility, viability, etc. of sperm after administration or intake of the composition.

In particular, the composition for improving sperm function of the present application essentially contains Lactobacillus rhamnosus, which has the most excellent improvement effect when comparing the effect of improving sperm function of various strains of Lactobacillus.

Specifically, the inventors of the present application analyzed sperm motility and antioxidant capacity in seminal fluid before and after refrigeration using various Lactobacillus species (see Example 1), and found that Lactobacillus rhamnosus compared to other species increased sperm motility. this is the lowest In addition, compared to other species, Lactobacillus rhamnosus has the highest antioxidant activity.

This is more clearly disclosed in FIG. 1 . It can be seen that L. rhamnosus, an essential component of the composition of the present application, has the smallest difference in sperm motility before and after refrigeration, at -1.1, compared to other Lactobacillus species.

Based on this, even if they belong to the same Lactobacillus genus, the increase or decrease in sperm motility is in a range that cannot be predicted by those skilled in the art. The composition of the present application, which is a small species of Lactobacillus, is derived from L. rhamnosus, which is most effective for sperm motility.

Conventionally, a combination of Lactobacillus, Bifidobacterium, and Streptococcus has been used to improve sperm function. Alternatively, Lactobacillus rhamnosus and Lactobacillus reuteri were used in combination. Conventionally, there was no awareness to select strains that are optimal for improving sperm function.

In order to confirm this, the inventors of the present application analyzed sperm motility, viability, concentration, etc. using a composition combining various types of probiotics (see Example 3), and as a result, the composition containing Lactobacillus rhamnosus of the present application It was confirmed that the rate of increase in motility, viability, and concentration was lower than when using (see Example 2).

That is, the inventors of the present application have selected Lactobacillus rhamnosus as the best probiotics for improving sperm function after painstaking efforts.

In the following, Lactobacillus rhamnosus, which is an essential component of the present application, will be described.

1) Lactobacillus rhamnosus

The composition of the present application includes Lactobacillus rhamnosus (hereinafter referred to as L. rhamnosus) as an essential component.

The L. rhamnosus is optimized for improving sperm function.

In particular, the L. rhamnosus can significantly improve sperm function compared to any one or more selected from L. plantarum, L. acidophilus, and L. reuteri. At this time, the sperm function is the same as described above.

For example, L. rhamnosus may have a higher sperm motility increase rate than L. plantarum, L. acidophilus and L. reuteri.

For another example, the L. rhamnosus may have a higher antioxidant activity increase rate than L. plantarum, L. acidophilus and L. reuteri.

As another example, the L. rhamnosus may have higher intestinal microbial diversity than L. plantarum, L. acidophilus and L. reuteri.

In one embodiment, the L. rhamnosus of the present application has i) a low rate of change in sperm motility; ii) increase in antioxidant capacity;

The strain of L. rhamnosus may be NBRC 3425, PL60, CECT 8800, KCTC13636BP, GR-1, JC1225, etc., but is not limited thereto.

In one embodiment, the composition of the present application includes Lactobacillus rhamnosus NBRC 3425.

The NBRC 3425 can significantly improve sperm function compared to any one or more selected from among PL60, CECT 8800, KCTC13636BP, GR-1, and JC1225.

Meanwhile, the composition may include metabolites of the L. rhamnosus.

At this time, the metabolite may be a substance produced by the L. rhamnosus itself or a substance produced by other factors. At this time, substances produced by other factors refer to substances secreted or metabolized due to substances activating L. rhamnosus.

For example, the metabolite may include hydrogen peroxide, bacteriocin, luterin, reutericyclin, pyroglutamic acid (PCA), and the like.

The metabolite may be contained in the culture medium in which the L. rhamnosus is cultured or in probiotics separated from the culture medium after culturing the L. rhamnosus in the culture medium.

For example, the composition may consist of L. rhamnosus and bacteriocins.

For another example, the composition may be composed of L. rhamnosus, MRS medium, and bacteriocin.

The L. rhamnosus may be included in 1% to 100% of the total weight in the composition.

For example, L. rhamnosus is present in 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, selected from 100% Can be included in one weight.

In one embodiment, the composition of the present application may contain 5% to 95% of L. rhamnosus based on the total culture medium.

In addition, the L. rhamnosus may be included in 10 ⁵ CFU / g to 10 ¹² CFU / g in the composition.

For example, the L. rhamnosus in the composition 10 ⁵ CFU / g, 10 ⁶ CFU / g, 10 ⁷ CFU / g, 10 ⁸ CFU / g, 10 ⁹ CFU / g, 10 ¹⁰ CFU / g, 10 ¹¹ It may be included in one concentration selected from CFU/g and 10 ¹² CFU/g.

In some embodiments, the composition of the present application may contain 10 ⁸ CFU/g to 10 ¹² CFU/g of L. rhamnosus.

2) Prebiotics

The composition may further include prebiotics.

The prebiotics may help the growth of the probiotics or further add or promote the improvement of sperm function of the L. rhamnosus.

The prebiotics are conjugated linoleic acid (CLA), polyunsaturated fatty acid (PUFA), oligosaccharide, inulin, polyphenol, phytochemical, resistant starch (resistant starch), etc., but is not limited thereto.

For example, conjugated linoleic acid may include cis-9 CLA, trans-11 CLA, trans-10 CLA, cis-12 CLA, and the like.

Polyunsaturated fatty acids are fatty acids that contain two or more double bonds. For example, polyunsaturated fatty acids may include linoleic acid, arachidonic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and the like.

Oligosaccharide is a form of 3 to 10 monosaccharides connected, which is not decomposed by digestive enzymes and acts similarly to dietary fiber to go down to the large intestine and become a source of nutrients for beneficial bacteria in the intestine to help improve the large intestine environment. For example, oligosaccharides include fructo-oligosaccharide (FOS), isomalto-oligosaccharide (IMO), galacto-oligosaccharide (GOS), xylo-oligosaccharide (XOS), Maltooligosaccharide (MOS), gentiooligosaccharide (GnOS), chito-oligosaccharide, cello-oligosaccharide, soy oligosaccharide, pectin oligosaccharide (POS), etc. can be included

In one embodiment, the probiotics of the present application may be oligosaccharides.

In one embodiment, the probiotics of the present application may be FOS or/and GOS.

The probiotics may be included in an amount of 1% to 100% of the total weight in the composition.

In certain embodiments, the composition of the present application may include FOS in a weight ratio of 1% to 50%.

In any other embodiment, the composition of the present application may include FOS in a weight ratio of 5% to 30%.

The composition may consist of L. rhamnosus and FOS.

In certain embodiments, the composition of the present application may comprise between 10 ⁸ CFU/g and 10 ¹² CFU/g of L. rhamnosus and FOS.

In certain other embodiments, the composition of the present application may comprise 5% to 95% L. rhamnosus and 0.5% to 50% FOS.

The composition composed of L. rhamnosus and prebiotics of the present application can exert a remarkable effect on improving sperm function than the composition composed of multiple types of probiotics and multiple types of prebiotics.

Figure 3 uses a composition consisting of L. rhamnosus and FOS (hereinafter referred to as Synbiotics-1), and Figure 8 uses a composition consisting of 3 types of probiotics and 2 types of prebiotics (hereinafter referred to as Synbiotics-2). This is the result of confirming the sperm motility, viability and concentration of the dog.

It can be confirmed that the rate of increase before and after ingestion of Synbiotics-1 was higher than that before and after ingestion of Synbiotics-2 in terms of sperm motility, viability, and concentration of dogs.

That is, since the effect of improving sperm function does not significantly increase by mixing various types of probiotics, it can be seen that a sufficient effect can be obtained with only the optimal L. rhamnosus for improving sperm function.

The biggest feature of this application is that it selects and uses L. rhamnosus, which is the most excellent for improving sperm function among various probiotics.

Meanwhile, the L. rhamnosus and prebiotics may be included in an amount of 1% to 100% of the total weight in the composition.

For example, the L. rhamnosus and prebiotics of the present application may be included in 5% to 95% of the total weight in the composition.

The L. rhamnosus and prebiotics may be included in the composition in a molar ratio, volume ratio, or concentration ratio of 1:1 to 10:1.

For example, the L. rhamnosus and prebiotics are 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1: 9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2:5, 2:7, 2:9, 2:11, 3:1, 3:2, 3:4, 3:5, 4:1, 4:3, 4:5, 5: 1, 5:2, 5:3, 5:4, 6:1, 6:4, 6:5, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 8:1, 8:3, 8:5, 8:6, 8:7, 9:1, 9:2, 9:4, 9:5, 9:7, 9:8, 10: 1, 10:3, 10:4, 10:6, 10:7, 10:8, may be included in any one molar ratio (molar ratio), volume ratio or concentration ratio selected from 9.

The composition may be in liquid form, powder form, semi-solid form, encapsulated form, etc., but is not limited thereto.

Depending on the purpose of use, the composition may be first prepared in the form of a suspension, which is a kind of liquid form, and then made into a powder form.

For example, it may be prepared in a powder form after preparing in a suspension form for concentrating the active ingredient of the composition or for user convenience or ease of storage.

In addition, the liquid form may have viscosity.

The liquid form may be further purified and concentrated to remove impurities and increase the content of active ingredients.

For example, the purification method may be centrifugation, filtration, sedimentation, ethanol precipitation, water precipitation, chromatography, etc., but is not limited thereto.

The concentration method may be an evaporation concentration method, a vacuum concentration method, a freeze concentration method, a membrane separation concentration method, etc., but is not limited thereto.

The powder form may be freeze-dried, vacuum-dried, spray-dried, far-infrared ray-dried, tunnel-type oven-dried, or the like.

Freeze drying is a method of sublimating and drying frozen food ice under reduced pressure, vacuum drying is a method of removing moisture under reduced pressure, and spray drying is a method of evaporating moisture using hot air while spraying a liquid.

The composition can be used for applications such as food, food additives, feed, feed additives, pharmaceutical supplements, and therapeutic agents.

In one embodiment, the composition is a pharmaceutical composition for improving sperm function.

The pharmaceutical composition may further include conventional pharmaceutically acceptable carriers, excipients, diluents, preservatives, and other adjuvants.

For example, the composition may further include distilled water, physiological saline, xylitol, malitol, starch, acacia, rubber, gelatin, magnesium, sorbitol, mannitol, sodium benzoate, and the like.

For example, when formulating the composition, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

The pharmaceutically acceptable carrier, excipient, diluent, preservative, and other preservatives added to the composition may be different depending on which part of the body of the subject to apply the composition to. In this case, the subject's body part may be oral, muscular, subcutaneous, intravenous, abdominal, etc., but is not limited thereto.

In particular, the composition of the present application may be prepared in a form for increasing intestinal reach or fixation, and may further include pharmaceutically acceptable carriers, excipients, diluents, preservatives, and the like to achieve its purpose.

For example, the composition may be a composition for oral administration. At this time, the composition may be prepared as a solid formulation or liquid formulation. For example, when the composition is a solid formulation, the composition may further include tablets, pills, powders, granules, capsules, excipients, and the like. For example, the excipient may be starch, calcium carbonate, sucrose or lactose, gelatin, and the like. For another example, when the composition is a liquid formulation, the composition may include various excipients, such as a wetting agent, a sweetening agent, a fragrance, a preservative, etc., in addition to water, a sterilized aqueous solution, and liquid paraffin. The liquid preparations may be suspensions, internal solutions, emulsions, syrups, and the like.

Again, this application uses L. rhamnosus, an optimal Lactobacillus species capable of improving sperm function. Since Lactobacillus species are widely known to have a useful function in improving the intestinal environment, the composition of the present application has the advantage of improving sperm function and at the same time increasing the diversification of intestinal microorganisms and antioxidant activity.

The composition can increase sperm motility by 5% to 100%.

For example, the composition of the present application can increase sperm motility by 20% to 90%.

The composition can increase sperm viability by 5% to 100%.

For example, the composition of the present application can increase sperm viability by 40% to 90%.

The composition can increase the concentration of sperm by a factor of 2 to 10.

For example, the composition of the present application can increase sperm concentration by 2 to 5 times.

As another example, the composition of the present application can increase Actinobacteria, Corynebacterium, Phocaeicola coprocola, etc. related to antioxidant activity.

For another example, the composition of the present application can reduce intestinal harmful bacteria such as Streptococcus and Clostririum perfringens.

Hereinafter, a method for preparing the composition will be described in detail.

In the method for producing the above composition, the composition composed of Lactobacillus rhamnosus of the present application has an effect of improving sperm function as well as diversifying intestinal microflora or/and increasing antioxidant activity according to the condition of sperm of a subject in need of improving sperm function. You can adjust the proportions or weight to help. Therefore, the weight, CFU, etc. of Lactobacillus rhamnosus are not intended to limit the scope of the present application in any way.

2. Method for preparing a composition for improving sperm function

Another aspect of the present application relates to a method for preparing the composition for improving sperm function.

As described above, the composition for improving sperm function,

It consists of one type of probiotics with excellent sperm function improvement effect.

That is, the composition for improving sperm function is composed of L. rhamnosus.

At this time, optionally the composition may further include prebiotics.

Description of the composition is the same as described above.

For example, the preparation method of the composition includes:

i) preparing L. rhamnosus;

ii) adding the Lactobacillus rhamnosus to the culture medium at a weight ratio of 5%;

iii) culturing Lactobacillus rhamnosus in a culture medium; and

iv) optionally purifying the cultured Lactobacillus rhamnosus from the culture medium;

In another example, the method for preparing the composition,

prepared Lactobacillus Rhamnosus NBRC 3425;

includes

In another example, the method for preparing the composition,

prepared L. rhamnosus;

mixing the L. rhamnosus with prebiotics;

includes

As another example, the method for producing the composition,

prepared Lactobacillus rhamnosus NBRC 3425;

mixing the Lactobacillus rhamnosus NBRC 3425 with prebiotics;

includes

The above i) preparing L. rhamnosus; preparing commercially available Lactobacillus rhamnosus; Or culturing Lactobacillus rhamnosus in a culture medium; may include.

Preparing the commercially available Lactobacillus rhamnosus; may be prepared by the Lactobacillus rhamnosus bacteria itself or included in a culture medium at a specific weight ratio.

The Lactobacillus rhamnosus may be included in a weight ratio of 1% to 10% in the culture medium. For example, the Lactobacillus rhamnosus of the present application may be included in a weight ratio of 5% to 10% in the culture medium.

The culture medium may be LB medium, MRS medium, BL medium, etc., but is not limited thereto. At this time, the culture medium may be a solid medium, a liquid medium, and the like.

In one embodiment, the method for preparing the composition of the present application includes preparing by adding Lactobacillus rhamnosus to a culture medium at a weight ratio of 5%.

Cultivating Lactobacillus rhamnosus in a culture medium; Then, selectively purifying the cultured Lactobacillus rhamnosus from the culture medium; can be performed.

The purification may be centrifugation, filtration, sedimentation, ethanol precipitation, water precipitation, chromatography, etc., but is not limited thereto.

mixing the L. rhamnosus with prebiotics; or/and mixing the Lactobacillus rhamnosus NBRC 3425 with prebiotics;

Preparing prebiotics; may further include.

Preparing the prebiotics; may be prepared by preparing commercially available prebiotics or by mixing various kinds of probiotics according to the user's purpose.

For example, preparing prebiotics; can be prepared by mixing FOS and GOS in an appropriate ratio. At this time, the appropriate ratio may be a ratio of 1:1, 1:2, 1:3, 2:1, 2:3, and the like.

When the prebiotics are combined with L. rhamnosus or Lactobacillus rhamnosus NBRC 3425, they may be combined in a specific ratio. At this time, the order of mixing is not limited.

For example, any one selected from L. rhamnosus or Lactobacillus rhamnosus NBRC 3425; and probiotics; is 1:1, 1:2, 1:3, 1:4, 2:1, 2:3, 3:1, 3:2, 4:1, 4:3, 4:5, 5: 1, 5:2, 5:3, 5:4, 6:1, 6:4, 6:5, 6:7, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 8:1, 8:3, 8:5, 8:7, 8:9, 9:1, 9:2, 9:3, 9:4, 9:5, 9: 7, 9:8, 9:10, 10:1, 10:3, 10:4, 10:6, 10:7, 10:8, 10:9, etc. may be mixed, but is not limited thereto. .

Also, mixing the L. rhamnosus with prebiotics; Or/and mixing the Lactobacillus rhamnosus NBRC 3425 with prebiotics; optionally, culturing in a culture medium; may be further performed. This may further promote the growth of Lactobacillus rhamnosus or further enhance the survival of Lactobacillus.

3. Methods for Improving Sperm Function

Another aspect of the present application relates to a method for improving sperm function.

The method for improving sperm function,

administering a composition for improving sperm function containing Lactobacillus rhamnosus to a subject;

includes

The subject may be a subject whose sperm function is lower than that of a normal person or a subject whose sperm function is within a normal range but is further improved. In this case, the target may be mammals, fish, birds, and ungulates.

For example, the mammal may be a human, dog, cat, mouse, or rabbit.

For example, the fish may be zebrafish or the like.

For example, the birds may be chickens, parrots, and the like.

For example, the ungulate may be a cow, goat, camel, or the like.

The term "administration" in this application means introducing the pharmaceutical composition of this application to a subject by any suitable method.

The administration site may be oral, vaginal, testicular, intravenous, abdominal, muscle, arterial, heart, subcutaneous, mammary gland, mucous membrane, etc., but is not limited thereto.

The method for improving sperm function of the present application includes administration to the oral cavity among various administration sites.

That is, the composition for improving sperm function of the present application includes orally administering the composition.

In the case of oral administration, 1 mL to 20 mL of the composition may be administered at a time, but is not limited thereto.

In one embodiment, when the composition is orally administered, 5 mL may be administered at a time.

In the case of oral administration, the number of administrations of the composition may be 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc., but is not limited thereto.

At this time, the administration interval may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, and the like. At this time, continuous administration or non-continuous administration may be performed.

The composition may be administered in a dose of 10 mg to 1000 g, but is not limited thereto.

For example, the composition of the present application may be administered in a dose of 100 g to 1000 g.

For example, in the case of adults, the composition can be administered into the body in an amount of 1 ml to 1000 ml once, and can be administered in a dose of 10 ⁶ CFU/ml/kg to 10 ¹⁰ CFU/ml/kg. The administration interval may be 1 to 3 times a day, and in the case of administration 3 times a day, it may be administered once every 6 to 8 hours.

The effect of improving sperm function using the method for improving sperm function of the present application can be confirmed through sperm concentration, motility, viability, DNA integrity, diversification of intestinal microorganisms, and increased antioxidant activity, but is not limited thereto.

For example, the effect of improving sperm function using the composition for improving sperm function of the present application can be confirmed through a sperm concentration test. At this time, the sperm concentration may be increased by 2 to 10 times after administration of the composition compared to before administration.

For another example, the sperm function improving effect using the composition for improving sperm function of the present application can be confirmed through a sperm motility test. At this time, the sperm motility may be increased by 5% to 95% after administration of the composition compared to before administration.

As another example, the effect of improving sperm function using the composition for improving sperm function of the present application can be confirmed through a sperm viability test. At this time, the viability of the sperm may be increased by 5% to 95% after administration of the composition compared to before administration.

The administration may be determined in consideration of the patient's age, weight, general health condition, diet, whether or not to consume hair loss medicine, whether to consume steroids, the treatment period, and the like.

The pharmaceutical composition of the present application may be administered in combination with other treatments designed to improve sperm function, such as adjuvants and antioxidants known in the art. In addition, the composition of the present application may be coded and administered with a protein designed to increase the survival rate in the intestine due to the nature of probiotics.

To summarize the above, in the method for improving sperm function, the fact that the sperm function improving effect is different according to various probiotics suggests that the selection of probiotics is important.

As described above, the method for improving sperm function of the present application i) uses L. rhamnosus, which is optimal for improving sperm function; ii) not only improving sperm function, but also exhibiting effects of diversifying intestinal microflora and increasing antioxidant activity; has technical significance. This is thought to be able to contribute a lot in that there is no hassle of culturing and mixing each strain to use various types of probiotics, improving sperm function as well as improving intestinal function due to diversification of intestinal microorganisms. .

[Form for Implementation of Application]

Hereinafter, the present application will be described in more detail through examples.

These examples are only for explaining the present application in more detail, and it will be apparent to those skilled in the art that the scope of the present application is not limited by these examples.

experimental material

The following experimental materials were used in the following examples.

Lactobacillus plantarum (L.plantarum) is Lactobacillus plantarum KACC 11451;

Lactobacillus acidophilus (L. acidophilus) is Lactobacillus acidophilus KACC 12419;

Lactobacillus reuteri (L. reuteri) is Lactobacillus reuteri KACC 11452;

Lactobacillus rhamnosus (L.rhamnosus) is Lactobacillus rhamnosus NBRC 3425;

FOS (Kal, Nutraflora FOS Powder)　;

GOS (Genofocus, GF galacto oligo basic) 　;

MRS medium (Kisanbio, MB-M1025).

Example 1-1: Measurement of changes in sperm function

1) Sperm concentration and motility analysis

After injecting 10 ul of semen prepared for sperm motility analysis on a standard count 4 chamber slide (Leja Products B.V., Netherlands), place the slide glass on a microscope connected to a computer, and then use a computer asstised sperm analyzer (computer asstised sperm analyzer) Sperm concentration and motility were analyzed.

2) Analysis of sperm viability

For sperm viability analysis, mix 5-10 ul of recovered semen with the same amount of eosin-nigrosin staining reagent on a warm slide glass, spread the mixture thinly on a new slide glass, and dry it in the air. 200 sperm were counted under a microscope and classified into sperm with intact membranes and sperm with incomplete membranes.

3) Sperm DNA integrity analysis

To analyze sperm DNA integrity, 20 ul of semen collected on a slide glass was spread thinly, dried in air, and then fixed in 3% buffered glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 30 minutes. Then, the slide glass was stained with 5% aqueous aniline blue solution mixed with 4% acetic acid (pH 3.5) for 5 minutes, and 200 sperm were counted under a microscope. Sperm stained blue were classified as abnormal (irmmature chromatin).

Example 1-2: Antioxidant activity measurement

In order to analyze the antioxidant capacity of the Lactobacillus species culture medium, % inhibition was obtained using the DPPH Antioxidant capacity assay kit (bioquochem, Asturias, Spain) according to the manufacturer's protocol. First, a standard solution (trolox) was diluted to 0, 100, 200, 300, 400, or 500 uM with Reagent A, and the standard solution or Lactobacillus species culture was reacted with Reagent B, and absorbance was measured at 517 nm. When the absorbance values obtained from the standard solution were named S1 (blank), S2, S3, S4, S5, and S6, the standard curve for % inhibition was drawn using the following formula, and the % inhibition value of the Lactobacillus species culture medium Rescued.

% inhibition = (1 - Sn/S1) x 100

Experiment Result 1-1: Measurement of changes in sperm function

When various types of Lactobacillus speceis cultures were added to dog refrigerated semen medium, changes in sperm motility before and after refrigeration showed the least decrease in motility, VCL, VAP, VSL, LIN, STR, ALH, and BCF in Lactobacillus rhamnosus (Table 1). .

Group Time Motility (%) VCL (um/sec) VAP (um/sec) VSL (um/sec) LIN (%) STR (%) ALH (um) BCF (Hz) L. plantarum 0h 98.5 130.0 68.3 49.4 37.0 69.6 3.3 14.0 16h 93.3 102.9 56.8 35.5 32.1 57.9 2.6 12.3 increase (%) -5.3 -20.9 -16.9 -28.2 -13.2 -16.8 -20.0 -12.2 L. acidophilus 0h 83.4 112.8 58.5 44.4 36.2 68.2 2.8 13.1 16h 79.5 107.2 55.9 34.8 29.5 56.9 2.8 11.5 increase (%) -4.7 -5.0 -4.4 -21.7 -18.5 -16.6 -1.8 -12.3 L. reuteri 0h 99.1 150.2 80.0 64.1 41.1 76.4 3.6 16.4 16h 93.8 105.0 56.4 36.0 31.3 58.2 2.6 12.7 increase (%) -5.4 -30.1 -29.5 -43.9 -23.7 -23.8 -28.1 -22.6 L. rhamnosus 0h 99.0 135.3 72.4 50.9 36.6 67.6 3.4 14.1 16h 98.0 132.9 70.6 45.9 33.0 61.7 3.3 13.7 increase (%) -1.1 -1.8 -2.5 -9.8 -9.8 -8.7 -1.5 -2.7

Motility in Table 1 is graphically illustrated in FIG. 1 .

Experimental results 1-2: antioxidant activity measurement results

As a result of DPPH antioxidant activity analysis, as shown in the table below, among the Lactobacillus species used in the experiment, Lactobacillus rhamnosus showed the highest antioxidant activity (Table 2). Therefore, it was judged that the antioxidant ability of Lactobacillus rhamnosus could contribute to protecting sperm. The values in Table 2 are graphically illustrated in FIG. 2 .

Group % inhibition L. plantarum 1.3 L. acidophilus 7.6 L. reuteri 10.4 L. rhamnosus 13.7

Through Example 1, it was found that among various Lactobacillus species, L. rhamnosus had the least change in sperm motility and the highest increase in antioxidant activity. This is that the present inventors derived that L. rhamnosus is optimal for improving sperm function through comparative experiments in order to achieve the purpose of using probiotics that are optimal for improving sperm function. Compared to L. plantarum, L. acidophilus, and L. reuteri, L. rhamnosus derived from this example showed a sperm motility increase and decrease rate of more than two times, and its antioxidant capacity was more than ten times higher. Therefore, it can be confirmed that L. rhamnosus is the most excellent strain for improving sperm function among various Lactobacillus species.

Example 2: Analysis of intake efficacy of selected Lactobacillus rhamnosus

In order to increase the efficacy of the selected Lactobacillus species, dogs were fed with prebiotics that could be food for probiotics.

For this purpose, SWANSON's L. rhamnosus with FOS product was used.

The following method was used to identify the Lactobacillus rhamnosus strain of the product.

strain identification

After streaking SWANSON's L. rhamnosus with FOS product on MRS agar medium, a single colony was inoculated into MRS broth. After culturing at 37° C. for 18-48 hours, the obtained culture medium was washed twice using 1X PBS. Then, gDNA was extracted using the G-spin genomic DNA extraction kit for bacteria (Intron, cat#17121). Then, a polymerase chain reaction (PCR) was performed to analyze the 16S rRNA gene sequence using the following primer set (Table 3).

Name Sequence (5’→ 3’) NO. Rhamnosus_Forward Primer AGAGTTTGATCMTGGCTCAG SEQ ID NO: 1 Rhamnosus_Reverse Primer TACGGYTACCTTGTTACGACTT SEQ ID NO: 2

Afterwards, 10 pmol of primer and 10 ng of DNA (template DNA) in PCR premix kit (Maxime, cat#25165) containing i-StarTaq DNA Polymerase 2.5U, dNTPs 2.5mM, Reaction Buffer 1x, Gel Loading buffer 1x was mixed. The mixed solution was denatured at 95 ° C for 3 minutes, and then PCR was performed by repeating the process of maintaining at 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute 35 times. The PCR product was loaded into ABI 3730xl DNA Analyzer to obtain sequencing results after removing dNTPs and reactants that did not participate in the reaction by a method recommended by Applied Biosystems. Sequencing was performed using Applied Biosystems' BigDye(R) Terminator v3.1 Cycle Sequencing Kit. At this time, the following sequencing primer set (Table 4) was used.

Name Sequence (5’→ 3’) NO. Seq_Forward Primers GGATTAGATACCCTGGTA SEQ ID NO: 3 Seq_Reverse Primer CCGTCAATTCMTTTRAGTTT SEQ ID NO: 4

As a result of sequencing, it was a strain having the sequence of SEQ ID NO: 5. As a result of analyzing SEQ ID NO: 5, it was shown in FIG. 11. 11, it can be seen that the Lactobacillus rhamnosus strain of SWANSON's L. rhamnosus with FOS product matches the nucleotide sequence of Lactobacillus rhamnosus NBRC 3425 with 100% coverage and 100% identity.

SWANSON's L. rhamnosus with FOS product in capsule form was fed to dogs for 6 weeks. At the time of feeding, 1 capsule was fed per day, and at this time, the capsule was opened and only the contents were sprinkled on the feed. That is, dogs were fed a composition consisting of Lactobacillus rhamnosus NBRC 3425 and FOS for 6 weeks, and semen and feces were collected before feeding, 3 weeks, and 6 weeks. In semen samples, changes in electron concentration, motility, viability, and DNA integrity were analyzed before and after lactobacillus feeding, and changes in intestinal microflora before and after lactobacillus feeding were analyzed in feces.

Example 2-1: Semen collection and transport & measurement of changes in sperm function

로 실험실로 운송하였다. 실험실로 운송한 직후 700 g, 1min 동안 원심분리 후 상층액을 제거하였다.Semen was collected from dogs by the resin rubbing method, and immediately after collection, the supernatant was removed after centrifugation at 700 g for 1 min. Primary medium (Tris-extender 1:1 (v/v)―distilled water, tris (hydroxymethyl) aminomethane 24 g/L, citric acid 14 g/L, fructose 8 g/L, kanamycin sulfate 0.15 g/L; pH 6.6 , 290 mOsm) was added and the supernatant was removed after centrifugation at 500 g for 5 min. And the secondary medium (54% (v/v) Tris-extender, 40% (v/v) egg yolk, and 6% (v/v) glycerol) was added to

was transported to the laboratory. Immediately after transport to the laboratory, the supernatant was removed after centrifugation at 700 g for 1 min.

The same methods as described above were used for sperm concentration and motility analysis, sperm viability analysis, and sperm DNA integrity analysis with the obtained semen.

Example 2-2: Measurement of intestinal microbial changes

1) DNA extraction from feces

After extracting genomic DNA from feces, the concentration and quality of the extracted DNA were checked using PicoGreen (Invitrogen, cat.#P7589) to perform meta-analysis. PCR amplification using a primer set (Table 5 below) specific to the V3-V4 section, which is the longest and most used for biological classification and identification, among the 16s rRNA V1-V9 sections with a length of about 1,500 bp used for microorganism identification was performed.

Name Sequence (5’→ 3’) NO. Microme_Forward Primer CGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG SEQ ID NO: 6 Microme_Reverse Primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC SEQ ID NO: 7

2) Library preparation for intestinal microbial DNA in feces

Library production was carried out by the secondary PCR method of attaching short DNA fragments (barcodes) containing primers for sequence analysis, and Nextra XT index kit was used. Before sequencing, the quality of the library, such as size and concentration, was finally checked using the Agilent Technologies 2100 Bioanalyzer, and samples that passed the quality were sequenced using the Sequencing By Synthesis (SBS) technology of the Illumina Miseq® System.

3) Intestinal microbial sequencing

In order to analyze the produced sequencing data and confirm the gut microbiota, the raw data produced by sequencing is purified, and then NCBI (https://www.ncbi.nlm.nih.gov/ ) was used to analyze the species information (kingdom, phylum, class, order, family, genus, species) of the gut microbiome using the 16s rRNA gene database of ) and analyze the diversity of the gut microbiome through read counts.

4) Statistical analysis

All results were expressed as mean ± SEM, and p < 0.05 was analyzed as a significant difference. Using GraphPad Prism 5, repeated measures ANOVA was performed for sperm analysis according to lactobacillus feeding periods of 0, 3, and 6 weeks, and paired tests were performed for sperm analysis according to lactobacillus feeding periods of 0 weeks and 6 weeks. In addition, the Friedman test was performed for the analysis of intestinal microbes according to the lactobacillus feeding period of 0, 3, and 6 weeks, and the Wilcoxon signed rank test was performed for the analysis of the intestinal microflora according to the 0, 6-week lactobacillus feeding period.

Experimental result 2-1: Results of measuring changes in sperm function of the composition combining selected Lactobacillus rhamnosus and FOS

Compared to before intake (week 0), it was confirmed that motility, VAP, and LIN gradually increased at 3 weeks and 6 weeks after ingestion of Lactobacillus rhamnosus and FOS (Table 6). VCL, VSL, STR, WOB, and ALH also showed a tendency to increase, although there was no statistically significant difference.

Groups Motility (%) VCL (μm/s) VAP (μm/s) VSL (μm/s) Linearity (%) Straightness (%) WOB (%) ALH (μm) 0w 78.1 ± ^3.1b 106.2 ± 12.7 57.8 ± ^5.8ab 36.0 ± 5.8 32.1 ± ^2.0ab 58.1 ± 4.3 54.9±1.0 2.8 ± 0.3 3w 95.4 ± ^1.5ab 111.8 ± 10.4 61.5 ± ^4.7b 36.4 ± 3.9 30.9 ± ^0.5b 55.1±1.4 55.1±1.0 2.9 ± 0.2 6w 99.6 ± 0.1 ^a 131.8 ± 11.0 79.0 ± 7.2 ^a 54.5 ± 4.7 38.4 ± 1.5 ^a 63.3 ± 1.6 58.2 ± 1.4 3.1 ± 0.2 a, b indicate statistically significant differences (P < 0.05).

The motility of Table 4 is shown graphically in FIG. 6 (a).

In addition, sperm concentration, viability, DNA integrity, and acrosomal integrity were all significantly increased at 6 weeks after ingestion (6w) compared to before (0w) ingestion of Lactobacillus rhamnosus and FOS (Table 7). The viability of Table 5 is graphically depicted in FIG. 6(b).

Groups Conc (M/mL) Viability (%) Membrane Integrity (%) Acrosomal integrity (%) 0w 191.5 ± ^67.4b 47.4 ± ^2.9b 78.6 ± ^7.9ab 87.4 ± ^1.2b 3w 491.2 ± ^61.3ab 61.2 ± ^4.7ab 83.8 ± ^2.7b 97.4 ± ^0.9ab 6w 617.2 ± 19.7 ^a 81.4 ± 3.1 ^a 95.0 ± 0.6 ^a 99.6 ± 0.2 ^a a, b indicate statistically significant differences (P < 0.05).

In addition, the number of deformed sperms showed a tendency to decrease at 6 weeks (6w) after intake compared to before intake (0w) of Lactobacillus rhamnosus and FOS (Table 8).

Groups Droplets (%) Coiled tail (%) Bent tail (%) 0w 12.2 ± 8.3 10.0 ± 2.5 5.4±1.2 3w 7.8 ± 5.7 7.0 ± 2.4 4.4±1.1 6w 7.0 ± 4.9 4.8±1.4 3.6 ± 1.2

Experimental results 2-2: Results of measurement of changes in intestinal microorganisms of the composition combining selected Lactobacillus rhamnosus and FOS

It was confirmed that the diversity of the composition and types of intestinal microorganisms changed at 3 and 6 weeks after ingestion compared to before (0 week) ingestion of Lactobacillus rhamnosus and FOS (FIG. 4).

In addition, as a result of analyzing the taxanomy abundance of intestinal microorganisms at 3 and 6 weeks after ingestion compared to before (0 week) ingestion of Lactobacillus rhamnosus and FOS, the ratio of Actinobacteria reported to have an antioxidant effect at the phylum level was It gradually increased, and significantly increased at 6 weeks after intake (2.6 ± 1.1%) compared to before intake (0.5 ± 0.1%) (FIG. 5).

Among them,

The results of analysis at the Genus level, which is a lower level, also showed that Corynebacterium, reported to have an antioxidant effect, significantly increased at 6 weeks after ingestion (1.7 ± 0.7) than before (0.0 ± 0.0%) ingestion of Lactobacillus rhamnosus and FOS. (FIG. 6(a)). These changes affect the reduction of harmful bacteria along with a significant increase in Phocaeicola coprocola that regulates the intestinal ecosystem (Fig. 7(a)), Streptococcus at the Genus level (Fig. 6(b)) and Clostririum perfringens at the Species level. Each significantly decreased at 6 weeks after intake compared to before intake (FIG. 7 (b)).

Through Example 2, a composition containing L. rhamnosus, which is the most excellent strain for improving sperm function derived from Example 1, and prebiotics that serve as food for probiotics, that is, a composition containing L. rhamnosus and FOS, also produced sperm There was an excellent effect on the increase in motility, viability, concentration, etc.

Therefore, the following Example 3 was conducted to determine whether the strain was excellent in improving sperm function even when L. rhamnosus, which is the most excellent strain for improving sperm function, was not included.

Example 3: Analysis of intake efficacy of a composition composed of various types of lactobacilli and various types of prebiotics

The sperm function improvement effect of the composition composed of Lactobacillus rhamnosus and FOS selected in this application and the sperm function improvement effect of the composition composed of various types of Lactobacillus and various types of prebiotics were compared and analyzed.

To this end, the different types of Lactobacilli and the different types of prebiotics were constructed as follows (hereinafter referred to as multi-type compositions):

Lactobacillus acidophilus KACC 12419, Lactobacillus reuteri KACC 11452, Lactobacillus salivarius KACC 10006, FOS and GOS.

The mixing ratio of the above strains is as follows:

L. reuteri 1.3 x 10^9 CFU/ml

L. acidophilus 3.0 x 10^9 CFU/ml

L. salivarius 2.7 x 10^8 CFU/ml

L. reuteri, L. acidophilus, and L. salivarius were each inoculated with a colony or stock in MRS broth medium and then cultured at 37°C for 18-48 hours. After incubation, 1 ml each was mixed. Thereafter, only the strain was purified, and the purification proceeded as follows. After culturing, the culture medium was washed twice using 1X PBS. Three types of Lactobacilli were put into a tube of 1 ml each, and then centrifuged to obtain a pellet. Then, the pellet was dissolved in bottled water to make it liquid, and glycerol was added at a concentration of 20%. One tube was manufactured and stored frozen so that it could be fed once, and after thawing at room temperature before feeding, it was sprinkled on feed.

The multi-species composition was fed to young dogs and old dogs for 6 weeks, respectively, and semen and feces were collected before feeding, 3 weeks, and 6 weeks. In semen samples, changes in electron concentration, motility, viability, and DNA integrity were analyzed before and after lactobacillus feeding, and changes in intestinal microflora before and after lactobacillus feeding were analyzed in feces.

The experimental methods used for semen collection, measurement of changes in sperm function, and changes in intestinal microflora were analyzed in the same manner as in Example 2.

Experimental result 3-1: Results of measuring changes in sperm function of compositions combining multiple types of lactobacilli and multiple types of prebiotics

Compared to before (week 0) ingestion of the multi-species composition, sperm motility increased at 6 weeks after ingestion compared to before ingestion. However, there was no statistically significant difference in the results of VCL, VAP, VSL, LIN, STR, WOB, and ALH at 6 weeks after intake compared to before intake (Table 9).

Groups Motility (%) VCL (μm/s) VAP (μm/s) VSL (μm/s) Linearity (%) Straightness (%) WOB (%) ALH (μm) 0w 92.0 ± 3.3 ^a 94.1 ± 14.6 53.0 ± 7.5 32.9 ± 4.8 ^a 33.5 ± 2.9 57.3 ± 3.6 56.7±1.4 2.5 ± 0.3 3w 95.9 ± ^2.3ab 106.0 ± 9.9 61.0 ± 5.1 42.7 ± ^2.8b 38.6 ± 3.3 65.0 ± 3.3 57.2 ± 2.1 2.6 ± 0.2 6w 99.6 ± ^0.3b 105.4 ± 13.0 60.5 ± 6.8 35.7 ± ^2.5ab 35.0 ± 2.6 58.3 ± 3.1 57.9±1.3 2.9 ± 0.3 a, b indicate statistically significant differences (P < 0.05).

The sperm motility of Table 9 is graphically illustrated in FIG. 8(a).

In addition, acrosome integrity was significantly increased at 6 weeks after ingestion (6w) compared to before (0w) ingestion of the multitype composition, but there was no significant change in sperm concentration or viability (Table 10). The viability of the sperm of Table 10 is graphically shown in FIG. 8(b).

Groups Conc(M/mL) Viability (%) Acrosomal integrity (%) 0w 485.1 ± 90.0 71.4 ± 6.8 90.0 ± 1.3 ^a 3w 501.4 ± 99.3 71.2 ± 3.6 92.4 ± ^2.5ab 6w 639.4 ± 113.5 80.0 ± 2.1 97.8 ± ^0.6b a, b indicate statistically significant differences (P < 0.05).

In addition, the number of deformed sperm tended to decrease at 6 weeks after ingestion (6w) compared to before (0w) ingestion of the multi-type composition, but there was no statistically significant difference (Table 11).

Groups Head (%) Droplets (%) Coiled tail (%) Bent tail (%) 0w 0.4 ± 0.4 2.0±1.5 3.6 ± 1.2 11.2 ± 6.7 3w 0.0 ± 0.0 0.0 ± 0.0 1.2 ± 0.5 2.8±1.4 6w 0.0 ± 0.0 0.0 ± 0.0 0.8 ± 0.5 2.0 ± 0.6

Experimental result 3-2: Results of measurement of changes in intestinal microbes of compositions combining multiple types of lactobacilli and multiple types of prebiotics

Compared to before (week 0) ingestion of the multi-species composition in dogs, no change in the composition and diversity of intestinal microorganisms was observed at 6 weeks after ingestion (FIG. 9).

As a result of analyzing the taxanomy abundance of intestinal microorganisms at 6 weeks after ingestion compared to before (0 week) ingestion of the multi-species composition, the ratio of Actinobacteria reported to have antioxidant effects at the phylum level (0.3 ± 0.1% vs. 0.8 ± 0.2%), there was no statistically significant difference (FIG. 10).

In Example 3, when the composition consisting of Lactobacillus acidophilus KACC 12419, Lactobacillus reuteri KACC 11452, Lactobacillus salivarius KACC 10006, FOS and GOS was used, sperm motility, viability, concentration, etc. were improved compared to L. rhamnosus and FOS as in Example 2. It can be seen that the composition composed of the composition does not show a superior effect as much as the effect of improving sperm motility, viability, concentration, etc. That is, it suggests that it is important to use a specific strain having an excellent effect on improving sperm function.

Taken together, L. rhamnosus derived by the inventors is not only optimal for improving sperm function, but also improves sperm function in that it shows a better effect than those combining various Lactobacillus strains other than L. rhamnosus. The selection of probiotics that is optimal for the purpose for the purpose is of great technical significance.

<110> MJBIOGEN CORP. <120> A composition for improving function of sperm and its use <130> CP21-127 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> Rhamnosus_Forward Primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> artificial sequence <220> <223> Rhamnosus_Reverse Primer <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 18 <212> DNA <213> artificial sequence <220> <223> Seq_Forward Primer <400> 3 ggattagata ccctggta 18 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> Seq_Reverse Primer <400> 4 ccgtcaattc mtttragttt 20 <210> 5 <211> 1483 <212> DNA <213> artificial sequence <220> <223> Sequencing Strain <400> 5 cgctggcggc gtgcctaata catgcaagtc gaacgagttc tgattattga aaggtgcttg 60 catcttgatt taattttgaa cgagtggcgg acgggtgagt aacacgtggg taacctgccc 120 ttaagtgggg gataacattt ggaaacagat gctaataccg cataaatcca agaaccgcat 180 240 tagttggtga ggtaacggct caccaaggca atgatacgta gccgaactga gaggttgatc 300 ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt 360 ccacaatgga cgcaagtctg atggagcaac gccgcgtgag tgaagaaggc tttcgggtcg 420 taaaactctg ttgttggaga agaatggtcg gcagagtaac tgttgtcggc gtgacggtat 480 ccaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540 cgttatccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa 600 agccctcggc ttaaccgagg aagtgcatcg gaaactggga aacttgagtg cagaagagga 660 cagtggaact ccatggttag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 720 aggcggctgt ctggtctgta actgacgctg aggctcgaaa gcatgggtag cgaacaggat 780 tagataccct ggtagtccat gccgtaaacg atgaatgcta ggtgttggag ggtttccgcc 840 cttcagtgcc gcagctaacg cattaagcat tccgcctggg gagtacgacc gcaaggttga 900 aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960 aacgcgaaga accttaccag gtcttgacat cttttgatca cctgagagat caggtttccc 1020 cttcgggggc aaaatgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1080 ggttaagtcc cgcaacgagc gcaaccctta tgactagttg ccagcattta gttgggcact 1140 ctagtaagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc 1200 ccttatgacc tgggctacac acgtgctaca atggatggta caacgagttg cgagaccgcg 1260 aggtcaagct aatctcttaa agccattctc agttcggact gtaggctgca actcgcctac 1320 acgaagtcgg aatcgctagt aatcgcggat cagcacgccg cggtgaatac gttcccgggc 1380 cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccgaagccg gtggcgtaac 1440 ccttttaggg agcgagccgt ctaaggtggg acaaatgatt agg 1483 <210> 6 <211> 49 <212> DNA <213> artificial sequence <220> <223> Microme_Forward Primer <400> 6 cgtcggcagc gtcagatgtg tataagagac agcctacggg nggcwgcag 49 <210> 7 <211> 55 <212> DNA <213> artificial sequence <220> <223> Microme_Reverse Primer <400> 7 gtctcgtggg ctcggagatg tgtataagag acaggactac hvgggtatct aatcc 55

Claims (20)

As a strain, containing only Lactobacillus rhamnosus as an active ingredient,
A composition for improving sperm function.
According to claim 1,
The composition, characterized in that the Lactobacillus rhamnosus is Lactobacillus rhamnosus NBRC 3425.
According to claim 1,
The composition further comprises a prebiotics.
According to claim 3,
In the composition, Lactobacillus rhamnosus and prebiotics are contained in a ratio of 1: 1 to 10: 1.
According to claim 3,
The composition, characterized in that the prebiotics are oligosaccharides.
According to claim 5,
The oligosaccharide is at least one selected from fructooligosaccharide (FOS), isomaltooligosaccharide (IMO), galactooligosaccharide (GOS), maltooligosaccharide (MOS), xylooligosaccharide (XOS), and gentiooligosaccharide (GnOS) Composition, characterized in that.
According to claim 1,
The composition is characterized in that at least one selected from a powder, liquid form.
According to claim 1,
The composition characterized in that the Lactobacillus rhamnosus is included in the composition at a concentration of 10 ⁷ CFU / g to 10 ¹² CFU / g.
According to claim 1,
The composition, characterized in that the composition may further include a metabolite of the Lactobacillus rhamnosus.
According to claim 9,
Composition, characterized in that the metabolite is at least one selected from hydrogen peroxide, bacteriocin, luterin, reutericyclin, and pyroglutamic acid (PCA).
A method for preparing the composition of claim 1,
prepared Lactobacillus Rhamnosus NBRC 3425;
How to include.
According to claim 11,
The method according to claim 1, wherein the composition is a culture medium.
According to claim 12,
The culture medium is characterized in that any one or more selected from LB medium, MRS medium, BL medium.
According to claim 12,
The method characterized in that the Lactobacillus rhamnosus is contained in a weight ratio of 5% to 10% in the culture medium.
administering the composition of claim 1 to a subject;
A method for improving sperm function comprising a.
According to claim 15,
Wherein the subject is an individual.
According to claim 15,
The method characterized in that the administration is oral administration.
According to claim 15,
The method characterized in that the administration of 100 mg to 10000 mg of Lactobacillus rhamnosus at a time.
According to claim 15,
The method increases antioxidant activity; And intestinal microbial diversification efficacy; characterized in that it comprises a method.
According to claim 15,
The functions include sperm number, concentration, motility, viability, straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), linearity (LIN), Straightness (STR), Amplitude of lateral head displacement (ALH), Beat-cross Frequency (BCF), Membrane Integrity, Acrosomal integrity, Antioxidant capacity, Intestinal A method characterized in that at least one selected from microbial diversification is improved.

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