Intestinal microbiota improver

Abstract

To provide a food or pharmaceutical material effective in improving intestinal microbiota as well as bowel movement.SOLUTION: Provided is an intestinal microbiota improver for increasing the occupancy of Enterococcus bacteria in the intestine, the intestinal microbiota improver comprising a plant-based lactic acid bacterial fermentation product of silk protein as an active ingredient, where the plant-based lactic acid bacterium is Lactococcus lactis.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an intestinal flora improving agent using fermented silk extract. [Background technology]

[0002] More than 95% of the cocoon threads spun by silkworms are composed of two types of proteins called "sericin" and "fibroin," with fibroin at the center and surrounded by sericin. Fibroin is a fibrous protein that is processed into silk thread and used extensively, and its properties as a functional food ingredient have also attracted attention. In the early 1990s, its effect of improving liver function in model rats was reported (Non-Patent Document 1), and more recently, its effectiveness in colitis model rats through the improvement of intestinal barrier function, wound healing effects, and anti-inflammatory effects have been found (Non-Patent Document 2).

[0003] Furthermore, while most of sericin has been removed and discarded during the silk thread degumming process, in recent years its functionality has gradually become clear, and currently it is being used in cosmetics for its moisturizing and wrinkle prevention properties (Non-Patent Document 3), and as an additive to serum-free culture media for its cell proliferation-promoting and cell death-inhibiting properties (Non-Patent Documents 4 and 5). It has also been reported that part of the sericin structure functions as a resistant protein (Non-Patent Document 6), and by taking advantage of its indigestibility, it has been reported to have functions in the body such as intestinal regulating properties (Non-Patent Documents 6 and 7) and improving the intestinal environment (Non-Patent Document 8).

[0004] On the other hand, since fibroin and sericin have large molecular weights, which pose problems in digestion and absorption, attempts have been made to use fermentation products obtained by fermenting fibroin and sericin with plant lactic acid bacteria (Lactobacillus plantarum) (Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2022-107155 [Non-patent literature]

[0006] [Non-Patent Document 1] Kiyoshi Hirabayashi, Chemistry. 47, 25-28 (1992) [Non-Patent Document 2] Rodoriguez-Nogales, A. et al., Int. J. Pharm. 511, 1-9 (2016) [Non-Patent Document 3] Rainer, V. Fragr. J. 26, 70-74 (1998) [Non-Patent Document 4] Terada, S. et al., Cytotechnology. 40, 3-12 (2002) [Non-Patent Document 5] Sasaki, M. et al., Biotechnol. Appl. Biochem. 42, 183-188 (2005) [Non-Patent Document 6] Norihisa Kato et al., Journal of the Japanese Society of Nutrition and Food Science. 53, 71-75 (2000) [Non-Patent Document 7] Sasaki, M. et al., Food Sci. Technol. Res. 6, 280-283 (2000) [Non-Patent Document 8] Okazaki, Y. et al., J. Nutr. 141, 1975-1981 (2011) Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing a food or pharmaceutical material that is useful for improving the intestinal flora and bowel movements. [Means for solving the problem]

[0008] In view of the above problems, the present inventors have conducted extensive research and have found that silk protein is a plant lactic acid. It has been discovered that lactic acid bacteria fermentation product (fermented silk extract) fermented using the bacterium Lactococcus lactis has the effect of increasing the occupancy rate of Enterococcus bacteria in the intestines, making it useful as an agent for improving the bacterial flora and bowel movements.

[0009] That is, the present invention relates to the following 1) to 7). 1) An agent for improving intestinal flora, which increases the occupancy rate of bacteria of the genus Enterococcus in the intestines, comprising as an active ingredient a plant lactic acid bacteria fermentation product of silk protein, wherein the plant lactic acid bacteria is Lactococcus lactis. 2) A laxative containing as an active ingredient a plant lactic acid bacteria fermentation product of silk protein, wherein the plant lactic acid bacteria is Lactococcus lactis. 3) A food for improving intestinal flora, which increases the occupancy rate of Enterococcus bacteria in the intestines and contains as an active ingredient a plant lactic acid bacteria fermentation product of silk protein, wherein the plant lactic acid bacteria is Lactococcus lactis. 4) A food for improving bowel movements, containing as an active ingredient a plant lactic acid bacteria fermentation product of silk protein, wherein the plant lactic acid bacteria is Lactococcus lactis. 5) The agent according to 1) or 2), or the food according to 3) or 4), wherein the plant lactic acid bacterium is Lactococcus lactis BM32-1 strain. 6) An agent according to 1) or 2), or a food according to 3) or 4), wherein the silk protein is a mixture of fibroin and sericin. 7) The agent or food according to 6), wherein the mixture ratio of fibroin and sericin (sericin / fibroin) is 0.20 to 0.60 by mass. Effect of the Invention

[0010] According to the intestinal flora improving agent or laxative improving agent of the present invention, it is possible to increase the occupancy rate of bacteria of the genus Enterococcus in the intestine and improve the laxative function. [Brief description of the drawings]

[0011] [Figure 1-1] Analysis results of fecal microbiota (left: relative occupancy rate of each group before and after intake, right: change in relative occupancy rate after intake). [Figure 1-2] Analysis results of fecal microbiota (left: relative occupancy rate of each group before and after intake, right: change in relative occupancy rate after intake). [Figure 1-3] Analysis results of fecal microbiota (left: relative occupancy rate of each group before and after intake, right: change in relative occupancy rate after intake). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The plant lactic acid bacteria fermentation product of silk protein of the present invention is a fermentation product obtained by fermenting silk protein with the plant lactic acid bacteria Lactococcus lactis. In the present invention, such a fermentation product is also called "fermented silk extract".

[0013] Silk protein is a general term for the proteins that make up silk threads, and is mainly composed of fibroin and sericin. The silk protein used for fermentation in the present invention may be either fibroin or sericin, but is preferably a mixture of both. For a mixture of fibroin and sericin, the mixing ratio of fibroin and sericin (sericin / fibroin) is, from the viewpoint of improving the bacterial flora or bowel movements, preferably 0.20 or more, more preferably 0.30 or more, even more preferably 0.35 or more, by mass, and is preferably 0.60 or less, more preferably 0.50 or less, even more preferably 0.45 or less.

[0014] Sericin and fibroin are obtained by extracting and purifying them from cocoons using known methods. For example, a sericin aqueous solution can be obtained by immersing cocoons in water and extracting them by heating, and a fibroin aqueous solution can be obtained by scouring the sericin extraction residue with an alkali such as sodium carbonate, soap, or sodium hydroxide, or a protease that decomposes sericin, and then placing the residue in a calcium chloride aqueous solution, dissolving the residue by heating, and then removing the calcium chloride by dialysis.

[0015] In the present invention, silk protein can be used as a fermentation raw material in a form suitable for fermentation, such as a solution (e.g., a sericin aqueous solution, a fibroin aqueous solution, or a mixed solution thereof), a concentrated solution thereof, or a powder thereof, or these can be diluted to an appropriate concentration with distilled water or the like.

[0016] In the present invention, the plant lactic acid bacteria is Lactococcus lactis. lactis), preferably Lactococcus lactis BM32-1 strain. The BM32-1 strain was isolated by the present inventor from the flowers of Euonymus sieboldianus var and deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary Center on October 4, 2023 under the name NITE P-03986.

[0017] The method of inoculating silk protein with plant lactic acid bacteria is not particularly limited, and examples thereof include a method of mixing bacterial cells with silk protein in a form suitable for fermentation, a method of mixing bacterial cells with water in which silk protein in a form suitable for fermentation is dissolved or suspended, a method of contacting a culture solution in which bacterial cells are cultured in an appropriate liquid medium with silk protein in a form suitable for fermentation, a method of culturing plant lactic acid bacteria in the above liquid medium in which silk protein in a form suitable for fermentation is dissolved or suspended, etc. In addition, various media such as fruit juice medium, vegetable juice medium, milk medium, skim milk medium, or medium containing milk components, and semi-synthetic medium not containing them can be used as the medium, and specific examples thereof include reduced skim milk medium obtained by reducing and heat-sterilizing skim milk, skim milk medium with yeast extract added, MRS medium, GAM medium, etc.

[0018] In the fermentation, a loopful of the plant lactic acid bacteria is inoculated into a seed culture medium and cultured for a predetermined time to obtain a seed culture liquid, and in the main culture, about 1 to 2% of the seed culture liquid relative to the volume of the main culture medium is inoculated, and then cultured at a temperature of 15 to 45°C for 12 to 72 hours, preferably 24 to 48 hours, to obtain a lactic acid bacteria fermentation liquid. Fermentation can be either aerobic fermentation or anaerobic fermentation, and anaerobic fermentation is preferred. In the case of aerobic fermentation, static fermentation is preferred to agitation fermentation. The obtained fermented product can be used as it is after heat sterilization (usually at 60 to 85°C for 2 to 60 minutes) as necessary, but it is also possible to use the supernatant obtained by removing insoluble matter from the fermented product by means of centrifugation or the like. Furthermore, it is also possible to use the supernatant obtained by removing the bacterial cells and the like by filtration.

[0019] As shown in the examples described below, when a beverage containing the plant lactic acid bacteria fermentation product (fermented silk extract) of silk protein obtained in this way is continuously taken by a human for 12 weeks, the frequency of spontaneous defecation increases. In addition, in fecal bacterial flora analysis, the occupancy rate of Enterococcus and Clostridium bacteria significantly increased, and the increase in the occupancy rate of Enterococcus bacteria was particularly remarkable, and the increase in the bacteria correlated with the increase in the frequency of defecation. Therefore, the plant lactic acid bacteria fermentation product of silk protein of the present invention can be an intestinal flora improving agent and a bowel movement improving agent that increase the occupancy rate of Enterococcus bacteria in the intestines, and can be used to improve the intestinal flora by increasing the occupancy rate of Enterococcus bacteria in the intestines and to improve bowel movements. Such an intestinal flora improving agent and a bowel movement improving agent can be used as a medicine or food that is effective for improving the intestinal flora and bowel movements by increasing the occupancy rate of Enterococcus bacteria in the intestines, or as a material to be added to a medicine or food. The food is a food that improves the intestinal flora or laxative effect by increasing the occupancy rate of Enterococcus in the intestine. Based on the concept of improving health, it is possible to produce health functional foods that are labeled as such, such as functional foods, foods for specified health uses, and foods with nutrient functions.

[0020] In the present invention, "increasing the occupancy rate of bacteria of the genus Enterococcus in the intestine" means increasing the content rate of bacteria of the genus Enterococcus in the intestine by activating the metabolism of the bacteria of the genus Enterococcus, etc. Therefore, improving the intestinal flora to increase the occupancy rate of bacteria of the genus Enterococcus in the intestine refers to changing the intestinal bacterial flora so that the physiological functions of bacteria of the genus Enterococcus are more effectively exerted. Examples of bacteria of the genus Enterococcus include Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, and Enterococcus durans. In the intestinal flora improvement of the present invention, it is more preferable that the occupancy rate of bacteria of the genus Clostridium is increased together with bacteria of the genus Enterococcus. Examples of bacteria of the genus Clostridium include Clostridium butyricum.

[0021] In the present invention, "improving bowel movement" preferably refers to increasing the number of bowel movements, and includes recovery, improvement, or enhancement of a state in which bowel movement function is impaired, prevention of impaired bowel movement function, and maintenance of a good state of bowel movement. Here, impaired bowel movement function includes constipation and diarrhea. By improving bowel movements, it is expected that the time it takes for ingested food to pass through the intestines will be shortened, and intra-intestinal and abdominal pressures will be reduced.

[0022] Examples of dosage forms for pharmaceuticals containing the plant lactic acid bacteria fermentation product of silk protein of the present invention include oral administration using tablets, capsules, granules, powders, syrups, etc., or parenteral administration using injections, suppositories, inhalants, transdermal agents, topical agents, etc., with oral administration being the preferred form. Such pharmaceutical formulations in various dosage forms can be prepared by appropriately combining other pharma- ceutical acceptable excipients, binders, bulking agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavorings, fragrances, coating agents, carriers, diluents, etc., within limits that do not interfere with the action of the plant lactic acid bacteria fermentation product of the silk protein of the present invention.

[0023] In addition, examples of food forms containing the plant lactic acid bacteria fermentation product of the silk protein of the present invention include beverages such as milk drinks, fermented milk, fermented fruit juice, fermented vegetable juice, fruit or vegetable juice drinks, carbonated drinks, tea drinks, alcoholic drinks, and soft drinks, as well as various food compositions such as jelly foods, various snacks, baked goods, cakes, chocolate, jam, bread, gum, candy, soups, pickles, and tsukudani, as well as supplements in the same form as the oral administration formulations described above (tablets, capsules, syrup, etc.).

[0024] The content of the plant lactic acid bacteria fermentation product of silk protein of the present invention in the above-mentioned medicines and foods is not particularly limited and may be appropriately adjusted according to the daily dosage, etc. As an example, when used as a preparation for oral administration, the content in the preparation is 0.01 to 50% by weight, preferably 0.05 to 10% by weight, in terms of dry solid content.

[0025] The subjects to whom the above medicines or foods are to be ingested or administered include those with impaired bowel movement function and those at high risk of impaired bowel movement function.

[0026] The intestinal flora improving agent or laxative improving agent of the present invention can be administered once a day or in several divided doses. The amount of administration or dosage may vary depending on the condition, weight, sex, age, or other factors of the subject. The daily administration or dosage amount for an adult (60 kg) is about 100 mg / kg. Lactis (viable bacteria count) is 10 7 cfu or more is preferable, 10 10 cfu or more is preferable, 10 11 More preferably, 10 cfu or more. 12 The number of days for which the virus is ingested is preferably 5 days or more, more preferably 2 weeks or more, even more preferably 4 weeks or more, and particularly preferably 8 weeks or more. The present invention will now be described in detail, but the present invention is not limited to the following examples. EXAMPLES

[0027] Manufacturing Example 1 Preparation of silk protein fermented by plant lactic acid bacteria (fermented silk extract) (1) Preparation of silk protein 1) The cocoons were immersed in water and heated in an autoclave (105℃) for 60 minutes, and then filtered through a 100μm sieve to obtain an aqueous sericin solution. 2) The sericin extraction residue or cocoons (1 part by mass) obtained in 1) was immersed in a 0.5% aqueous sodium carbonate solution (30 parts by mass), heated, washed with water, and immersed in a 0.25% aqueous citric acid solution to neutralize (alkaline scouring). After that, fibroin (1 part by mass) was added to a 50% aqueous calcium chloride solution (20 parts by mass) and dissolved by heating, and the calcium chloride was removed by dialysis to obtain an aqueous fibroin solution.

[0028] (2) Preparation of fermented silk extract A culture solution (Table 1 below) containing the sericin aqueous solution and the fibroin aqueous solution obtained in (1) above was prepared, and pre-cultured lactic acid bacteria (Lactococcus lactis BM32-1 strain) was added to the culture tank (1% added relative to the liquid volume in the culture tank) and cultured at 28°C for 48 hours. In preparing the culture solution, the fibroin solution was diluted with water to adjust the solid content to 2%. The skim milk powder solution was sterilized in an autoclave (100°C, 10 minutes) and added to the culture tank. All ingredients except the skim milk powder solution were mixed in the culture tank and sterilized (100°C, 10 minutes).

[0029] [Table 1]

[0030] Production Example 2 Preparation of fermented silk extract-containing beverage A fermented silk extract-containing beverage (Table 2 below) containing the fermented silk extract obtained in Production Example 1 was prepared. All ingredients except the fermented silk extract were sterilized in an autoclave (100°C, 10 minutes), cooled, and then mixed. The beverage contained approximately 2 x 10 Lactococcus lactis BM32-1 strain. 12 Contains viable cells (100 ml). [Table 2]

[0031] For the placebo beverage, a control fermented extract was produced by fermenting in the same manner as in Production Example 1 using the control culture medium shown in Table 3 below instead of the culture medium shown in Table 1, and a beverage containing this (Table 4 below) was prepared. Each ingredient was sterilized in an autoclave (105°C, 10 minutes), cooled, and then mixed and prepared.

[0032] [Table 3]

[0033] [Table 4]

[0034] Test Example 1: Effects of fermented silk extract on bowel movements and intestinal flora 1. Method The clinical trial was conducted using a double-blind, randomized, placebo-controlled, parallel-group comparative design: eligible subjects who met the criteria were enrolled, stratified by sex (male or female), and then assigned to the test or control diet group using a blocked randomization method. (1) Target audience From the recruited subjects, 50 men and women were selected as subjects. They had no history of allergies or hypersensitivity to silk or dairy products, did not regularly consume foods containing sericin or fibroin, were aged between 20 and 74 years, and were free of illnesses with spontaneous bowel movements less than five times per week.

[0035] (2) Dosage, etc. The subjects were instructed to take one bottle (100 ml) of either the beverage prepared in Preparation Example 2 or a placebo beverage at any time during the day every day for 12 weeks.

[0036] (3) Check the frequency of spontaneous bowel movements The mean number of spontaneous bowel movements (times / week) was analyzed.

[0037] (4) Intestinal (fecal) bacterial flora analysis Fecal samples were collected using a stool collection tube FS-0003 (TechnoSuruga Laboratory Co., Ltd.) filled with 4 ml of RNAlater solution (Thermo Fisher Scientific, Waltham, MA, USA). The samples were collected before and after the intake period within 3 days from the test date (weeks 0 and 12, respectively). Fecal DNA extraction was performed according to a previously reported method (Morita H, et al., Microb. Environ., 22, 214-222 (2007), Kim SW, et al., DNA Res., 20, 241-253 (2013)). The collected feces was uniformly suspended in RNAlater solution in a collection tube, and the suspension was filtered through a 100 μm mesh filter (Sysmex-Partec) to remove undigested food fragments. 2 ml of the filtrate was centrifuged at 9000 × g for 15 min at 4 °C to obtain a pellet, which was resuspended in 5 ml of phosphate-buffered saline (PBS) (pH 7.4) and washed by centrifugation under the same conditions. After further washing with TE10 buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8.0) under the same conditions as above, the pellet was placed in 650 μl of TE10 buffer (chicken egg white, Sigma-Aldrich) containing 23 mg / ml lysozyme. The cells were resuspended and the suspension was incubated at 37°C. After 1 hour of reaction, 40 μl of achromopeptidase solution (Wako Pure Chemical Industries, 50 U / μl stock prepared in TE10 buffer) was added and incubated for another 30 minutes. The cells were lysed by adding 100 μl of 10% (w / v) SDS solution and 10 μl of proteinase K solution (Wako Pure Chemical Industries, 400 U / ml, recombinant) and incubated at 55°C for 1 hour. Denatured proteins were removed by extraction with phenol / chloroform / isoamyl alcohol (25 / 24 / 1), and DNA pellets were obtained by ethanol precipitation. The obtained DNA sample was further treated with ribonuclease (RNase A, NIPPON GENE CO., LTD) and purified by ethanol precipitation. Microbial flora analysis based on 16S rRNA was outsourced to Seibutsu Giken Co., Ltd. The Illumina Miseq sequencing platform and Miseq Reagent Kit v3 (Illumina Inc.) was used, and the protocol included with the kit was used. We performed a comprehensive analysis of the V3-V4 region sequences according to the rules.

[0038] 2.Results (1) Average spontaneous bowel movement frequency As shown in Table 5, the control diet group experienced only a slight increase (1.1 times the baseline frequency), whereas the test diet group experienced a significant increase (1.4 times the baseline mean), i.e., the difference between the changes in mean stool frequency was significant (p=0.017).

[0039] [Table 5]

[0040] (2) Fecal microbiota analysis The results of analyzing the change in the relative occupancy rate of the bacterial flora before and after the 12-week intake period (change in the composition of the bacterial flora based on the V3V4 region sequence of 16S rDNA) are shown in Figures 1-1, 1-2, and 1-3. In the figures, the left side shows the relative occupancy rate of each group before and after intake, and the right side shows the change in the relative occupancy rate after intake. p values ​​were calculated using the Mann-Whitney U test.

[0041] Genera that showed significant differences between the two groups included Parabacteroides, Streptococcus, Enterococcus, Clostridium, and Anaerotruncus, and among classifiable species, Parabacteroides distasonis and Clostridium cocleatum were identified. Among these genera, the ones that showed significant changes before and after ingestion in the test diet group were the genus Enterococcus (p<0.001) and the genus Clostridium (p=0.007). Of these, the change in the genus Enterococcus was particularly notable, and it was speculated that this change may have had a large effect on the frequency of bowel movements.

[0042] In fact, eight subjects whose bowel movement frequency increased significantly after taking the test diet (showing an increase greater than the average among the 25 subjects) were considered high responders, and when the correlation between the increase in bowel movement frequency and the increase in the relative occupancy rate of Enterococcus bacteria was examined, a very strong correlation was found (Spearman's rank correlation coefficient ρ = 0.857, p < 0.01).

Claims

1. An agent for improving intestinal flora, which increases the occupancy rate of bacteria of the genus Enterococcus in the intestines, contains as an active ingredient a plant lactic acid bacterium fermentation product of silk protein, wherein the plant lactic acid bacterium is Lactococcus lactis.

2. A laxative containing as an active ingredient a plant lactic acid bacterium fermentation product of silk protein, wherein the plant lactic acid bacterium is Lactococcus lactis.

3. A food for improving intestinal flora, which increases the occupancy rate of bacteria of the genus Enterococcus in the intestines, contains as an active ingredient a plant lactic acid bacteria fermentation product of silk protein, wherein the plant lactic acid bacteria is Lactococcus lactis.

4. A food for improving bowel movements, comprising as an active ingredient a plant lactic acid bacteria fermentation product of silk protein, wherein the plant lactic acid bacteria is Lactococcus lactis.

5. The agent according to claim 1 or 2, or the food according to claim 3 or 4, wherein the plant lactic acid bacterium is Lactococcus lactis BM32-1 strain.

6. 5. The agent according to claim 1 or 2, or the food according to claim 3 or 4, wherein the silk protein is a mixture of fibroin and sericin.

7. The agent or food according to claim 6, wherein the mixture of fibroin and sericin has a mixing ratio (sericin / fibroin) of 0.20 to 0.60 by mass.

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