KR20190002036A - Prebiotics comprising blueberry extract for proliferating intestinal probiotics the use thereof - Google Patents

Abstract

The present invention relates to probiotics for proliferating beneficial bacteria in the intestines, comprising a blueberry extract, and uses thereof. In particular, the blueberry extract of the present invention was tested for effects thereof on the proliferation of beneficial bacteria in the intestines, Lactobacillus acidophilus ATCC 4356 (Experimental example 1), on the proliferation of harmful bacteria in the intestines (Experimental example 2), on the total content of polyphenols to investigate factors that proliferate beneficial bacteria in the intestines (Experimental example 3), for DPPH radical scavenging ability (Experimental example 4), for ABTS radical scavenging ability (Experimental example 5), and for sugar composition analyses (Experimental example 6). According to the present invention, the blueberry extract has potent effects on proliferating beneficial bacterial in the intestines and suppressing the growth of harmful bacteria in the intestines; has potent DPPH free radical and ABTS free radical scavenging activities, thus exhibiting excellent electron donation ability; and promotes the proliferation of beneficial bacteria in the intestines, probiotics, in terms of the total contents of polyphenols and sugars. The probiotics comprising the blueberry extract has the advantage of regulating all beneficial bacteria in the intestines, and at the same time providing potent antioxidant actions due to excellent electron donation ability, and thus may be beneficially used in a pharmaceutical composition, a functional health food, and a functional health supplement.

Description

Prebiotics for the proliferation of intestinal beneficial bacteria including blueberry extract and their uses {Prebiotics comprising blueberry extract for proliferating intestinal probiotics the use thereof,

The present invention provides a prebiotics for proliferation of beneficial bacteria in the intestines containing blueberry extract and uses thereof.

[1] Jung Hye-mi et al., Effects of jujube extracts on the growth of intestinal microorganisms and antioxidant activity 2011, J Korean Soc Food Sci Nutr 40 (4), 500 ~ 508

[Document 2] Manning TS. Gibson GR. 2004. Prebiotics. Best Practice & Research Clinical Gastroenterology. 18 (2): 287-298

[Literature 3] Cardona et al., 2013, Journal of Nutritional biochemistry, 24, 1415-1422

[Patent Literature 4] Jeong et al., Nutritional Component Analysis and Antioxidant Activity of Domestic Commercial Blueberries and Raspberries, Journal of the Korean Society of Food Science and Nutrition, 37 (11), 1375-1381, 2008

[Literature 5] Martineau LC et al. Antidiabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait. , Phytomedicine.13 (9-10): 612-623, 2006

J Agric Food Chem., 31: 54 (11): 3773-8, 2006). [

[Literature 7] Sellappan et al. Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries., J Agric Food Chem. 50 (8): 2432-8. 2002).

[Literature 8] Orrhge, K. et al., 2000, Bifidobacteria and lactobacilli in human health, Drugs Exptl. Clin. Res., 26, 95-111

[Literature 9] Fuller, K., 1989, Probiotics in man and animals, J. Appl. Bacteriol., 66, 365-378

[Patent Document 10] Korean Patent Publication No. 10-2015-0075447

[Literature 11] Xie et al. Effects of Ligustrum robustum on gut microbes and obesity in rats, World J Gastroenterol. 2015, 21 (46): 13042-13054

About 400 bacteria are present in the human colon and feces, and more than 90% of them are anaerobic strains. They maintain a constant balance to form microorganisms in the intestines. They are affected by the health conditions of the human body, stress, aging physiological changes, as well as foods, medicines and the like introduced from the outside. In order to improve physical health and longevity, more intensive enteric bacteria should be retained in the intestinal microflora and fewer harmful bacteria should be maintained. In 2001, the Joint Expert Committee of the World Health Organization (FAO) and the International Food Agency (FAO) defined probiotics as 'healthy germs when they consume modest amounts of living microbes' (JH Lee, 2011, J Korean Soc Food Sci Nutr 40 (4), 500 ~ 508) This means a probiotic agent that can help improve intestinal microorganisms. On the other hand, prebiotics refers to substances that can be used in intestinal useful microorganisms and can help the growth and function of intestinal yeasts. Previously known prebiotics were nonpolar polysaccharides and oligosaccharides and were not degraded by human enzymes but were available to microorganisms (Manning TS, Gibson GR 2004. Prebiotics. Best Practice & Research Clinical Gastroenterology. 18 (2): 287-298). Recently, the concept of prebiotics has been broadened as polyphenols have been known to have beneficial effects on intestinal microorganisms (Cardona et al., 2013, Journal of Nutritional biochemistry, 24, 1415-1422).

The blueberries used in the present invention are distributed mainly in Southeast Asia and have about 400 species of shrubs belonging to the North American species of Ericaceae Vaccinium (Jeong et al., Domestic commercial blueberries and raspberries , Journal of the Korean Society of Food Science and Nutrition, 37 (11), 1375-1381, 2008). In 2002, New York Times, one of America's leading daily newspapers, selected blueberries for the world's top 10 longevity foods. Blueberry has been shown to be effective against diabetes mellitus (Martineau LC et al., Anti-diabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait., Phytomedicine.13 (9-10): 612-623, 2006) (Flavonoids) and phenols (phenolic compounds), which are known to be effective in the production of flavonoids and phenolic compounds, Compunds and antioxidants have been reported to be effective in preventing disease and preventing aging (Sellappan et al., Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries, J Agric Food Chem. 50 8): 2432-8, 2002).

On the other hand, lactic acid bacteria are widely used as probiotic bacteria. They are widely found in nature and are easily found in the intestines and fermented foods of humans and animals. Food and Drug Administration (Orrhge, K. et al., 2000, Bifidobacteria and lactobacilli in human health, Drugs Expt. Clin. Res., 26, 95-111). Lactic acid bacteria attach to the intestinal epithelial cells and become parasitic, thereby improving the properties of intestinal microflora. Thus, the intestinal microflora such as the stabilization of intestinal microflora, the reduction of the formation of decay products due to the inhibition of harmful bacteria and the prevention of disease, immune activation, It helps a lot. Lactic acid bacteria have several metabolic characteristics that inhibit the growth of various pathogenic microorganisms and pathogenic microorganisms because lactic acid bacteria produce organic acid, hydrogen peroxide, reuterin, diacetyl, acetaldehyde, bacteriocin, etc. as metabolic products (Fuller, K., 1989, Probiotics in man and animals, J. Appl. Bacteriol., 66, 365-378; Korean Patent Publication No. 10-2015-0075447)

Although probiotics are currently used as health functional foods, they are also used for therapeutic purposes. Specifically, they increase the production of immunoglobulin A antibody and reduce rotavirus shedding, shortening the duration of diarrhea, And a therapeutic agent for inflammatory bowel disease such as ulcerative colitis or Crohn's disease, a therapeutic agent for abdominal bloating or constipation, a therapeutic agent for irritable colitis, a therapeutic agent for radiation-induced enteritis, a therapeutic agent for Helicobacter infection, etc., Has been reported to be effective as a therapeutic agent for inflammatory bowel disease. (Korean Patent Laid-Open No. 10-2008-0075971)

None of the above documents, however, teach or disclose any content of the proliferative prebiotics of the enteric bacterium, including blueberry extract, and their uses.

In order to develop a prebiotics for intestinal health from natural products, the inventors of the present invention conducted experiments (Experimental Example 1) on the effect of the present blueberry extract on the proliferation of enterobacteriaceae Lactobacillus acidophilus ATCC 4356, The total polyphenol content (Experimental Example 3), the DPPH radical scavenging activity test (Experimental Example 4) and the ABTS radical scavenging activity experiment (Experimental Example 2) were conducted in order to examine the effect on the proliferation of harmful bacteria, 5), and the sugar assay (Experimental Example 7). As a result, the blueberry extract of the present invention strongly proliferated the intestinal beneficial bacteria, showed strong growth inhibition effect against intestinal harmful bacteria, and DPPH free radical and ABTS free It was confirmed that the radical scavenging activity showed a strong electron donating ability and the total polyphenol content and sugar content of the probiotic bacteria The present invention has been completed.

In order to achieve the above object, the present invention provides a prebiotics for promoting the proliferation of beneficial bacteria in the intestines containing blueberry extract.

The term " beneficial bacteria in the intestine " as defined herein means a strain that is equivalent to " probiotics ", which helps digestion and absorption and inhibits the growth of harmful bacteria in the intestines to maintain the intestinal environment in a desirable state. As a representative strain, preferably, any lactic acid bacteria widely used as probiotics can be used as a commercially available strain such as, for example, American Type Culture Collection (ATCC), Korea Biotechnology Research Institute This is a strain available from the Korean Collection for Type Cultures (KCTC), the Korean Culture Center of Microorganisms (KCCM) and the Korean Agricultural Culture Collection (KACC) Specifically, microorganisms belonging to the genus Lactobacillus, Bifidobacterium, Bacillus, Streptococcus and Enterococcus; and microorganisms belonging to the genus Lactobacillus, Bifidobacterium, Bacillus, Streptococcus, Enterococcus; Preferably Bifidobacterium, Lactobacillus or Streptococcus, more preferably Bifidobacterium or Streptococcus; More preferably Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis spp., Bifidobacterium spp., Bifidobacterium spp., Bifidobacterium spp. lactis, Bifidobacterium adolescentis, Bifidobacterium pseudocatenulatum, Bifidobacterium catenulatum or Bifidobacterium infantis, and the like. Bifidobacteria, such as B. thermophilum; Bifidobacterium sp .; Lactobacillus plantus, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus casei, Lactobacillus casei ssp. Paracasei, Lactobacillus rhamnosus, Lactobacillus spp. Lactobacillus delbrueckii ssp. Lactobacillus delbrueckii ssp. Bulgaricus, Lactobacillus delbrueckii ssp. Lactobacillus delbrueckii ssp., Lactobacillus delbrueckii ssp., Lactobacillus delbrueckii ssp. Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus reuteri, L. brevis, L. cellobiosus, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp. , Lactobacillus crispatus, Lactobacillus support (Lb. GG), Lactobacillus Loose zone soniyi (Lb. johnsonii), Lactobacillus lactis (Lb. lactis), Lactobacillus flow Terry (Lb. reuteri), Lactobacillus spp, including Lactobacillus salivarius (Lb. salivarius) species; Bacillus subtilis strains such as Bacillus cereus toyoi or Bacillus cereus strains; Leuconostoc species, such as Leuconostoc citreum and Leuconostoc mesenteroides; Pediococcus species such as Pediococcus acidilactici and Pediococcus pentosaceus; Pediococcus species such as Pediococcus pentosaceus; Propionibacterium species such as Propionibacterium acidifaciens, Propionibacterium acidipropionici, etc .; Streptococcus thermophilus, S. < RTI ID = 0.0 > S. cremoris, S. < RTI ID = 0.0 > S. infantarius, S. et al. S. intermedius, S. < RTI ID = 0.0 > S. lactis, S. Streptococcus species such as S. salivarius subsp. Thermophilus strains; Enterococcus species such as Enterococcus faecalis and E. faecium; Enterococcus species such as Enterococcus faecalis and E. faecium; A Saccharomyces species such as Saccharomyces cerevisiae and Saccharomyces boulardii, preferably a strain belonging to the genus Lactobacillus or Bifidobacterium, most preferably a strain belonging to the genus Escherichia, Lactobacillus acidophilus ( Lactobacillus acidophilus ), most preferably, the Lactobacillus sp. Strain is selected from the group consisting of Lactobacillus acidophilus ATCC 4356, Lactobacillus acidophilus ( KCTC No. 3166), Bacillus acidophilus Bacillus cereus ( KCTC No. 13123), Bifidobacterium and a sub sp .longum longum, KCTC No.3128) or Streptococcus species (Streptococcus sp, KCTC No.5644).

The extracts as defined herein are preferably water, water, or a mixed solvent of water and ethanol, more preferably water or 10 to 10% by weight of water, alcohol, C ₁ to C ₄ lower alcohols such as methanol, ethanol, To 99% (v / w) water and ethanol mixed solvent, more preferably water or 20-80% (v / w) water and ethanol mixed solvent.

Hereinafter, the method for obtaining the extract of the present invention will be described in detail.

The extract of the present invention is obtained by washing, drying and chopping blueberries, respectively, and extracting purified water (about 1 to 200 times volume (v / v), preferably 10 to 100 times volume , Water, alcohol, or water, or a mixture of water and 10-99% of water, alcohol, lower alcohol of 1 to 4 carbon atoms, acetone, chloroform, methylene chloride, hexane or a mixed solvent thereof, (v / w) water and ethanol mixed solvent, more preferably water or 20-80% (v / w) water and ethanol mixed solvent, and extraction by cold extraction, hot water extraction, ultrasonic extraction, The method for obtaining the extract is carried out by carrying out a reflux cooling extraction method carried out at a temperature of 50 to 300 DEG C, preferably 60 to 290 DEG C for 1 to 24 hours, preferably for 2 to 12 hours, Stage 1; The extract obtained above is filtered with a filter, concentrated and lyophilized for 1 day to 24 days, preferably 2 days to 10 days, to obtain the extract of the present invention.

Accordingly, the present invention provides a pharmaceutical composition, a health functional food and a health supplement food for promoting the proliferation of a beneficial bacterium containing the blueberry extract prepared by the above production method and the above production method as an effective ingredient.

Experimental Example 1 Influence on the proliferation of lactobacillus acidophilus ATCC 4356 as a target of the present blueberry extract according to the present invention (Experimental Example 2) (Example 3), DPPH radical scavenging activity test (Experimental Example 4), ABTS radical scavenging activity test (Experimental Example 5), and sugar assay experiment (Experimental Example 6) to examine the intracellular beneficial microbial growth factor , It was confirmed that the blueberry extract of the present invention strongly proliferated the beneficial bacteria in the intestines and showed a strong inhibitory effect on the harmful bacteria in the intestines and strong DPPH free radical and ABTS free radical scavenging activity, The total polyphenol content and the sugar content were found to promote the proliferation of probiotics, which are useful bacteria in the intestines. Thus, the extracts containing blueberry extract By prebiotics as well as the advantage of being able to control both the intestinal yuikgyun it also acts as a powerful antioxidant benefits can also provide its high electron donating property, it is useful as a pharmaceutical composition, functional food and dietary supplements.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for the prevention and treatment of a disease caused by promoting the proliferation of a beneficial bacterium in the intestines containing the active ingredient or the growth of a harmful bacteria in the intestine.

"Diseases caused by proliferative intestinal bacteria" as defined in the present invention include, but are not limited to, for example, antibiotic-associated diarrhea, ulcerative colitis, inflammatory bowel disease such as Crohn's disease, abdominal bloating, constipation, acute or chronic irritable colitis, Radiation-induced enteritis, Helicobacter infection, and the like.

The present invention also provides a health functional food for preventing and improving diseases caused by promoting the proliferation of beneficial bacteria in the intestines containing the active ingredient or the growth of harmful bacteria in the intestines.

The composition of the present invention contains the herbal extract in an amount of 0.01 to 99% by weight based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, including, for example, oral and rectal, or intravenous.

The present invention also provides a health functional food for preventing and improving diseases caused by promoting the proliferation of beneficial bacteria in the intestines containing the active ingredient or the growth of harmful bacteria in the intestines.

&Quot; Health functional food " as defined herein means food prepared and processed using raw materials or ingredients having functionality useful to the human body in accordance with Law No. 6727 on Health Functional Foods. &Quot; Functional " Structure and function of the nutrient to control or physiological effects, such as to obtain a beneficial effect for health is intended to eat.

The health functional food for the prevention and improvement of the disease caused by the promotion of the proliferation of the beneficial bacteria in the intestines or the proliferation of the intestinal bacteria in the intestines according to the present invention preferably comprises 0.01 to 95% by weight, preferably 1 to 80% .

Furthermore, the composition of the present invention may be a health food for the purpose of promoting the proliferation of enteric bacteria in the intestines or for the treatment of diseases caused by the growth of harmful bacteria in the intestines or for enhancing the testicular function.

In addition, for the purpose of prevention and improvement of diseases caused by promoting the proliferation of beneficial bacteria in the intestines or the growth of harmful bacteria in the intestines, there may be used pharmaceutical dosage forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions and syrups, It can be manufactured and processed into health functional foods in the form of health drinks.

In addition, the present invention provides a health supplement for prevention and improvement of a disease caused by promoting the proliferation of a beneficial bacterium in the intestines containing the active ingredient or the growth of a harmful bacteria in the intestines.

The main ingredient of the health supplement defined herein means that the content of the active ingredient with respect to the weight of the whole composition ranges from about 30% to 99%, preferably from 50% to 99%, more preferably from 70% to 99% do.

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

When the extract according to the present invention is used as a food additive, the extract may be directly added or used together with other food or food ingredients, and may be suitably used according to a conventional method. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the extract of the present invention can be added to foods or beverages for the purpose of preventing disease caused by promoting the proliferation of beneficial bacteria in the intestines or the growth of harmful bacteria in the intestines. At this time, the amount of the extract in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, .

The present invention also provides a food or food additive for preventing or improving a disease caused by promoting the proliferation of a beneficial bacterium in the intestines containing the active ingredient or the growth of a harmful bacteria in the intestines.

Examples of the products listed in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, coloring matter, licorice extract, crystalline cellulose, guar gum, Sodium laurate, sodium glutamate preparation, noodles-added alkaline agent, preservative agent, tar pigment preparation and the like.

 Examples of the functional food containing the extract of the present invention include confectionery ice creams such as bread, rice cake, dried fruit, candy, chocolate, chewing gum and confectionery, ice cream products such as ice cream, ice cream powder, low fat milk, Processed products such as processed oil, goat milk, fermented oil, butter oil, concentrated oil, yogurt cream, butter oil, natural cheese, processed cheese, milk powder, milk products, meat products such as hamburger meat products, ham , Fish oil products such as sausages, bacon, etc. Fish products such as noodles, noodles, noodles, noodles, noodles, luxury noodles, improved noodles, noodles such as frozen noodles, pasta, vegetable beverages, Seasonings such as beverages such as soy sauce, miso, kochujang, chunchu, chonggukjang, mixed berries, vinegar, sauce, tomato ketchup, curry, dressing, Lean, shortening, and pizza.

The extract according to the present invention, which is added to foods containing beverages in the course of manufacturing the health functional food, can be appropriately added or decreased as needed.

It was confirmed that the blueberry extract of the present invention strongly proliferated the intestinal beneficial bacteria, showed strong growth inhibition effect against intestinal harmful bacteria, showed strong DPPH free radical and ABTS free radical scavenging activity and showed excellent electron donating ability. Total polyphenol It is confirmed that promoting the proliferation of probiotics, which are beneficial bacteria in the intestines, in terms of content and sugar content, not only has the advantage of controlling all the beneficial bacteria in the intestines by the prebiotics including blueberry extract, And can be used as a pharmaceutical composition, a health functional food and a health supplement food.

Figure 1 shows a process for preparing blueberry extract;
FIG. 2 shows the effect of each extract of blueberry on the proliferation of Lactobacillus acidophilus ATCC 4356 (1a) (2a) and FIG. 2b shows the effect of BL30E on the growth of Lactobacillus acidophilus ATCC 4356 (1a) On the proliferation;
Figure 3 shows the inhibitory effect on Staphylococcus aureus by addition of BL30E (3a); FIG. 3B shows the Listeria exhibit an inhibitory effect on monocytogenes ;
Figure 4 shows the antioxidative activity of the blueberry extract by DPPH free radical scavenging ability;
Figure 5 shows the antioxidative activity of blueberry extract by ABTS free radical scavenging ability.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Example 1. Preparation of blueberry extract

1-1. Preparation Example of Water Extract (see Fig. 1)

The water was added to the dried blueberry (organic Lovepam, Sunchang, Jeollabuk-do) at a volume of 20 times volume (v / v) and concentrated by reflux extraction at 250 ° C for 3 hours. The concentrated aqueous blueberry extract was filtered through a filter net, concentrated with a vacuum concentrator (1 ton, manufactured by Myeongil Il) and lyophilized with a freeze dryer (PVTFD10R, Ilshin Lab) for 5-7 days to obtain the water extract of the present invention Hereinafter referred to as BLW ) was obtained and used as a sample in the following experimental example while being stored in a freezer.

1-2. Of 30% ethanol extract Manufacturing example

The solution was concentrated by conducting a reflux extraction method in which 30% ethanol (30% w / v) of the weight of dry blueberries (Organic Lovepam, Sunchang, Jeollanamdo) was distilled at 250 ° C. The 30% ethanol extract of the concentrated blueberry was filtered through a filter net, concentrated with a vacuum concentrator (1 ton, manufactured by Myeongil Il) and lyophilized for 5 to 7 days with a freeze dryer (PVTFD10R, % Ethanol extract (hereinafter referred to as & quot ; BL30E & quot ;) was obtained and used as a sample in the following experimental example while kept in a freezer.

1-3. Of 70% ethanol extract Manufacturing example

The concentrated solution was subjected to a reflux extraction process in which 20% by volume (w / v) of 70% ethanol was added and the mixture was distilled at 250 ° C for 3 hours, which was weighed in dry blueberries (Organic Lovepam, The 30% ethanol extract of the concentrated blueberry was filtered through a filter net, concentrated with a vacuum concentrator (1 ton, manufactured by Menier), and lyophilized for 5 to 7 days with a freeze dryer (PVTFD10R, (Hereinafter referred to as & quot ; BL70E & quot ;) was obtained and used as a sample in the following experimental example while being kept in a freezer.

Experimental Example 1. Effect on the proliferation of beneficial bacteria in the intestines

In order to confirm the effect of the sample of the present invention obtained in Example 1 on the growth of beneficial bacteria in the intestines, the method disclosed in the literature was applied as follows (Chung Hye-mi et al. Active 2011, J Korean Soc Food Sci Nutr 40 (4), 500-508 ).

1-1. Experimental strain

The strains were distributed from KCCM (Korean Culture Center of Microorganisms, Korea). Lactobacillus acidophilus ATCC 4356 (ATCC: American Type Culture Collection) was used as the enteric bacteria. Lactobacilli MRS broth (288130, BD difco, USA) was used as a growth medium for culture of intestinal beneficial bacteria. The strain was used after being activated by subculture three times or more before use in the experiment.

1-2. Experimental course

The number of generated colonies was counted, and the average number of colonies was multiplied by a dilution factor to calculate the number of viable cells per mL of the culture. 1 (v / w)% each of blueberry extract powder (BLW, BL30E, BL70E) was added to 5 mL MRS broth and sterilized.

The test strain (Lactobacillus acidophilus, ATCC 4356) was inoculated in a sterile 5 mL MRS broth medium to 1% inoculated to the MRS broth by adjusting the bacterial concentration to an optical density value of 0.60 at 660 nm, followed by incubation at 37 ° C , And the number of bacteria was measured at 0, 20 and 72 hours. As a control, MRS broth without added blueberry extract was used.

1-3. Experiment result

The relationship between the blueberry extract and the proliferation of intestinal beneficial bacteria is shown in Fig. In the case of Lactobacillus acidophilus, the blueberry extract was not effective for the initial proliferation until 35 hours, but it was effective for the proliferation after 35 hours. BL30E logarithms were not significantly different from BLW and BL70E, but BL30E showed 5.68 CFU / mL at the stationary phase (5.43 CFU / mL) ( P <0.05) higher than control group without blueberry ( P <0.05) And 5.94 CFU / mL at 72 hours, which were significantly higher than those of the control group (5.43 CFU / mL). As shown in FIG. 2B, BL30E showed a significantly higher number of bacteria than the control group.

Experimental Example 2: Effect on the proliferation of enteric bacteria in intestines

In order to confirm the effect of the sample of the present invention obtained in Example 1 on the growth of harmful bacteria in the intestines, the method described in the literature was applied as follows (Xie et al., Effects of Ligustrum robustum on gut microbes and obesity in rats , World J Gastroenterol. 2015, 21 (46): 13042-13054)

2-1. Experimental course

Harmful bacteria is Listeria monocytogenes Regensburg (Listeria monocytogenes ATCC 19111), Staphylococcus aureus ATCC 6538p). For Listeria monocytogenes ATCC 19111, Staphylococcus aureus ( Staphylococcus aureus ) was added to sterile Brain Heart Infusion liquid medium (237500, BD difco, USA) ATCC 6538p) was inoculated 1% in Soybean-Casein Digest liquid medium (211768, BD difco, USA) and cultured at 37 ° C in an anaerobic environment.

The inhibition rate was measured by inoculating 1% in Brain Heart Infusion broth after adjusting the bacterial concentration to the optical density value of 0.70 at 600nm. The blueberry extracts contained 0.5, 1, 5, 10 % (v / w).

The culture was performed in a volume of 200 μl on a 96-well plate, and the cells were cultured for 24 hours at 37 ° C in an incubator (JISICO). The absorbance was measured at a wavelength of 600 nm using a microplate spectrophotometer (epoch, BioTek) Respectively.

In the control group, each liquid medium to which no sample was added was used, and the inhibition rate was expressed by the inhibition efficiency (% inhibition) of the sample at each concentration according to the following equation (1).

2-2. Experiment result

The relationship between the blueberry extract and the growth of enteric bacteria in the intestine is shown in Fig. Staphylococcus aureus ( Staphylococcus aureus) ATCC 6538p) showed an inhibitory effect of 42.8% and 65.4% when BL30E was added at 5 and 10%, respectively, as shown in FIG. 3A. In the case of Listeria monocytogenes ATCC 19111, , And 10%, respectively, the inhibitory effect was 279.8% and 98.4%, respectively.

Experimental Example 3. Measurement of total polyphenol content

In order to confirm the total polyphenol content of the sample of the present invention obtained in Example 1 above, the Folin Deinis method (AOAC) using a phenol compound, which reacts with phosphomolybdic acid and exhibits blue color. , Association of official analytical chemists, Official methods of analysis. 15 ^th ed., Washington D C. for Cd 8-35 (1990)) was subjected to colorimetric quantitative by some modification.

To 20 μl of the blueberry extract, 80 μl of distilled water was added, and 20 μl of Folin-Ciocalteu reagent (F9252, Sigma, USA) was mixed. After incubation for 5 minutes, 140 μl of 7% Na ₂ CO ₃ (S7795, Sigma, USA) was added, and the absorbance was measured at 750 nm in a dark room at room temperature for 1 hour. Gallic acid (G7384, USA) was used.

Table 2 shows the total polyphenol contents of the blueberry according to the extraction method. When gallic acid was used as a reference material, the polyphenol content of BLW was 137.31 mg / g, that of BL30E was 275.12 mg / g and that of BL70E was 206.17 mg / g. there was. The polyphenol content of blueberry was the highest in BL30E.

Blueberries  The total polyphenol concentration in the extract Blueberry extract Total polyphenol (mg GAE / g) ^{1 )} BLW 137.31 ± 2.00 BL30E 275.12 + - 2.97 BL70E 206.17 ± 14.70 ¹⁾ : When gallic acid was used as a standard, total polyphenol

Experimental Example  4. DPPH  Radical Scatters  exam

Methods such as Blois to confirm the effects of the DPPH radical scavenging activity test of samples of the present invention obtained in Example 1 (Bllis MS et al, Nature , Antioxidant determination by the use of stable free radicals 181:.. 1199 -1200 (1958)).

5-1. Experimental course

The scavenging activity for DPPH (α, α-diphenyl-β-picrylhydrazyl) radicals was tested as follows. The absorbance was measured at 515 nm using a microplate spectrophotometer (Epoch, BioTek Instruments, Inc., Winooski, VT, USA) after adding 380 μl of 100 μl DPPH solution to 20 μl of blueberry extract and reacting in a dark room at room temperature for 30 minutes . Vitamin C (ascorbic acid, SMB00390, Sigma, USA) was used as a control.

5-2. Experiment result

The DPPH method is based on the ability of DPPH, a stable free radical, to react with a hydrogen donor (H-donor). DPPH is a relatively stable free radical, which is reduced by antioxidants and discolored, so it is widely used for measuring antioxidant efficacy. The antioxidant activity of DPPH free radical scavenging activity was ascertained by using ascorbic acid, a natural antioxidant, as a control, as shown in FIG. When the concentration of the control was 250 μM, the antioxidant activity of BL30E was 69.6%, which was 92.7% of EGCG, which was the highest among BLWE of 58.4% and BLW of 53.2%.

Experimental Example 6: ABTS radical scavenging ability test

In order to confirm the effect of the sample ABTS radical scavenging activity test of the present invention obtained in Example 1, such as Re (Re R et al, Free Radical Biol Med 26:. 1231-1237 (1999)) was tested according to the.

6-1. Experimental course

The scavenging activity for the ABTS (2,2'-azino-bis) (3-ethylbenzothiazoline-6-sulphonic acid) radical was tested as follows. ABTS ⁺ radical solution was prepared by dissolving 7 mM ABTS (A1888, Sigma, USA) and 2.45 mM potassium persulfate in 7 mM potassium phosphate solution at a ratio of 70: 1. Absorbance was measured at 734 nm using a spectroscope (microplate spectrophotometer, Epoch, BioTek Instruments, Inc., Winooski, VT, USA) after mixing 10 μl of blueberry extract with 390 μl of ABTS ⁺ radical solution.

6-2. Experiment result

The ABTS method shows representative radical scavenging activity, and EGCG (epigallocatechin gallate) was used as a control, and the result of antioxidative activity by ABTS free radical scavenging was shown in Fig. When the concentration of EGCG was 10 mg / mL, the radical scavenging activity of 96.5% was exhibited. The antioxidant activity of BL30E was 92.14%, which was 83.75% of that of BL70E and 70.78% of that of BLW. Therefore, blueberry extract not only promotes beneficial bacteria in the intestines but also has antioxidant activity.

Experimental Example  7 BL30E's  Comparison per configuration

For comparison of the sugar composition of the present invention obtained in Example 1, it was commissioned by an authorized analysis institute (Korea Bioanalytical Research Institute, Ltd., 519, Daungchon-dong, Jungwon-gu, Seongnam-si, Gyeonggi-do). Fructose, glucose, sucrose, maltose, Were analyzed.

Table 2 shows the result of the sugar content analysis of BL30E. As a result of the analysis, excretion was 37.68 g / 100 g and glucose 35.89 g / 100 g, and sucrose, maltose and lactose were not detected.

BL30E's Comparison of party configurations extract fruit sugar glucose saccharose Maltose Lactose Blueberry 30% extract 30.62 g / 100 g 28. 69 g / 100 g - - -

As a result of the above-mentioned experiment, it was confirmed that the effect of the present invention on the proliferation of the intestinal bacilli Lactobacillus acidophilus ATCC 4356 (Experimental Example 1), the effect on the proliferation of enteric bacteria in the intestinal tract (Experimental Example 2) The total polyphenol content (Experimental Example 3), the total flavonoid content (Experimental Example 4), the DPPH radical scavenging activity test (Experimental Example 5), the ABTS radical scavenging ability (Experimental Example 6), and sugar composition analysis experiment (Experimental Example 7). As a result, the blueberry extract of the present invention strongly proliferated the intestinal beneficial bacteria and had a strong growth inhibitory effect against intestinal harmful bacteria, DPPH free radical and ABTS free radical scavenging activity showed strong electron donating ability and it was confirmed that promoting the proliferation of probiotics in the intestines in terms of total polyphenol content, total flavonoid content and sugar content.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but will be specifically described.

Preparation Example 1. Preparation of powder

Blueberry extract (BLW) ----------------------------------- 20 mg

Lactose ------------------------------------------------- - 100 mg

Talc ------------------------------------------------- --- 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation Example 2. Preparation of tablets

Blueberry extract BL30E) --------------------------------- 10 mg

Corn starch -------------------------------------------- 100 mg

Lactose ------------------------------------------------- - 100 mg

Magnesium stearate ------------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation Example 3. Preparation of capsules

Blueberry extract (BL70E) -------------------------------- 10 mg

Crystalline cellulose --------------------------------------- 3 mg

Lactose --------------------------------------------- 14.8 mg

Magnesium stearate 0.2 mg &lt; RTI ID = 0.0 &gt;

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation Example 4. Preparation of injection

Blueberry extract (BLW) ---------------------------------- 10 mg

Mannitol ------------------------------------------------ 180 mg

Sterile sterile distilled water used - 297 mg

Na ₂ HPO ₄ , 12H ₂ O ------------------------------------------- 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation Example 5. Preparation of a liquid preparation

Blueberry extract (BL30E) --------------------------------- 20 mg

Isseong Party ------------------------------------------------ - 10 g

Mannitol ------------------------------------------------- --- 5 g

Purified water ------------------------------------------------- - Suitable amount

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Of health food  Produce

Blueberry extract ( BL70E ) ------------------------------- 1000 mg

Vitamin mixture --------------------------------------------

Vitamin A Acetate ------------------------------------- 70 [mu] g

Vitamin E ----------------------------------------------- 1.0 Mg

Vitamin B1 --------------------------------------------- 0.13 mg

Vitamin B2 - 0.15 mg

Vitamin B6 ---------------------------------------------- 0.5 mg

Vitamin B12 --------------------------------------------- 0.2 g

Vitamin C ------------------------------------------------ 10 mg

Biotin ------------------------------------------------- - 10 [mu] g

Nicotinic acid amide 1.7 mg &lt; RTI ID = 0.0 &gt;

Folic acid ------------------------------------------------- --- 50 μg

Calcium pantothenate ------------------------------------------ 0.5 mg

Inorganic mixture --------------------------------------------

Ferrous sulfate ------------------------------------- 1.75 Mg

Zinc oxide ---------------------------------------------- 0.82 mg

Magnesium carbonate ------------------------------------------ 25.3 mg

Potassium monophosphate --------------------------------------------- 15 Mg

Secondary Calcium Phosphate --------------------------------------------- 55 Mg

Potassium citrate ---------------------------------------------- 90 mg

Calcium carbonate ----------------------------------------------- 100 Mg

Magnesium chloride - 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation Example 7. Preparation of health drink

Blueberry extract ( BLW ) ----------------------------------- 1000 mg

Citric acid ------------------------------------------------- 1000 mg

oligosaccharide ------------------------------------------------- 100 g

Plum concentrate ------------------------------------------------ - 2 g

Taurine ------------------------------------------------- ---- 1 g

Purified water was added.

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, &Lt; / RTI &gt;

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (9)

Prebiotics for promoting the proliferation of beneficial bacteria in the gut containing blueberry extract. The method according to claim 1,
Wherein the intestinal beneficial bacteria are microorganisms belonging to the genus Lactobacillus, Bifidobacterium, Bacillus, Streptococcus, or Enterococcus, . The method according to claim 1,
Wherein the extract is an extract which is soluble in C ₁ to C ₄ lower alcohols such as water, alcohol, methanol, ethanol, butanol or a mixed solvent thereof. A pharmaceutical composition for preventing and treating diseases caused by promoting the proliferation of a beneficial bacterium in the intestines containing the active ingredient or the growth of a harmful bacteria in the intestines. 5. The method of claim 4,
A disease caused by the proliferation of the enteric bacteria in the intestine &quot; is an inflammatory bowel disease such as an ulcerative colitis accompanied by antibiotic-associated diarrhea, vomiting, vomiting, nausea, Crohn's disease, abdominal bloating, constipation, acute or chronic irritable colitis, , &Lt; / RTI &gt; Helicobacter infection. A health functional food for preventing or ameliorating a disease caused by promoting the proliferation of a beneficial bacterium in the intestines containing the active ingredient or the growth of a harmful bacteria in the intestine. The method according to claim 6,
The health functional food may be a pharmaceutical dosage form of a powder, a granule, a tablet, a capsule, a pill, a suspension, an emulsion or a syrup, or a health functional food in the form of a tea bag, an oil- A health supplement for preventing or ameliorating a disease caused by promoting the proliferation of beneficial bacteria in the intestines containing the active ingredient or the growth of a harmful bacteria in the intestines. A food additive for preventing or ameliorating a disease caused by promoting the proliferation of a beneficial bacterium in the intestines containing the active ingredient or the growth of a harmful bacteria in the intestines.

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