KR20080075971A - Probiotics combined preparations - Google Patents

Abstract

A probiotic composition comprising lactic acid bacteria is provided to increase immunity, normalize balance of intestinal flora, prevent and alleviate allergic disease such as skin allergy and food allergy, improve diabetes, control body weight, prevent adult disease such as hypertension, and enhance health of intestines. A probiotic composition comprises lactic acid bacteria containing 70-95 wt.% of at least one Lactobacillus sp. selected from Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus casei and Lactobacillus plantarum and 1-15 wt.% of at least one Bifidobacterium sp. selected from Bifidobacterium longum and Bifidobacterium breve, 0.1-10 wt.% of the extract of Acanthopanax senticosus and 0.1-10 wt.% of the extract of Perilla frutescens var. acuta leaf, and is formulated as powder, granule or capsule. Further, the lactic acid bacteria additionally comprise Streptococcus thermophilus.

Description

PROBIOTICS COMBINED PREPARATIONS}

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the results of viability evaluation in artificial gastric juice for each probiotic contained in a probiotic complex preparation of the present invention.

Figure 2 is a graph showing the results of viability evaluation in artificial serous for each probiotics contained in the probiotic complex formulation of the present invention.

The present invention relates to a probiotic complex formulation, and more particularly, by combining and combining lactic acid bacteria and herbal medicines such as thorny scabies and / or perilla leaves, cumulatively expressing each characteristic to stimulate the immune system and intestine The present invention relates to a probiotic complex formulation comprising a mixture of lactic acid bacteria and herbal medicines, which has a beneficial effect on the improvement of general health through normalization of the bacterial flora.

There are many fungi living in the human body, especially about 300 kinds of bacteria and about 100 trillion bacteria exist in the intestine, and its total weight is about 1kg, which is much more than about 60 trillion human cells. Known. The intestinal flora, which is a collection of these intestinal bacteria, has a balanced ratio of beneficial and harmful bacteria to the human body, and the fact that the intestinal flora is closely related to the health and various diseases of animals, including humans, has been epidemiological investigations and studies. Is becoming increasingly apparent.

When the human health as yuikgyun belonging to the group of lactic acid bacteria (乳酸菌群) typical Bifidobacterium (Bifidobacterium genus) and Lactobacillus genus (Lactobacillus genus ) predominantly balances and coexists, while the balance between beneficial and harmful bacteria is compromised when quantitative or qualitative changes in the foods consumed, antibiotic use, overwork, sleep deprivation, mental stress, or changes in environmental conditions. As the number of harmful bacteria increases, it is known that various types of diseases can be caused, such as diarrhea, gastritis, chronic fatigue or rough skin, liver failure, hypertension or atherosclerosis.

On the other hand, probiotics (probiotics) has an etymological meaning that is opposed to antibiotics (antibiotics), which means antibiotics and is defined as a microbial agent or microbial component to help balance the microorganisms in the intestines, Lactobacillus bacteria and bifidus bacteria Bacillus species collectively called lactic acid bacteria is typical.

Probiotic functions known to date inhibit the growth of pathogens in the intestines, produce substances that control the immune capacity of intestinal epithelial cells or immune cells, compete with pathogens to inhibit the growth of pathogens, and secondary antibiotic treatment or external It is known to maintain the homeostasis of gut flora against environmental changes, and the effect of probiotics is to increase the number of enteric microorganisms in Bifidobacterium or Lactobacillus, thereby reducing the incidence of intestinal homeostasis. The evidence has been demonstrated through various experiments. However, not all probiotics are always useful, and intestinal bacteria of the same genus may have clinical effects or not at all, and some may worsen the clinical course.

In addition, there has been much discussion about whether probiotics should be present in the form of live bacteria in order to be effective in the body, but probiotics that have been sterilized by radiation irradiation in recent years have also been proven to improve intestinal inflammation in certain animal models. It is generally recognized that the effect is different depending on the fungal species regardless of whether the fungi are killed or not.

Probiotics are currently used primarily as dietary supplements, but are also partly used for therapeutic purposes. Specifically, probiotics are used to increase the production of immunoglobulin A antibodies and to reduce rotavirus shedding, thereby reducing the duration of diarrhea. It has been actively applied as a prophylactic and therapeutic agent for inflammatory bowel disease such as ulcerative colitis or Crohn's disease, for bloating or constipation for the stomach, for the treatment of irritable colitis, for the treatment of irritable colitis, for the treatment of radiation induced enteritis, and for the treatment of Helicobacter infection It is reported to be effective as an agent for treating inflammatory bowel disease.

Various lactic acid bacteria formulations have been conventionally proposed. As a typical example, Korean Patent Laid-Open Publication No. 1996-0034401 proposes a lactic acid bacterium formulation using Bifidobacterium longum having excellent resistance to acids and bile acids. Publication No. 2001-0081822 discloses a lactic acid bacterium preparation using specific Lactobacillus acidophilus and Lactobacillus casei having antibacterial activity against Helicobacter pylori . Korean Patent Publication No. 2002-0012894 proposes a lactic acid bacterium preparation using the new strain Lactobacillus clearans and Enterococus faecalis , and Korean Patent Publication No. 2004-0111434 discloses Lactobacillus. salivarius (Lactobacillus salivarius) the oral micro sample of the active ingredient And it describes a la normalization lactic acid bacteria preparations, Korea Patent Publication No. 2004-0084285 discloses a mentions the specific Lactobacillus casei Lactobacillus preparation for inhibiting rotavirus infection by the (Lactobacillus casei).

However, the above-mentioned prior art only suggests a formulation consisting of a specific one or two kinds of lactic acid bacteria, and none of them suggests or suggests to include herbal medicine ingredients.

On the other hand, most lactic acid bacteria products such as calpis and yogurt are widely consumed as beverages or dressings in recent health-oriented trends, but they have been consistently steadily for several months to years. Even if eating can not realize the improvement of health or symptoms are not satisfied enough so far for those who expect a certain effect, so that they are only comforted or simply treated as a favorite food.

Therefore, the first object of the present invention is to provide a probiotic complex preparation that can obtain intestinal health promoting effect in a relatively short period of time.

A second object of the present invention is to provide a probiotic complex preparation that can obtain an immune enhancing effect in addition to the first object of the present invention described above.

The third object of the present invention is to provide a probiotic complex preparation which can obtain the anti-allergic effect and the prevention and alleviation of atopic symptoms in addition to the first and second objects of the present invention described above.

According to one preferred aspect of the present invention for smoothly achieving the first to third objects of the present invention, there is provided a probiotic complex preparation comprising a lactic acid bacterium and a thorn stem extract or perilla leaf extract.

According to another aspect of the present invention, the lactic acid bacteria in the above-described aspect is Lactobacillus ashdophyllus ( Latobacillus acidophilus ), Lactobacillus fermentum , Lactobacillus casei , and at least one Lactobacillus plantarum selected from the group consisting of Lactobacillus plantarum and Bifidobacterium longgum ( Bifidobacterium) longum ) and Bifidobacterium breve , a probiotic complex preparation is a complex lactic acid bacterium of at least one Bifidobacterium selected from the group consisting of.

According to another aspect of the present invention, there is provided a probiotic complex preparation, which is a complex lactic acid bacterium further comprising Streptococcus thermophilus .

According to another aspect of the present invention, in the above-described aspect Lactobacillus ashdophyllus ( Latobacillus acidophilus), Lactobacillus momentum spread (Lacotbacillus fermentum), Lactobacillus Kasei (Lactobacillus casei) and, Lactobacillus plan tareom (Lactobacillus plantarum) with 70-95% by weight of at least one species selected from the group consisting, preferably from 75 92 wt%, more preferably 80-90 wt%; Ronggeom Bifidobacterium (Bifidobacterium longum ) and 1 to 15% by weight, preferably 5 to 10% by weight, more preferably 6 to 8% by weight of at least one species selected from the group consisting of Bifidobacterium breve ; There is provided a probiotic complex formulation, a complex lactic acid bacterium comprising 0.1-10% by weight, preferably 2-8% by weight, more preferably 2.5-6% by weight, respectively.

Hereinafter, the present invention will be described in detail.

In general, probiotics promote the exercise of the intestine to perform the formal functions of digesting food and absorbing nutrients, inhibiting the production of harmful substances and simultaneously decomposing them, synthesizing vitamins or amino acids, or intestinal infection of pathogens. There is an excellent health promotion effect, such as showing an immune enhancing effect that protects 感染, an anticancer effect, an antiallergic effect, or a combination of these effects.

According to the inventors, the stool was collected and examined from 700 patients with atopic dermatitis, and the average number of intestinal bacteria was only about one thousandth of that of the average healthy person. It fell down and the harmful bacteria were dominant.

Therefore, the present inventors believe that intestinal bacteria and immune mechanisms are closely related, and thus, instability of the immune apparatus, reduction of intestinal bacteria, or inequality of intestinal bacteria deteriorate the function of each system and organs, thereby causing atopic symptoms. It was inferred that this could be an important factor.

In newborns, atopic symptoms can be developed when breastfeeding, due to the small amount of lactic acid bacteria in the intestine, which in turn can lead to an excess of immune. Therefore, if you do not normalize the balance between the strains between the intestines and strains, it may not be possible to reduce the stress, such as itching and rashes, so atopic symptoms are not alleviated to a satisfactory level.

From the above results, infants, children, and adolescents who are suffering from atopy, as well as adults, may eat foods (fermented foods, plant fiber) that can increase the number of bacteria by sufficiently growing intestinal bacteria, that is, atopic symptoms. May help alleviate

The present inventors have concentrated on the broad spectrum of lactic acid bacteria spectrum, various herbal ingredients, and various combinations thereof, and have studied the effects of enhancing immune function, anti-allergic effect, and preventing or relieving atopy and its symptoms. It is early.

The probiotic complex formulation according to the present invention is Lactobacillus fermentum ( Lacotbacillus fermentum ), Latobacillus acidophilus , Lactobacillus casei , Lactobacillus plantarum , or a plurality of species by any combination thereof, Bifidobacterium longgum ( Bifidobacterium longum ) and / or Bifidobacterium breve , and optionally, lactic acid bacteria of Streptococcus thermophilus ; Consisting of thorns and / or perilla leaf extracts.

The composition of the above-described probiotic complex formulation composition of the present invention will be described.

Lactobacillus fermentum effectively inhibits the attachment of histamine to receptors on tissues or vascular cells by neutralizing histamine by glycoproteins on the cell surface of lactic acid bacteria. As a result, it suppresses excessive histamine reactions and helps allergic reactions and symptoms of atopic dermatitis. Clinical trials have shown that the above-mentioned lactic acid bacteria have anti-allergic effects as well as general antihistamine drugs, but have no side effects. In addition, the anti-allergic effect is the same in both live and dead bacteria.

In addition, six to 18 months of age at the University of Western Austrailia were administered Lactobacillus fermentum and placebo twice daily for sixty- six or more children with severe atopic dermatitis. Compared to the post-war SCORAD (Scoring Atopic Dermatitis Card: Objective Indicators of Atopic Symptoms) records, atopic symptoms in the Lactobacillus fermentum- treated group have been reported to be significantly reduced.

Subsequently, Latobacillus acidophilus is a type of Lactobacillus bacteria that has a rod shape and is acid resistant bacteria. It is a microorganism that inhibits the growth of intestinal rot microorganisms and Helicobacter pylori, enhances mineral absorption, enhances intestinal immune function, anticancer effect, lowers cholesterol, and synthesizes vitamin B group.

In addition, Lactobacillus casei has anti-diabetic and acute diarrhea inhibitory effects and has been reported to be used as an adjuvant for anticancer metastasis.

On the other hand, Lactobacillus plantarum ( Lactobacillus plantarum ) is a kimchi fermented lactic acid bacteria to inhibit the proliferation of Listeria bacteria, it is widely used for reproductive fermentation.

In addition, Streptococcus thermophilus , which may optionally be used in the present invention, is a high temperature bacterium having an optimal growth temperature of 40 ° C. or higher, compared to other conventional lactic acid bacteria having an optimal growth temperature of about 37 ° C. It has excellent lactic acid production ability, antioxidant activity, and ability to inhibit rotavirus, a virus that causes acute diarrhea in infants, and has antibiotic-sensitive properties.

In addition, Bifidobacterium longum ( Bifidobacterium longum ) produces a group of vitamin B, biotin, has been reported to improve the liver function, improve the oblique thyroid disorder, inhibit the fat peroxidation.

In addition, Bifidobacterium breve has a protective effect against influenza virus infection and an immunomodulatory effect, and is known to be excellent in inhibiting rotavirus infection.

In the present invention, a plurality of species by any one or combination of the above-mentioned species of the genus Lactobacillus is 70 to 95% by weight, preferably 75 to 92% by weight, more preferably based on the total weight of the probiotic complex preparation. Preferably it comprises 80 to 90% by weight, any one or both species of the above-mentioned species of bifidus 1 to 15% by weight, preferably 5 to 10% by weight, more preferably based on the total weight of the probiotic complex formulation Preferably 6 to 8% by weight, and when added, Streptococcus thermophilus is 0.1 to 5% by weight, preferably 0.3 to 3% by weight, more preferably 0.5 based on the total weight of the probiotic complex formulation It contains -5 weight%.

The lyophilized powder of the above-mentioned strain is generally 0.1x10 ^{9 to} 8.0x10 ⁹ cfu / g, particularly 0.3x10 ^{9 to} 3.5x10 ⁹ cfu / g.

Meanwhile, one of the effective herbal plant extract ingredients in the probiotic complex preparation composition of the present invention is an extract of Eleutherococcus senticosus or Acanthopanax nticosus, also called Siberian ginseng (siberian sinseng). In particular, abundantly contained Eleutheroside E and Acanthocide D and the like.

Prickly Ogapi is a low-temperature and grotesque plant that lives in Siberia, northeastern China, Manchuria, high altitudes over 600m above sea level, and Hokkaido. According to the recent research results, it is reported that Korean prickly pears have 4 and 6 times higher content of eleuteroside E and acantoside D than Siberian prickly pears or Chinese prickly pears, respectively.

The thorny ogapi used in the present invention is not particularly limited even if any one of thorny ogapi, mingashiogai, jirisan ogapi, island ogapi, white hair ogapi and the like is used herein. It means, but it is preferably thorny ogapi, folk house ogapi, white hairy ogapi.

It is effective in preventing the disease by extracting the extract with tonic effect and nonspecific adaptogen effect, and it is effective against the increase of adrenal weight and spleen contraction caused by stress in the restraint stress experiment using rats. It is reported that there is no side effect such as insomnia, unlike ginseng, because it has a slight effect on reducing the content of ascorbic acid in the adrenal glands and does not excite people (Lloydia, Vol. 32, No. 1, 1969). . In addition, labor force improvement and gonadotropin have been reported. In other words, thorns are effective in preventing and treating geriatric and modern diseases, as well as anti-inflammatory, fatigue, anti-stress, central nervous system excitement and muscle strengthening detoxification effect, improving body's immunity and resistance, enhancing energy, improving diabetes, It has various effects such as improving liver function, and it helps to prevent adult diseases such as diabetes, weight control and hypertension by fat metabolism, carbohydrate metabolism, and water metabolism, and removes inflammatory diseases such as rhinitis, sinusitis and arthritis. It is known to have an analgesic effect.

In addition, it is perilla frutescens var. Acuta which can be used as another one of the effective herbal plant extract ingredients in the probiotic complex preparation composition of the present invention, which is native to China and is distributed in Korea and Japan. It is a perennial herb of Lamiaceae, especially the perilla leaves contain about 0.5% of essential oils with a unique scent, and its main component is 55% of phenylaldehyde essential oils. Perillaaldehyde, limonene, pinene, etc. It is contained.

Perilla leaf is also called as Japonica in oriental medicine, and its phenylaldehyde component makes the intestinal comfortable and has a neurostable effect and anorexia nervosa. Like lactic acid bacteria, it has an effect on food poisoning, gastritis or enteritis, and it is known to promote the secretion of sweat, prevent colds, and reduce cough by smoothing blood circulation.

In the probiotic complex preparation composition of the present invention, the above-mentioned prickly pear extract and perilla leaf extract extract these plants while boiling water, ethanol, or any mixed solvent thereof in a temperature range of 70 to 100 ° C. according to the type of solvent used therein. The pack of three fragments of suture is put in and extracted for about 2 to 12 hours, usually 3 to 8 hours. At this time, it can also be boiled by adding 0.1 to 0.5% citric acid. The extract is freeze-dried after filtration using a filter paper having a pore size of about 0.3 ~ 0.8㎛.

Said prickly pear extract or perilla leaf extract is contained in an amount of 0.1 to 10% by weight, preferably 2 to 8% by weight, more preferably 2.5 to 6% by weight, based on the total weight of the probiotic complex preparation, which is a complex lactic acid bacterium.

The probiotic complex preparation according to the present invention may be in a powdered or granulated formulation, or a liquid form including a capsule form thereof, or a suitable sweetener, etc., but is usually in a powdered or granulated formulation, or a capsule form thereof. In addition, prickly pear extract or perilla leaf extract is an extract by water, ethanol, or a mixed solvent thereof, or a citric acid addition solvent, which may likewise be in powdered, granulated, or liquefied form, but usually It may be in powder or granule form.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended to illustrate the present invention but are not intended to limit the present invention.

Example 1 Composition

Lactobacillus, prickly pear and perilla leaf extract for preparing the probiotic complex formulation composition of the present invention was purchased commercially from Mediogen. All of the lactic acid bacteria described above can be easily obtained commercially as a standard bacterial group, and these strains can all be obtained from KCTC (Korea Biotechnology Research Institute).

The accession numbers of these lactic acid bacteria strains available from KCTC are illustrated in Table 1 below.

KCTC Strain Deposit No. of Probiotic Strains Genus and species KCTC strain number Lactobacillus casei 2180 Lactobacillus fermentum 3112 Lactobacillus acidophilus 3140 Lactobacillus plantarum 3103 Streptococcus thermophilus 3658 Bifidobacterium longum 3421 Bifidobacterium breve 5081

Example 2: Cultivation and preparation method of each lactic acid bacteria and extract powdering of herbal ingredients

The lactic acid bacteria were inoculated into 250 ml of liquid MRS broth medium and shaken aerobicly for 36 hours at 37 ° C. The cultured bacteria were inoculated again in 1 L of liquid MRS broth medium and incubated at 37 ° C. for 48 hours. The culture solution was centrifuged to obtain approximately 16 g of bacteria. Subsequently, the process of washing with physiological saline 800m and centrifuging was repeated twice. The obtained bacterium was placed in 500 ml of a solution consisting of 20 g of skim milk, 30 g of soluble starch, and 0.5 g of sodium glutamate, mixed thoroughly, and lyophilized by a conventional method to prepare lactic acid bacteria. Viii) 54 g was obtained. The final bacterial content was determined by CFU (colony forming unit) method, and in culture, Lactobacillus was 2.5 × 10 ⁸ cfu / ml, Bifidobacteria was 0.5 × 10 ⁸ cfu / ml, and lyophilized powder was Lactobacillus. In the case of 2.5 × 10 ⁹ cfu / g, bifidus bacteria contained 0.5 × 10 ⁹ cfu / g.

Herbal plant material, prickly pear and perilla leaf extract powder can be obtained by the following method.

First, the material was ground to a size of 2 × 2 mm, sealed in a cotton bag, and then sterile distilled water was added to the extraction vessel, and 0.3% citric acid was added thereto. When the water began to break, the pulverized material was added and extracted for 5 hours while maintaining the state of breaking. The material remaining on the bag was compressed, combined with the extract, and filtered using a filter paper having an average pore size of 0.5 µm, and then the filtrate was lyophilized in the usual manner.

The lyophilized lactic acid bacteria and herbal plant extract materials were formulated into compositions of various compositions according to the content ratios as in Example 3 described later.

Example 3: Formulation

Probiotics, herbal plant extracts used in the experiment was used after mixing so that the total amount to 1000mg according to the composition ratio (weight%) shown in Table 2 below.

Composition of combinations containing lactic acid bacteria and herbal plant extracts Formulation example Probiotic powder Herbal Ingredient Extract Powder Lactobacillus Bifidus Go away Persimmon blade Formulation Example 1 100% - - - Formulation Example 2 92.6% 7.4% - - Formulation Example 3 - - 100% - Formulation Example 4 - - - 100% Formulation Example 5 - - 50% 50% Formulation Example 6 88% 7% 5% - Formulation Example 7 88% 7% - 5% Formulation Example 8 83.8% 6.7% 5% 5%

o Lactobacillus and Streptococcus bacteria: at least 0.5 × 10 ⁹ cfu / g of lactic acid bacteria per gram of the formulation

o Bifidus bacteria: Each bifidus bacteria 0.25 × 10 ⁹ cfu / g per 1g of the test combination

Test Example 1: Evaluation of viability of lactic acid bacteria in artificial gastric juice

The viability in the artificial gastric juice of the lactic acid bacteria used in the present invention was measured. Artificial gastric juice (pH 1.2) was prepared by mixing 1 ml of distilled water with 7 ml of HCl in 2 g of NaCl according to the composition of KFDA disintegration test method. Since the survival rate of lactic acid bacteria is generally very low at pH 1.2, a certain amount of bacteria were plated in Agar 1.5% MRS broth solid medium at 15 minute intervals for 1 hour. The cells were incubated at 37 ° C. per day and the viable count was counted by colony counter.

Test Example 2 Evaluation of Viability of Lactic Acid Bacteria in Artificial Bile

Since lactic acid bacteria have to be resistant to bile in order to survive in the human body, in order to confirm this, artificial bile solutions having the same effect as bile were made according to the composition of KFDA disintegration test method. The composition was made into 1 L of 6.8 g KH ₂ PO ₄ , 4.72 ml of 5N NaOH mixed with distilled water. At 30 and 60 minutes of experiment, a certain amount of cells were plated in agar 1.5% MRS broth solid medium. The cells were incubated at 37 ° C. per day and the viable count was counted by colony counter.

Test Example 3: Antibiotic Resistance Experiment

The probiotic was spread on an MRS plate solid medium using a spreader according to the test example, and then placed on a filter containing an antibiotic (6 mm in diameter) and incubated at 37 ° C. for 48 hours. The size of the growth inhibitory ring by antibiotics was measured, showing that the smaller the size of the inhibitory ring, the greater the resistance to antibiotics.

Table 3 below shows the degree of antibiotic resistance of probiotics.

Antibiotic Resistance Test Results for Lactic Acid Bacteria Antibiotics Diameter (mm) in Lactobacillus and Bifidobacteria plate Lactobacillus Bifidus Penicillin 32 mm 30 mm Oxacillin - - Cephalothin 24 mm 24 mm Erythromycin - - Tetracyclin 31 mm 29 mm Gentamycin 16 mm 15 mm Ciprofloxacin 34 mm 33 mm

Test Example 4: Cholesterol Degradation Capacity Test of Probiotic and Herbal Ingredient Combinations

Hydoxy Methyl Glutaric (HMG), Orotic Acid, Uric acid, etc., produced by fermenting lactic acid bacteria when probiotics are ingested, accelerates cholesterol breakdown and release and inhibits cholesterol resorption, especially Lactobacillus axido-fillers. Decomposes cholesterol directly.

Reported that lactic acid bacteria digested or adsorbed cholesterol as it grew in in vitro cholesterol supplemented medium (Gilliland, SE, CR Nelson and C. Maxwell. 1985. Assimilation of Cholesterol by Lactobacillus acidophillus.Appl.Environ.Microbio. 49: 377-381 ) And lactic acid bacteria deconjugate bile acids secreted into the small intestine by secreting bile salt hydrolase and converting them into free bile acids with glycine or taurine separated. (Klaver, FAM and R. van der Meer. 1993. The assumed assimilation of cholesterol by Lactobacilli and Bifidobacterium bifidum is due to their bile salt deconjugating activity.Appl. Environ. Microbio. 59: 1120-1124), Free bile acids have less resorption capacity than conjugated bile acids, so the amount of cholesterol that is reabsorbed decreases, thus increasing liver cholesterol demand. It is reported that the amount of cholesterol in the blood decreases.

Therefore, based on this report, the following test was made.

In vitro assay

Strains were incubated at 37 ° C. in MRS liquid medium containing 7.2 mg of soluble cholesterol. After culturing for 24 hours, 0.5 ml of the supernatant was separated by centrifugation (4 ° C., 13,000 rpm), and the separated supernatant was maintained at 60 ° C. for 10 minutes by adding 2 ml of 50% KOH and 3 ml of 95% ethanol. After cooling, 5 ml of hexane was added and mixed well. Then, 3 ml of distilled water was added, followed by thorough mixing. This was allowed to stand at room temperature for 15 minutes to cause delamination. At this time, the hexane layer was evaporated at 60 ° C. using nitrogen gas and 4 ml of o-phthalaldehyde reagent (0.5 mg o-phthalaldehyde / glacial acetic acid 1ml). Was added and left for 10 minutes. 2 ml of concentrated sulfuric acid was slowly added thereto, mixed well, and 10 minutes later, the absorbance (Optical Density) was measured at 550 nm.

Table 4 below shows cholesterol degradability in Formulation Examples 1 to 8 in Example 3 consisting of lactic acid bacteria and herbal plant extracts.

 Cholesterol Degradation Ability Test MRS MRS + Bile Control 7.1mg 5.95mg Formulation Example 1 6.26mg 5.81mg Formulation Example 2 5.70mg 5.65mg Formulation Example 3 6.82mg 5.95mg Formulation Example 4 6.85mg 5.94mg Formulation Example 5 6.85mg 5.95mg Formulation Example 6 5.62mg 5.57mg Formulation Example 7 5.60mg 5.57gm Formulation Example 8 5.48mg 5.42mg

※ Cholesterol content remaining after adding 7.2mg cholesterol

Test Example 5: Immune Boosting Effect by Oral Administration to Mice

Probiotics activate microphages that detect pathogens in the immune system to quickly detect bacteria and viruses, and promote lymphocyte division to prevent cancer cell proliferation and increase the production of IgA, an antibody in the blood. Increasing IgA production by promoting lymphocytic division is caused by intestinal B cells (Vitini et al., 2000. Gut mucosal immunostimulation by lactic acid bacteria. Biocell 24, 223-232). Increasing production has been shown to show enhanced immune resistance to infection by E. coli O157: H7 pathogenic bacteria, one of the food poisoning bacteria (Shu et al., 2002. Immune protection mediated by the probiotic Lactobacillus rhamnosus) HN001 (DR20) against Escherichia coli O157: H7 infection in mice.FEMS Immunology and Medical Microbiology 34, 59-64).

The epithelial cells of the small and large intestine allow the intestinal mucus to interact with the immune system and lactic acid bacteria (Perdigon et al., 2000 Intestinal pathway of internalisation of lactic acid bacterial and gut mucosal immunostimulation.Int Jour of Immunopathology and Pharmacology 13, 141-150), lactic acid bacteria affect the cytokine production of epithelial cells, the Th1 / Th2 cytokines are balanced by the generated cytokines (Borruel et al., 2003. Effects of non- pathogenic bacteria on cytokine secretion by human intestinal mucosa.American Jour of Gastroenterology 98, 865-870), immunostimulation by lactic acid bacteria, such as peptidoglycan, a component of the cell wall of lactic acid bacteria, and polysaccharides in and out of cells. (Gomez et al., 2002. Effect of bile on the lipid composition and surface properties of bifidobacteria. Jour of Applied Microbiology 93, 794-799) The test was carried out on the basis of the following.

Buy 6-6 week old BALB / c mice from Korea's Central Laboratory Animals and place them in separate boxes for 12 hours of light and darkness in a 25 ° C room. It was. Lactobacillus was orally administered in a group of 5 mice per each formulation. After dissolving the composition of each formulation in 1mg / μl in milk, 50 μl was administered once a day, 21 days.

(A) Observation of an increase in IgA producing cells in the small intestine of the mouse orally administered each composition of the formulation example

Mice were sacrificed by inhalation of carbon dioxide after oral administration of the composition in each formulation example for 21 days, the small intestine was extracted, fixed in 95% ethanol, and paraffin blocks were prepared. The paraffin block tissue was cut to 4 μm thickness and adhered to the slide glass, and paraffin was removed with xylene and rehydrated with ethanol. Blocking for 30 minutes at room temperature with 1% BSA solution, and dilution of anti-IgA monospecific antibody (α-chain specific) bound to Fluorescein isothiocyanate (FITC) to 1/100 in 0.01M PBS solution at 37 ° C The reaction was carried out for 30 minutes at. After washing three times with PBS buffer solution, it was observed by fluorescence microscope. The number of IgA producing cells was determined by measuring the number of fluorescence cells in the field of view of 10 zones observed at 1,000 × magnification.

Increase in IgA Producing Cells in Mouse Small Intestine After Oral Administration of Each Formulation Composition Treatment test example Number of IgA producing cells (mean + SEM), n = 5 Control 53 ± 1.3 ^{*, #} Formulation Example 1 63 ± 1.6 ^{*, #} Formulation Example 2 69 ± 1.5 ^{*, #} Formulation Example 3 56 ± 1.3 ^{*, #} Formulation Example 4 57 ± 2.1 ^{*, #} Formulation Example 5 57 ± 1.1 ^{*, #} Formulation Example 6 69 ± 2.5 ^{*, #} Formulation Example 7 71 ± 1.5 ^{*, #} Formulation Example 8 74 ± 1.1 ^{*, #}

* Mean value showed a significant difference compared to the control (P <0.001).

# There was a significant difference between Formulation Example 1 and Formulation Examples 2 to 8 (P <0.001).

(B) Observation of the increase of cytokine producing cells in the small intestine of the mouse orally administered each composition of the formulation example

As in the above experiment, each composition was orally administered to 5 mice per group according to the formulation example for 21 days. After 21 days, sacrifice was made in the same manner and tissue cuts were prepared. Rabbit anti-mouse IL-10, IFN- [gamma] polyclonal 1/100 dilution antibody was reacted at room temperature for 1 hour, and then washed twice with 0.01 M PBS. The reaction was carried out for 45 minutes at room temperature with 1/200 dilution of FITC bound goat anti-rabbit antibody. IL-10 and IFN-γ producing cells in the small intestine of the mouse were measured by fluorescence microscopy in the same manner as in the measurement of the number of IgA producing cells.

IL-10 is derived from Th2 cells and specifically has a Th1 cytokine inhibitory function and an anti-inflammatory cytokine function.

IFN- [gamma] is also called immune interferon because it is produced by CD4 T cells or CD8 T cells and regulates the immune response. IFN- [gamma] can act on T cells, B cells, neutrophils, NK cells, vascular endothelial cells and activate them, and like other interferons, can inhibit the growth of the virus.

Increase in cytokine producing cells in mouse small intestine after oral administration of each formulation composition Treatment test example Number of IL-10 producing cells (mean + SEM), n = 5 Number of IFN-γ producing cells (mean + SEM), n = 5 Control 32 ± 2.3 ^{*, #} 35 ± 1.8 ^{*, #} Formulation Example 1 44 ± 1.3 ^{*, #} 49 ± 2.3 ^{*, #} Formulation Example 2 52 ± 1.2 ^{*, #} 61 ± 1.9 ^{*, #} Formulation Example 3 36 ± 1.1 ^{*, #} 39 ± 1.6 ^{*, #} Formulation Example 4 38 ± 1.1 ^{*, #} 37 ± 1.4 ^{*, #} Formulation Example 5 39 ± 1.4 ^{*, #} 38 ± 1.0 ^{*, #} Formulation Example 6 53 ± 2.0 ^{*, #} 62 ± 1.0 ^{*, #} Formulation Example 7 52 ± 1.2 ^{*, #} 63 ± 1.5 ^{*, #} Formulation Example 8 55 ± 1.3 ^{*, #} 67 ± 1.1 ^{*, #}

* Mean value showed a significant difference compared to the control group (P <0.001).

# There was a significant difference between Formulation Example 1 and Formulation Examples 2 to 8 (P <0.001).

The probiotics used in the present invention show very good viability in artificial gastric juice and artificial intestinal fluid, so that a large number of lactic acid bacteria can survive in the intestine without being affected by gastric juice and bile. In addition, the antibiotic resistance evaluation resulted in more effective antibiotic resistance when incubated with bifidus bacteria than when only Lactobacillus bacteria were cultured.

Cholesterol degradability test results of compositions formulated with probiotics and herbal plant extracts showed that herbal plants extracts were mixed (formulation example 2) rather than adding probiotics to MRS and 7.2 mg cholesterol compounds (Formulation Example 2). Example 8 It was observed that cholesterol degrading ability was increased by about 14%. However, the cholesterol degradability of the composition containing only the herbal plant extract (Formulation Examples 3, 4, 5) was about 19% compared to the cholesterol degradability of the composition consisting of probiotics and herbal plant extract (Formulation Example 8).

After oral administration of the composition of each formulation example to the mouse, the number of IgA and cytokine producing cells was observed in the small intestine of the mouse to observe the immune enhancing effect. Example 1) Increased about 18% compared to the control group, but when only Lactobacillus bacteria and bifidus bacteria were administered together (Formulation Example 2) about 30% compared to the control group, when administered as a composition containing more thorn stem and perilla leaf ( Formulation Example 8 It showed a tendency to increase by about 40% compared to the control. However, when only herbal extracts were administered (Formulations 3, 4, and 5), only 7.5% increase compared to the control group.

The number of cytokines IL-10 and IFN-γ-producing cells was increased by about 37 to 40% compared to the control group when orally administered Lactobacillus alone (Formulation Example 1), but when Bifidobacteria were administered together (Formulation Example 2). IL-10 was increased by 62% and IFN-γ was increased by 74% compared to the control group, and when administered with a composition further including prickly pear and perilla leaf (Formulation 8), IL-10 was about 81% compared to the control group. , IFN-γ showed an increase of about 80-105%. In particular, when bifidus and herbal plant extracts were administered simultaneously, IL-10 increased by about 72% and IFN-γ by 91% compared to the control group. However, when only herbal extracts were administered (Formulations 3, 4 and 5), they increased by only 19% compared to the control group.

As described above, the present invention is lactic acid bacteria, especially Lactobacillus bacteria and bifidus contained in lactic acid bacteria foods known to exhibit intestinal function, immune enhancing effect, anticancer effect, anti-allergic effect, etc. By formulating the fungus, and herbal herb prickly pear and / or perilla leaf extract together, through the enhancement of immunity according to the cumulative synergistic expression of each characteristic and the normalization of the balance of intestinal flora, such as skin allergy, food allergy, etc. In addition to the prevention and alleviation of allergic diseases, it can be expected to have an excellent effect on the prevention of geriatric diseases such as diabetes improvement, weight control, high blood pressure and intestinal health, and the probiotic complex preparation according to the present invention is more effective for healthy people. Healthy gut flora in the intestines like unhealthy people Restore the flora give gives strengthen the immune system is effective in the improvement of anti-inflammatory diseases.

Claims (7)

Probiotics combined preparation consisting of lactic acid bacteria and barberry extract or perilla leaf extract. The method of claim 1, wherein the lactic acid bacteria Lactobacillus ash FIG filler's (Latobacillus acidophilus), Lactobacillus momentum spread (Lacotbacillus fermentum ), Lactobacillus casei , and at least one Lactobacillus bacterium selected from the group consisting of Lactobacillus plantarum ; Ronggeom Bifidobacterium (Bifidobacterium longum ) and Bifidobacterium breve , a probiotic complex formulation which is a complex lactic acid bacterium consisting of at least one Bifidobacterium bacterium selected from the group consisting of. The probiotic complex preparation according to claim 1 or 2, wherein the lactic acid bacterium is a complex lactic acid bacterium further comprising Streptococcus thermophilus . The method according to claim 1 or 2, wherein 70 to 95% by weight of the Lactobacillus fungus, 1 to 15% by weight of the Bifidobacterium, and psyllium creeper extract or perilla leaf extract, respectively, based on the total weight of the composition. Probiotics complex formulation containing 10% by weight. According to claim 3, Probiotics complex formulation containing 0.1 to 5% by weight of the Streptococcus thermophilus ( Streptococcus thermophilus ) based on the weight of the composition. The probiotic complex formulation according to claim 1 or 2, wherein the complex formulation is a powdered or granulated formulation, or a capsule thereof. The probiotic complex preparation according to claim 1, wherein said prickly pear extract or perilla leaf extract is powdered or granulated as an extract by water, ethanol, or a mixed solvent thereof, or a citric acid addition solvent thereof.

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