TW200817508A - Lactic acid bacterium for amelioration of lactose intolerance - Google Patents

Abstract

Disclosed is a lactic acid bacterium useful for the amelioration of lactose intolerance. More specifically, disclosed are: a method for screening a lactic acid bacterium capable of ameliorating lactose intolerance, characterized by selecting a bacterium having an enhanced enteroadherence property and an enhanced lactose-degrading enzyme activity from lactic acid bacteria belonging to the genus Lactobacillus; and a lactic acid bacterium provided by the method.

Description

200817508 IX. Description of the invention: [Technical field to which the invention pertains] This month is a lactic acid bacterium useful for improving lactose intolerance and its utilization. [Prior Art]. Sugar intolerance refers to a constitution in which the symptoms of abdominal pain and abdominal pain are easily caused by poor digestion of lactose in the milk taken. It is generally believed that the lactose intolerance (4) will become significant with an increase in age 'but it is also visible in early childhood. In order to alleviate the symptoms of lactose innocence, it is necessary to avoid ingesting products containing lactose, but if it is not possible to ingest a dairy product that is a good source of maternity, for example, it will increase the risk of osteoporosis in the elderly, and it will become a major obstacle in nutrition. . Osteoporosis threatens Japan's largest disease, which is rapidly becoming a long-term aging, and therefore reduces the bones of the elderly. <The significance of risk is particularly important to promote positive intake of dairy products. Lactose is usually decomposed by lactose-degrading enzymes produced in cells of the inner layer of the small intestine and absorbed. In another aspect, various microorganisms such as lactic acid bacteria are known to produce lactose-degrading enzyme, and lactose is decomposed. The manufacturer believes that the yogurt or lactic acid bacteria beverage containing live lactic acid bacteria or yeast is partially decomposed by lactose, so it is difficult to cause symptoms of lactose intolerance. Further, by continuous oral ingestion, these bacteria will be Slow proliferation in the intestines increases the ability of lactose to break down in the intestines, and as a result, lactose intolerance itself is also improved. It has been known from the past that lactose metabolism of lactic acid bacteria includes two kinds of lactose-degrading enzymes of galactosidase (β-gal) and phosphate-β-galactosidase (pj^gal). 119625.doc 200817508 " There is also a phosphate-PL sugar phase contained in the money a. The third lactose-degrading enzyme (Non-Patent Document 1) 2 considers that the lactic acid bacteria with higher activity of such lactolytic enzymes can effectively alleviate lactose intolerance. The symptoms of the strong ▲ are obvious, and the lactic acid bacteria in the parenteral lactic acid bacteria have the lactose-decomposing activity of the sputum: but it is generally considered to exist in the human intestine Such a lactic acid bacterium does not continue to produce a lactose-degrading enzyme in the intestinal tract of the human body, and it is considered that lactose-degrading enzyme activity of a component or higher is not obtained (Non-Patent Document 2). Therefore, in order to improve lactose intolerance by oral administration of fish (10), it is not only required to have lactose-decomposing enzyme activity (and lactic acid bacteria which can stably decompose lactose in the human intestinal tract, but there has been no significant progress in the exploration of the above-mentioned lactic acid bacteria. 4", and & know that there are bacteria with high intestinal adhesion in the intestinal lactic acid bacteria. As a method for exploring such a high intestinal adhesion, for example, there is a test for the human intestinal mucin The method of screening for lactic acid bacteria having high intestinal adhesion to various blood types of the human body (Patent Literature) This method is based on the following contents and reports, and is intended to cause lactic acid bacteria to be initially infected in the human body. The intestine 'is essential for its adhesion to the surface of intestinal epithelial cells; Takahashi et al.'s report on the use of colorectal mucin (EM) by the genus of the bacteria [using a coating coated with colorectal mucin (RCM) When the styrene beads were selected as the Lactobacillus acidophilus lactic acid strain, the surface protein (sLp) derived from the sputum sputum strain was also bound to the human mucous layer of the large intestine. Document 3, etc.; The chemical structure of the sugar bond constituting the human intestinal mucin is reported differently according to the ABO type blood type (for example, Non-Patent Document 4), etc. However, the screening method has not been applied to 119625.doc. 200817508 is suitable for use in the use of lactic acid bacteria of various blood types. [Patent Document 1] Japanese Patent Laid-Open Publication No. 2004-101249 [Non-Patent Document 1] Saito Tatsuo, Ito Katsuyuki, Yoshino Yuno, Yamazaki Yukiko, Human Intestine Discovery of a new approach to the lactose assimilation system in the origin of Lactobacillus gasseri (Glass), Biotechnology Society, (2001), 79(6), ρρ·172-173 [Non-Patent Document 2] Marteau P, Minekus M, Havenaar R, Huis (, in t Veld JH ^ Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: validation and the effects of bile·, J Dairy Sci, (1997) 80(6), pp · 103 1-1037 [Non-Patent Document 3] Takahashi N, Saito T, Ohwada S, Ota H, Hashiba H, Itoh T, ''A new screening method for the selection of Lactobacillus acidophilus group lactic acid b Acteria with high adhesion to human colonic mucosa·,,, Biosci Biotechnol Biochem, (1996), 60(9), pp·1434-U 1438 [Non-Patent Document 4] Blood type sugar chain structure on the genus of the wild genital and digestive tract mucin - Clarification of the Mechanism of Bacterial Infection, Biochemistry, Society of Japanese Society of Biochemistry, (1999) 71(4), ρρ· 274~277 ^ [Summary of the Invention] [Problems to be Solved by the Invention] An object of the present invention is to provide a A lactic acid bacterium that improves the ability of lactose intolerance. [Technical means for solving the problem] 119625.doc 200817508 The present inventors have actively studied the above-mentioned problems, and found that a part of Lactobacillus lactic acid bacteria has enhanced intestinal adhesion and lactose-degrading enzyme activity, and the knowledge is completed based on this knowledge. this invention. That is, the present invention includes the following. π 种 筛选 筛选 筛选 筛选 筛选 筛选 筛选 筛选 筛选 筛选 筛选 筛选 筛选 筛选 筛选 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺 寺In this method, it is better to use the surface plasma resonance analysis to determine the lactic acid bacteria in human intestinal mucin, human β-intestinal* protein and human type 0 intestinal mucin. At least one of the binding ability 'will indicate that the RU value of the binding ability is 1 〇〇 or more, and is screened as a bacteria having enhanced intestinal adhesion. In the T method, the lactose-degrading enzyme activity in the selected bacteria, preferably the lactose-degrading enzyme of at least one of 疋β_semisidase, phosphate-β-galactosidase, and glucosylglucosidase active. In the method, the Lactobacillus gasseri, or the Lactobacillus mucosae ® [2] lactic acid bacteria, which is selected by the above, is preferably a lactic acid bacterium of the genus Lactobacillus. (1) The method of obtaining intestinal adhesion and lactose-degrading enzyme activity together is enhanced. [3] A lactic acid bacterium, which is a strain of Lactobacillus kawaii 〇1^2836 (registered number BCRC 91035 1), Lactobacillus kawaii LL2948 strain (registered number bcrc 9103 53), and Lactobacillus mucosa LL2848 strain (registered) Any of the numbers bcrc 910352). 119625.doc 200817508 [4] A lactose intolerance improving agent comprising at least one lactic acid bacterium as described in [3] above. As the lactose intolerance improving agent, it is more preferable to include at least Lactobacillus kawaii OLL2836 strain (registered number BCRC 91〇351), Lactobacillus gasseri OLL2948 strain (registered number bCRC 910353), and Lactobacillus mucosa 〇1^ Three strains of lactic acid bacteria of the 2848 strain (registered number ^〇1^ 9103 52). [5] A beverage food comprising at least one lactic acid bacterium as described in [3] above. As the food and beverage, it is more preferable to include at least Lactobacillus kawaii OLL2836 strain (Accession No. BCRC 910351), Lactobacillus kawaii LL2948 strain (Accession No. BCRC 910353), and Lactobacillus mucosa LL2848 strain (Accession number BCRC 9). 103 52) Three lactic acid bacteria. The food or drink is preferably a baby food, a baby food, a breastfeeding women's food, an aged food, a patient food, a health-care food, a supplement, a fermented milk, or a lactic acid bacteria beverage. It is especially preferred to use the beverage food as a beverage food for improving lactose intolerance. [Effect of the Invention] According to the screening method of the present invention, a lactic acid strain which is advantageous for improving lactose intolerance can be effectively screened. Further, when the lactic acid strain of the present invention is used, it is possible to produce a medicament for improving lactose intolerance or various foods and beverages. The present specification contains the contents disclosed in Japanese Patent Application No. 2006-175897, which is the priority of the present disclosure. [Embodiment] 119625.doc -10- 200817508 Hereinafter, the present invention will be described in detail. 1. Method for screening lactic acid bacteria having the ability to improve lactose intolerance The present invention relates to a bacterium capable of screening for intestinal adhesion and lactose-degrading enzyme activity by lactic acid bacteria of Lactobacillus A method of improving the ability to improve lactic acid bacteria. In the method of the present invention, the "lactobacillus lactic acid bacterium" for screening means a bacterium classified into any bacterial species belonging to the genus Lactobacillus or Lactobacillus according to a usual taxonomic method. The taxonomic method is not particularly limited, and includes a previous morphological classification method for classifying known Lactobacillus or a classification based on physiological and biochemical traits, but for example, for bacteria that are not identified by the strain, the utilization is based on When a molecular phylogenetic classification method having a 16S rDNA sequence, particularly a homology analysis of a base sequence of the V3 region, is used, classification can be clearly performed, which is preferable. Specific examples of the bacterial species belonging to the lactic acid bacterium of the genus Lactobacillus include Lactobacillus bulgaricus (Lactobacillus bulgaricus, Lactobacillus delbrueckii subsp. bulgaricus), Lactobacillus delbrueckii (Lactobacillus delbrueckii, or Lactobacillus delbrueckii subsp. delbrueckii), acidophilic lactic acid. Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus helvelicus , Lactococcus 1 actis, Lactobacillus crispatus, hemolytic lactic acid 119625.doc 200817508 bacillus (Lactobacillus johnsonii), Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus mutagenes (Lactobacillus mucosae), etc., but is not limited to these. In the method of the present invention, lactobacillus lactic acid bacteria are preferred as intestinal lactic acid bacteria, and particularly preferred are Lactobacillus gasseri and Lactobacillus. "Lactobacillus lactic acid for screening" in the method

The bacterium may be a cell group or a mixture of cells containing a plurality of Lactobacillus lactic acid bacteria cells, or may be a collection of individual strains of Lactobacillus lactic acid bacteria. In the present invention, the intestinal lactic acid and lactase-degrading enzyme activities of the Lactobacillus lactic acid bacteria as described above are measured to screen the bacteria which are mutually enhanced. Here, the term "intestinal adhesion" refers to the ability of lactic acid bacteria to bind to the surface of intestinal epithelial cells of a subject. When measuring the intestinal adhesion of the lactic acid bacteria of the present invention, typically, the lactic acid bacteria can be measured for at least one of the human intestinal eight-type intestinal mucin, human B-type intestinal mucin, and human intestinal intestinal mucin. The binding ability of mucin, and the obtained measured value is used as a value indicating intestinal adhesion. The measurement of the binding ability of the lactic acid bacterium to the intestinal mucin can be carried out by, for example, the method of surface electropolymerization resonance analysis disclosed in Patent Document i and International Patent Application WO 2006/067940. When using surface plasmon resonance analysis, it is preferred to use a lactic acid strain having a RU value of 1 〇〇Ru# indicating the binding ability of lactic acid bacteria to intestinal mucin, and screening for a bacteria having enhanced intestinal adhesion. Alternate. / Below', more specifically describes the analysis system for the binding capacity of the intestinal mucin. Baixian' is for the preparation of blood type human intestinal mucin, taking the surface layer from the human intestinal tract of each blood type (type A, B type, and sputum type), using salt 119625.doc -12- 200817508 solubilizer such as bismuth Gel filtration is carried out, and the target is taken for purification by protein absorption (absorbance of 28 Å) and neutral sugar content. A detailed description of the gel filtration purification can be found, for example, in Purushothaman S. S. et al, "Adherence 〇f • Shigella dysenteriae 1 to Human Colonic Mucin. Curr.

Mi〇rC)bi〇1·' 42 (6) 'P· 381-387 (2001) Purification method of human large intestinal mucin. After purification, it is better to use anti-blood-type antigen antibodies, and f) 5 tolerate the discovery of blood group antigens in the prepared human intestinal mucin. As a specific example, the method disclosed in the examples can be mentioned. As the blood group antigen, a commercially available sugar chain probe (for example, manufactured by Biochemical Industries) or a glycoprotein. Blood Group A Trisaccaride-BSA (for example, Calbiochem & s) or a glycoprotein. Blood Group B Trisaccadde-BSA can be used. (For example, Calb1〇chem Co., Ltd.), etc., as an antigen, an anti-blood-type antigen antibody can be produced by various antibody production methods well known to the practitioner. In the screening method of the present invention, human intestinal mucin, human BJ intestinal mucin, human intestinal mucin, human intestinal mucin, respectively, are used as probes for surface plasma resonance spectroscopy Thereby, the ability of the lactic acid bacteria to bind to human intestinal mucin can be determined. When performing surface plasma resonance spectrum analysis, it can be used as a biosensor (biological intermolecular interaction analysis device) 2BIAC〇RE system (BIAC〇RE), for example, BIACORE1000. The BIACORE system is based on the principle of surface plasma resonance spectroscopy (spR). It can simultaneously monitor the interaction of biological molecules (bonding and dissociation) without using markers. Specifically, the intermolecular interaction between the ligand (also referred to as probe after 119625.doc -13 - 200817508) and the analyte was determined. In the present invention, lactic acid bacteria are used as the analyte system, and human intestinal mucin (type A, type b, and sputum type) is used as a ligand system. With regard to the principle of surface plasma resonance, a number of detailed instructions have been published. If the original '1' is used in the device to bind/dissociate the substance on the gold film, determine the change in the refractive index of the reflected light accompanying the change of the bond to the film. Thereby the amount of substance bound to the membrane is opened. More specifically, the device wipes a glass substrate with a gold film called a sensor sheet on the way of the f sample or the test weaving path, and the sample D is placed on the side. Waiting into the path, on the sensing sheet, using 稜鏡 or the like to make the polarizing of 760 into a wedge shape, and making it reflect on the gold film, monitoring the refractive index of the reflected light, then the surface electro-convergence resonance spectrum The change in angle is not proportional to the change in the amount of binding of the substance on the gold film. Therefore, in the BMCORE system, first, the probe is flowed into the human channel, the probe is fixed on the gold film of the sensing sheet, and then the portion to which the probe is not bound is blocked, and then the probe is combined with the probe. The substance (analyte) of the dissociation reaction flows into the channel' and monitors the refractive index of the reflected light over time, whereby the amount of binding of the S-out to the analyte can be varied therefrom. In this system, the angle of the surface plasma resonance spectrum (the change of M is defined as the hetero resonance unit, and the value of the resonance unit is set to the unit value (10)), indicating that the refractive index of the reflected light is here, 1 The resonance unit (RU) is equivalent to the mass change of J p曰g/1 mm2 on the surface of the sensing sheet... the amount of binding of the substance to the gold film on the sensing sheet can be terminated by the time before the combination of the substances (before the addition) The difference in the RU value at the time is calculated. If the slave system is used, the probe (in the present invention, each human intestinal mucin) can be obtained as the mass change (Pg) of the sensor piece, ie, the value. The amount of binding to the analyte (in the present invention, lactic acid bacteria), the amount of binding (RU value) corresponds to the ability of the analyte to bind to the probe. Furthermore, the BIAC〇RE is a sensing sheet that can be used in the system, and corresponds to a substance (probe) fixed on the sensing sheet, an immobilization method, a purpose of use, etc., for example, except for the most Standard _ (a glass film with a gold film (typically 50 nm) with a carboxymethyl dextran layer on its surface (typical 2 〇, i 〇 0 nm). In addition to the carboxyl group of the sugar layer and the amine group, sulfhydryl group, aldehyde group or carboxyl group of the ligand, and the ligand (probe) is immobilized on the sensing sheet, there are also ^^4, CM3 , ci, SA, ΝΤΑ, LI, ΗΡΑ, etc. In the present invention, in order to immobilize intestinal mucin as a probe to the sensor, for example, it can also be carried out according to the manufacturer's instructions for the BIACORE system. The immobilization method may be, for example, a method by physical adsorption or adsorption by covalent bonding. As an example, when CM5 (biac〇re company) is used as the L-sensing sheet, first, N-ethyldiaminopropyl carbodiimide hydrochloride (EDC) is mixed in a ratio of 1:1. N_hydroxysuccinimide (ΝΗ§) is a mixed reagent (EDC/NHS) flowing into the channel to activate the thioglycan in the carboxymethyl dextran on the sensor sheet, and then prepared for immobilization. A mixed solution of human intestinal mucin solution (each using A.HCM, B-HCM, 0-HCM) was added to the acetate buffer (pH 4.0), and further, it was flowed into the channel by amine coupling. The reaction causes the human intestinal mucin to covalently bond with the slowing base on the sensing sheet. Further, it is also preferred to use ethanolamine hydrochloride-NaOH (pH 8.5) to block the carboxy moiety of 119625.doc -15 - 200817508 on the sensor sheet not bound to the probe to prepare intestinal mucin ( HCM) Immobilized sensor. In the present invention, the amount of HCM immobilization in the sensor sheet is not particularly limited, but is preferably 1000 to 2000 RXJ. Then, the sample containing the lactic acid bacteria (analyte solution) was flowed into the channel, and the amount of binding of the HCM (probe) on each HCM immobilization sensor to the lactic acid bacteria (analyte) in the analyte solution was calculated. Although not limited, an example of the measurement conditions of BIAC 〇 RE1 可 which can be used for the analysis is shown below. , Electrophoresis buffer: HBS-EP buffer (pH 7.4) Addition amount of analyte solution (sample) · 2〇 μΐ Flow rate: 3 μΐ/min

/ 人没没· 2 5 C Regeneration solution·· 1 Μ 胍 胍 hydrochloride solution 5 μ1 According to the binding/dissociation curve obtained based on such measurement, the RU value after analyte binding is subtracted from the pre-analyte binding 2Ru The value of the ruler can be used as the binding amount (pg) of the lactic acid bacteria as the analyte and the γ mm of each intestinal mucin as a probe, that is, the binding of the lactic acid g to the intestinal mucin. value. The towel of the present invention exhibits (10) a value of more than (10) fat to at least one of human intestinal eight-type intestinal mucin, type B intestinal mucin, and sputum intestinal mucin, lactic acid g and intestinal mucin The binding ability is "increased intestinal adhesion" or "high material adhesion". The servant, the rib value can be changed according to the measurement conditions in the coffee system: ~. The temperature condition in the method is, for example, two. ~, C, preferably 3〇C is better 疋23~28. . . Further, the concentration of the analyte solution 119625.doc 200817508 (sample) in the present method is preferably 0.1 to 0.5 mg/mL, and the flow rate in the method is preferably 3 to 1 〇 μΐ/min. If it is in the above range, the RU value will not change even if the above various conditions are changed. Therefore, "1〇〇 RU" is used as the standard.

The standard can determine whether the intestinal adhesion is high. In addition, when a plurality of conditions such as the concentration of the analyte Z are outside the above range, the intestinal tract of the lactic acid bacteria is obtained when the binding energy I is the same as 100 RU when the RU value in the above condition is converted. Adhesion is enhanced. Further, in the method of the present invention, the higher the Ru value of the bacteria is, the higher the intestinal adhesion is, that is, the intestinal adhesion is enhanced as compared with the conventional lactic acid bacteria. In the present invention, 'better' is a lactic acid bacteria which exhibits a binding energy f to at least one human intestinal mucin, and which exhibits an RU value of more than 3 〇〇RU or more, particularly preferably 疋RU or more. , screening for bacteria with enhanced intestinal adhesion.进而 Further, in the screening method of the present invention, the above-mentioned Lactobacillus lactic acid bacterium is determined to be β-galactose__gal, the keto-galactose (iv), and the acid-based glucoside enzyme (P + gle) At least one lactose-degrading enzyme, the sugar cleavage enzyme 2 (lactolytic enzyme activity), _ select the strain with enhanced activity.

Ml::: 'According to the Μ method', suspend the freeze-dried cells in 〇··5 = series of rinsing, add toluene _ _ _ mixed and vigorously stir, if 1)] add to its suspension Ga lactose decomposing enzyme activity measurement, (ONPGal) dish acid into the heart of the basic semi-milk ° than glucoside measurement, heart two; ^ know 1 lactase enzyme activity 'night added o-nitrobenzene galactose吼119625.doc -17- 200817508 Phosphatase-6-phosphate (ONPGal-6P) phosphate buffer; or, if it is Ρ-β-glc lactase activity assay, add it to its suspension Phosphate buffer of o-nitrophenyl-β-D-glucoside-6-phosphate (ONPG1C-6P) was reacted at 37 ° C for 15 minutes. After the reaction, 〇.5 k Μ 碳酸 carbonate was added. Solution 'to stop the reaction, remove the bacterial components by centrifugation (6, 〇〇〇 x rPm, 丨〇 min, 4 ° C), using an absorbance photometer (eg 'Multiskan MS-UV (Labsystems, Helsinki, Finland) )), determine the absorbance of the supernatant at 405 nm. The free o-nitrobenzene (0ΝΡ) 1 μιηοΐ in 1 minute The unit is converted into the activity value, and the activity value per 1 mg of the cells and the activity value of the protein contained in each 1 mg of the culture supernatant are calculated from the activity values. Further, the protein is quantified. For example, a BCA Protein Assay Reagent Kit based on the BPa method (Pierce,

Rockford, Illinois, u.S. A), after reacting for 2 hours, the iy absorbance at 540 nm was measured using an absorptiometer (for example, Multiskan MS-UV). The calibration curve can be made using bovine serum albumin (BSA). Based on the calibration curve, the amount of protein contained in the supernatant can be calculated. According to the activity value per 1 mg of the β-ga Pj_gal and P_p_glc calculated in the above manner, and the activity value per mg of protein contained in the culture supernatant, the lactase activity can be screened. A lactic acid bacterium that is particularly high (the lactose-degrading enzyme activity is enhanced compared to common lactic acid bacteria). Although it is not limited to, but for example, regarding lactose-degrading enzyme activity of p_gal (also referred to as β-ga丨 activity in the present invention), it is particularly preferred to select 15 units or more per 1 mg of protein in the culture supernatant. It is (10) above the unit 119625.doc -18- 200817508. In addition, about Ρ-β-gal lactose decomposition yeast sound # ^, also known as Ρ β, fresh enzyme activity (the β-gal activity of the present invention), especially good It is the screening of mg protein prepared _ heart culture supernatant 1 Fritillaria does not exceed 15 units, preferably the strain. Who and „ 〇疋 〇疋 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 The supernatant, the mother of the night 1 mg protein is displayed on the π liquid shell.,,, can not afford 25 earlier than the above, better strains above. 疋, ', can not be out 50 early 兀Γ gen: tr: two In the method, in the above manner, the above-mentioned lactobacillus::... is more preferably Lactobacillus, Garcinia or Lactobacillus, and the membrane is measured: intestinal adhesion and lactose-degrading enzyme activity, and as a result, it is effective The cultivator selects a lactic acid strain which is enhanced by intestinal adhesion and lactose-degrading enzyme activity. The present invention also relates to a lactic acid strain which is thus screened, and the Lactobacillus lactic acid bacterium obtained in this way can be well proliferated in the intestinal tract. In addition, it is very useful for improving lactose deficiency. In the present invention, "lactose intolerance: improvement" means ingesting a beverage containing lactose (dairy product, etc.) When reducing lactose is not symptomatic The frequency of gastrointestinal symptoms (abdominal pain, diarrhea, abdominal feeling of hunger, etc.) appear in 'or completely prevent the onset or reducing the severity of symptoms. In addition, "lactose-free disease improvement ability" refers to the ability of lactic acid bacteria to improve lactose intolerance. In the present invention, by using the above-described screening method, it is possible to actually obtain Lactobacillus lactic acid bacteria of three strains which have significantly enhanced intestinal adhesion and lactose-degrading enzyme activity. These three lactic acid bacteria are Lactobacillus glabrata 〇LL2836 (later, also known as OLL2836 strain), Lactobacillus calyceii ◦22" heart strain (after 119625.doc -19-200817508, also known as OLL2948 strain), And Lactobacillus cloacae OLL2848 strain (hereinafter, also known as OLL2848 strain). These lactic acid strains are deposited in the Patent Microbiology Depository Center of the independent administrative legal person product evaluation technology infrastructure. The information related to these deposits is as follows: (1) Name of the depository institution : The administrative microbial deposit center of the independent administrative corporation product evaluation technology infrastructure (2) The liaison office of the depository agency: 2-5-8 r._% of the general manager of Kisatsuzu, Chiba Prefecture, Japan (3) Deposit period (original deposit) Period: June 9th, 2006 (4) Registration number and collection number: • Lactobacillus gasseri OLL2836 strain: Deposit number NITE BP-241 (original registered collection number NITE AP-241), • Lactobacillus gasseri OLL2948 strain: Accession number NITE BP-242 (original registered collection number NITE AP- 242), • Lactobacillus mucosae OLL2848 strain: Registered number NI TE BP_243 (original registered collection number NITE AP-243) These lactic acid strains were deposited with the Taiwan Food Industry Development Research Institute (FIRDI) on June 4, 2007 under the following registration number. OLL283 6 strain: storage number BCRC 91035 1 • Lactobacillus kawaii OLL2948 strain: storage number bcrc 910353 • Lactobacillus mucosa OLL284 8 strains · Deposit number BCRC 91〇352 The deposited OLL2836 strain, 〇LL2948 strain and 〇LL2848 Strain, 119625.doc -20- 200817508 Has the properties shown in Table 1 below [Table 1] Strain name OLL2836 strain OLL2948 strain OLL2848 strain Taxonomic location deposit: Registered number Lactobacillus bulgaricus BCRC 910351 Lactobacillus calivae lactic acid Bacillus BCRC 910353 BCRC 910352 — ·— —- The colony on the medium (BL medium) is round, translucent, smooth, flat, round, translucent, smooth, flat, round, smooth, conical, White ιί morphology Gram staining---'- lactic acid fermentation form aerobic developmental temperature source suitable medium oxygen-demanding bacilli positive homologous milk Acidic fermentation + 15 C is -, 45 ° C for + human feces separation

Lactobacilli MRS Broth — common anaerobic bacilli positive isotype lactic acid fermentation + at -15 ° C - at 45 ° C + from human feces

Lactobacilli MRS Broth Positive anaerobic bacillus positive + 15 ° C at +45 ° C for + human fecal separation

Lactobacilli MRS Broth common anaerobic intestinal adhesion lactase activity ', 'the adhesion of human B-type intestinal mucin and human intestinal gut protein is significantly better --- Ρ-β The activity of -gal is significantly higher and the adhesion to human intestinal mucin is significantly higher. The activity of Ρ-β-glc is significantly higher. The adhesion to human type A intestinal mucin was significantly higher. The activity of B-gal is significantly higher. 119625.doc 21 200817508 Table! The LactobacUli MRS Br〇th (MRS medium; for example, Difco, Ref. No. 288160) can be preferably used for culturing the medium of the lactic acid bacteria strain. Typical compositions of MRS medium are shown in Table 2. [Table 2] Composition of MRS medium

示1脒 Protein extract (Beef Extract) 10.0 g Yeast Extract 5.0 g Dextrose 20.0 g Polysorbate 80 io g Ammonium citrate 2.0 g Sodium acetate 5.0 g Magnesium sulfate 0.1 g Manganese sulphate 0.05 g sulphuric acid nitrogen second unloading 2.0 g Distilled water 1 L Three strains not shown in Table 1 are considered to have particularly high improvement in lactose intolerance, and can be particularly advantageously used in the present invention. 2. Lactose intolerance improving agent containing the lactic acid bacterium of the present invention The lactic acid bacteria of the present invention exerts potent lactose-degrading enzyme activity by oral administration, reaching the intestinal tract in a state of survival, and licking the intestinal tract. ... the fruit is that it can improve lactose intolerance. Further, in the mash produced by the lactic acid bacteria of the present invention, the decomposition rate of lactose 119625.doc -22-200817508 can be improved by the high lactose decomposition activity. Accordingly, the present invention also relates to a lactose intolerance improving agent comprising Lactobacillus lactic acid bacteria (lactic acid bacteria of the present invention) which is significantly enhanced by the intestinal adhesion and lactase-decomposing enzyme activity obtained by the above-described sieve method. The lactose intolerance improving agent of the present invention is effective for improving lactose intolerance by administering a test for lactose intolerance or suspected lactose intolerance, that is, reducing lactose intolerance Symptoms of digestive system symptoms (abdominal pain, diarrhea, abdominal hunger, etc.) occur frequently, or completely prevent the onset, or reduce the severity of symptoms of lactose intolerance. The lactose intolerance improving agent of the present invention also has an effect of preventing the onset of lactose intolerance. The lactose intolerance improving agent of the present invention comprises at least one sputum strain or the lactic acid bacteria of the present invention. The lactose intolerance improving agent of the present invention is preferably a lactic acid bacterium of the present invention, which comprises a strain of Lactobacillus delbrueckii ll2836 (registered number BCRC 910351) and a strain of Lactobacillus kawaii 〇1^2948 (a deposit number BCRC 910353). And at least one selected from Lactobacillus mucosa LL2848 strain (registration number BCRC9M352). In a particularly preferred embodiment, the lactose intolerance improving agent of the present invention comprises at least Lactobacillus kawaii OLL2836 strain (Accession No. BCRc 910351), Lactobacillus delbrueckii OLL2948 strain (Accession No. BCRC 91〇353), and mucosal lactic acid. Three lactic acid bacteria of the bacterium OLL2848 strain (registered number BCRC 910352). Combination The lactose intolerance improving agent containing the two lactic acid bacteria can be bound to intestinal mucin containing any blood group antigen of type A, B type, and sputum type, and therefore, is effective for a wide range of subjects. The lactose intolerance improving agent of the present invention may be a composition comprising the purified bacterial cell of the lactic acid bacteria 119625. doc -23 to 200817508 of the present invention, or may be a mash, a culture, or a bacterium prepared by using the lactic acid bacterium. This 辇▲ 4 wave shrinkage composition. The lactose intolerance improving agent of the present invention may also be a dead cell, and the thirsty moisturizing fungus may be a living cell, and the interdrip may be a dried fungus. More preferably, ❹ = 'tt' The lactose intolerance improving agent of the present invention comprises a composition of a viable lactic acid bacterium. The lactose intolerance improving agent of the present invention is not limited, and may be in any form such as a liquid makeup (4), a powder form, a granule form, a solid form, a capsule form or a sheet form. It is possible to impart a lactose T / positive effect to a beverage or a pharmaceutical composition by adding an appropriate amount of the sputum 4 of the present invention. The lactose intolerance improving agent of the present invention may contain, as an active ingredient, the lactic acid bacteria of the present invention or a culture thereof, a fermented product, or a concentrate thereof, or the like, or may contain a carrier or an additive which is allowed by the drug-killing preparation. Things. As the above-mentioned carrier and the addition of (IV) I, the organic solvent allowed in water and medicine, 'collagen, polyvinyl alcohol, polyethylene, 叛 乙烯基 vinyl polymer, sodium alginate, water-soluble dextran, m Methyl starch steel, pectin, Sanxian gum 'arabis gum, road protein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, nectar Other than the sugar alcohol, the sorbitol, the methylidene cellulose, the surfactant which can be used as a pharmaceutical additive, etc., an artificial cell structure, such as a liposome, etc. are mentioned. The additives to be used may be selected as appropriate or in combination depending on the dosage form of the preparation. The lactose intolerance improving agent of the present invention can be administered orally or parenterally (e.g., intragastric administration or enteral administration), and it is particularly preferred to administer it orally. The lactose intolerance improving agent of the present invention by Z may be a tablet, a granule, a solid preparation such as a powder, a pill or a capsule, a gel, or a liquid agent, and a county float. A dosage form such as a liquid preparation such as a syrup or a syrup. When used as a liquid preparation: It may be supplied at the time, and is intended to be a dried product which is re-dissolved when the lactose intolerance of the present invention is improved. ’ In the above dosage form, the 'oral preparation for oral administration may also contain an additive such as a pharmaceutically acceptable binder' excipient, a lubricant, a disintegrant, a wetting agent and the like. Further, the oral liquid preparation may contain a stabilizer such as a pharmaceutically acceptable stabilizer, a scenting agent, a preservative, a fragrance, a coloring agent, and the like. The dosage of the lactose intolerance improving agent of the present invention varies depending on the age, weight, route of administration, and number of administrations, and the manufacturer changes it in a wider range as appropriate. For example, in the case of the sister's mouth, the lactose contained in the lactose intolerance improving agent of the present invention is not more than mg/kg/day. The sugar-tolerance improving agent of the present invention can be administered once or repeatedly. Between j f and the object of the lactose-free disease improving agent of the present invention, there are no restrictions, such as human, domestic animal, smuggling, smuggling, smuggling, and testing (testing) animals. Further, as the subject of the present invention, whether or not the 疋 ' of the e 馇 为 为 ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' After being tested, "the sugar inlerance is improved and the body is better, the mammal is decreased because of the increase in age, or the mammal is not capable of decomposing lactose. The lactose intolerance of the present invention. Symptoms = the original worry is small, therefore, Yulian • User.../The use of the field can be very useful. H9625.doc -25- 200817508 3. Beverage food containing the lactic acid bacteria of the present invention The present invention also relates to a food or drink containing Lactobacillus which is significantly enhanced by the intestinal adhesion and lactase-decomposing enzyme activity obtained by the above screening method. Is a lactic acid bacteria. The beverage food of the present invention is not limited, and particularly preferred is a beverage food for improving lactose intolerance. The term "lactose intolerance improvement" means that a test substance exhibiting lactose intolerance or suspected lactose intolerance is used as a drug for the purpose of reducing the discomfort of the lactose intolerance (abdominal pain, diarrhea, abdomen). The frequency of hunger, etc.), or the complete prevention of the onset of 'or reduce the symptoms of lactose is not serious. In the present invention, the "beverage food" is not limited, and the beverage food containing the lactic acid bacteria of the present invention including the food, the food, and the functional food is not particularly limited. For example, as the lactic acid bacteria of the present invention, two-dot acid yeast milk (sour milk road, etc., lactic acid bacteria drink, :) cow: fruit water, etc.), tea-based beverage (green tea w), fruit, and glutinous beverage ( Beverages containing alcoholic beverages such as bananas, grapes, and other vegetable juices, and alcoholic beverages (beer, gas =:::;), carbonated beverages, and soft drinks. For the manufacturing method of the second melon, please refer to the existing gift beverage "_3" (Guanglin Co., Ltd.).例如 ' For example, the food of the lactic acid bacteria of the present invention is not particularly limited, and may be a fresh food inlet or a processed food, and the lactic acid bacteria invented as a remedy*, and fresh oysters reach the intestines in a living state; The product may be a food containing the lactic acid bacteria of the present invention, a yeast or a lactic acid bacteria drink product, and a yeast for producing yoghurt milk. It may be a milky road or a milky breast. 119625.doc -26- 200817508 As a beverage food having the lactic acid bacteria of the present invention, a functional food is particularly preferable. The "functional food" of the present invention means a food having a special ability for a living body, for example, a health food containing a specific health food (including a conditional special care [specific health food]) and a nutraceutical food. Such as mouthwash, special purpose foods, nutritional supplements, and health supplements: mouth, supplements (for example, spinners, coated tablets, sugar-coated tablets, capsules and liquid J, etc.) and beauty foods (for example) , diet food) and so on all the so-called healthy food. The functional food of the present invention, in addition, includes a health food to which a health claim based on the food specification of (10) X (FA〇/WH〇Contract Food Regulatory Commission) is applied. More specific examples of the better foods of the present invention include patients: food, maternal, breast milk for women, and powder for infants: foods for the elderly, such as food, ☆, gargle. The lactic acid bacteria described in this article slowly improve the intestinal environment by mild action. In ancient times, lactose decomposition in the intestinal tract can improve lactose intolerance, and it is also possible to reduce the symptoms by a point ± πτ, so it is especially good. It is used for the preparation of uncle or liquid milk for the treatment of sugar-tolerance of infants with weak physical strength ( 叮U Σ 疋 (for example, it can be added to the raw materials for milk for children) , 吱 咕 者 者 者 者 者 者 者 者 者 者 者 者 者 者 者 者 者 者 者 者 者 者 者 者 妇女 妇女 者 妇女 妇女 妇女 妇女 者 者 妇女 妇女 妇女 妇女 妇女 妇女 妇女 妇女 妇女 妇女 妇女 妇女 妇女 妇女 妇女 妇女Use 0 As a better functional food of the present invention, it can be exemplified to treat the milk of a test subject which is inferior to the sputum due to congenital or acquired causes. Supplements for children's maternity, feeding and patient supplements. Female supplements. Oral supplements for the elderly 119625.doc -27. 200817508 As a good example of a functional food containing the lactic acid bacteria of the present invention, Can be listed Health-care foods. The health-care function food system is designed not only for ordinary foods, but also for foods in the form of spinners and capsules, based on the integration of the domestic and international movements and the previous special health food system. Under this system, health-care functional foods include two types of specific insurance (4) (individual licensed type) and nutritious functional foods (standard type). Further, 'the conditional special insurance is also included. [Special health food types." In the pure food of the invention, it is preferred that the lactic acid bacteria of the present invention are introduced into the intestine to improve the lactose decomposition ability in the intestinal tract (preferably, continuous improvement), and as a result, it is resistant to milk. The effect of improving the disease. This = Ming = functional food, can also be used to improve the use of lactose intolerance. The purpose of the first purpose of the invention of functional foods (preferably specific protection, food or conditional special protection [ Specific health foods]), can also be revealed = not to mention the ability of lactose decomposition in the intestinal tract, thereby improving lactose intolerance =, the effect of symptoms. So reveal or show 'may also The display is recognized based on the health care function: the regulations specified in the product system. For example, the present invention can be considered to be "suitable for those who want to adjust the abdominal condition to maintain the abdominal condition well" and "adjust the intestinal environment". The functional food of the present invention may be a solid preparation of a tablet, a granule, a powder 'pill, a capsule, or the like, a liquid preparation, a suspension agent, and the like. The shape of the preparation such as a liquid preparation such as syrup or a linger can also be in the shape of a general beverage food (for example, mashed milk, beverage, powdered tea, snack, etc.) H9625.doc -28- 200817508 The lactic acid 4 and the gal in the invention are not particularly limited, and may be determined by two. The specific addition amount ' can be appropriately determined after considering the beverage or the desired taste or texture. However, it is generally preferred that the total amount of the lactic acid bacteria of the present invention is 添加 〜 1 〜 1 〇〇 in an amount of 0.5% by weight. in. - Preferably, the lactic acid bacteria of the present invention can be included in the food or beverage by any suitable method available to the manufacturer. For example, the lactic acid bacteria of the present invention can be directly mixed with a raw material of a beverage or food. The lactic acid bacteria of the present invention can also be coated,

Cover, soak or spray on beverages and foods. The lactic acid bacteria of the present invention can be uniformly dispersed into beverages and foods, and W ▲ r can also be dispersed unevenly in beverages and foods. It is also possible to adjust a capsule or the like to which the lactic acid bacteria of the present invention are added. The lactic acid bacteria of the present invention may also be coated with a film or a coating agent. Further, after adding an appropriate excipient or the like to the lactic acid bacteria of the present invention, it may be formed into a shape such as a key. The food or drink containing the lactic acid bacteria of the present invention may be further processed. The above-mentioned processed product φ is included in the specification of the present invention. Further, it is particularly preferable that the beverage food containing the milk food produced by using the lactic acid g of the present invention is not limited, and particularly preferred is a lactic acid bacterium of the present invention containing a living state. When the beverage or food of the present invention is produced, various additives such as those used in foods and drinks can also be used. The additive is not limited, and examples thereof include a coloring agent (such as sodium nitrosonate), a coloring material (such as yellow vibrating pigment, red, etc.), a fragrance (such as scented scent 7), and a sweetener (stevia, aspartame). Sweet, etc.), preservation materials (sodium acetate, hibiscus, etc.), ritual agents (sodium chondroitin sulfate, propylene glycol fatty acid esters, etc.), antioxidants (disodium edetate, vitamins (:, etc.), pH Adjusting agent 119625.doc 200817508 (citric acid, etc.), chemical seasoning (sodium inosine, etc.), viscosity increasing agent (such as Sanxian gum), expansion agent (calcium carbonate, etc.), antifoaming agent (calcium phosphate), etc. A drug (such as sodium polyphosphate), a nutrient enhancer (calcium enhancer, vitamin A, etc.), etc. Further, an Asian cockroach extract, a Siberian cockroach extract, an eucalyptus extract, an Eucommia tea extract, or the like may be further added. Functional raw materials.

The beverage food of the present invention contains at least one (1 strain or more) of the lactic acid bacteria of the present invention. In the beverage food of the present invention, it is preferred that the lactic acid bacteria of the present invention include Lactobacillus sinensis OLL2836 strain (registered number bcrc 91〇35i) and Lactobacillus kawaii LL2948 strain (registered number bcrc 91〇353). And at least one selected from the group consisting of Lactobacillus faecalis LL2848 strain (registration number BCRC 910352). In a particularly preferred embodiment, the beverage food of the present invention comprises at least Lactobacillus glabrata LL 2 8 3 6 strain (Accession No. BCRC 910351), Lactobacillus kawaii LL2948 strain (Accession No. BCRC 910353), and Lactobacillus mucosa. Three lactic acid bacteria of 〇LL2848 strain (registered number BCRC 910352). The beverage food containing the three lactic acid bacteria can be combined with intestinal mucin containing any blood group antigen of type A, B type, and sputum type, and thus is effective for a wide range of subjects. The subject to be ingested or administered the beverage or food of the present invention is not limited, and preferably includes humans, livestock (pig, horse, etc.), pet U sentences, | seedlings, etc., experimental (test) animals (mouse, A lactating animal such as a rodent or a rabbit such as a rat. Further, as the subject of the present invention, it is preferable whether it is a sugar-tolerance or a lactation intolerance of a lactose intolerance, an adult period = 2 ritual animals, X, as a beverage for ingesting or administering the present invention. The food sample 'more (4) is a mammal whose age is increased by W, etc. (4) The mammal whose decomposing energy can be reduced by 119625.doc -30· 200817508, or the genetic cause or environmental cause of lactose intolerance. [Examples] Hereinafter, the present invention will be more specifically described by way of examples. However, the technical scope of the present invention is not limited to the embodiments. [Example 1] Culture and preparation of test lactic acid bacteria As the test lactic acid bacteria used below, Meiji Dairy Co., Ltd. & lactic acid strain derived from human body, from American American ype culture C〇llecti〇n (ATCC) ), the Physicochemical Research Institute Microbial System Preservation Facility (JCM), the Agriculture, Forestry and Fisheries Provincial Animal Production Test Site (NIAI), and the British National Collections of Food Bacteria (NCFB) purchased lactic acid strains, and the inventors of the present invention used modified LBS medium. A total of 104 strains of lactic acid isolated from the feces of young children. Among the tested lactic acid bacteria, in addition to Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus gasseri, Lactobacillus crispatus In addition to various strains of Lactobacillus johnsonii and Lactobacillus mucosae, strains not identified by the strain are also included. Further, a strain revealed as 〇LL or MEP in the strain name indicates a strain held by Meiji Dairy Co., Ltd. The cells of the lactic acid bacteria to be tested were dispersed in a sterilized iOo/c^w/v) skim milk medium (Snow Printing Dairy Co., Ltd., Sapporo), and stored in the refrigerator at -8 (TC was used until use). 119625.doc -31 - 200817508 When cultured, each strain was subcultured (37C, 24 hours) 3 times by MRS medium (Difc〇, Detroit, MI, USA), and then borrowed *MRSL medium (2%) The MRS medium in which glucose was replaced with lactose was subcultured (37. 〇, 24 hours) twice. Then, 1% of the obtained bacterial culture solution was inoculated into the re-prepared MRSL medium, and thereafter, under 37 After culturing for 18 hours, the thus prepared culture solution (6, 〇〇〇Xg, 2 〇, 4 〇 〇 〇 之 , 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 清洗 , , , This was suspended in distilled water and then freeze-dried. [Example 2] Screening of intestinal adhesion strains From the above-mentioned test lactic acid strains, the binding to intestinal mucin was examined by the inventors of the present invention. Ability analysis system to screen for high intestinal adhesion (RU value above 100) The analysis system used is also disclosed in detail in the international application WO2006/067940. Specifically, surface electrochemical resonance spectroscopy is performed using a biosensor BIAC〇RE1〇〇〇 (BIAC〇RE&) Analyze, thereby determining the binding ability of lactic acid bacteria to human intestinal type A intestinal mucin, human B type intestinal mucin, and human intestinal intestinal mucin. 1. Preparation of analyte solution First, make each strain After the bacteria, 3-person was subcultured by MRS medium, and after incubation for 12 hours at 37 C, 5 μl of 1 was dispensed into the volume of i 5 volume. The culture solution was centrifuged (6, 〇〇〇 Ah, generation, (7) tiller) The cells obtained by removing the supernatant are washed in PBS buffer (positive value 7.2) for 2 times/month, suspended in distilled water, and subjected to cold roll drying. -EP buffer (〇.01 M HEPES (pH 7.4), 〇·15 M Naa, 3 119625.doc -32 - 200817508 mM EDTA, 0.005% Surfactant P2〇) modulate the bacteria into 〇" mg /ml concentration to prepare the analyte solution 2. Preparation of human intestinal mucin (HCM) and HCM immobilization Preparation of the test piece* Preparation of human intestinal mucin and immobilization into sensory tablets. Human type A intestinal tract. (from blood type A human large intestine), human B type intestinal tract (derived from blood type B human large intestine) ), and the human intestinal tract (from the large intestine of the blood type sputum type), was purchased from the Graduate School of Medicine, Tohoku University, Japan. Furthermore, the sample was taken through an ethics committee of the Graduate School of Medicine, University of Tohoku University, Japan, and patient consent was obtained. In the normal part of the large intestine of these intestines, the mucus layer is collected by surface scraping. The mucin mucin layer is degreased by Folch solvent (chloroform-methanol mixture (2.1 (v/v)), J. Folch et al.: J. Biol. Chem. (1957) 226 p 497-500) and diethyl ether. Thereafter, it was dried and extracted by a 4 Torr hydrochloric acid solution at 37 ° C for 2 hours. The extract (i is then purified by gel filtration. The gel filtration purification method is based on

Purushothaman S. S. et al, '’Adherence of Shigella dysenteriae 1 to Human Colonic Mucin. n Curr. Microbiol., 42 (6), p. 381-387 (2001) is a method for purifying human colorectal mucin. The mobile layer was treated with 4 Μ guanidine hydrochloride solution, and the column used for gel filtration chromatography was Toyopearl HW-65F (90 cm x 2.6 cm, Tosoh.

Tokyo· japan). For the obtained column extract, the neutral sugar was measured by the phenol sulfuric acid method (the absorbance at 490 nm was measured after the reaction), and the absorbance at 280 nm was measured for the protein, and the protein absorption was observed (28 〇 119 625.doc) -33 - 200817508 Absorbance), and the peak of the highest neutral sugar content is selected. Further, the large intestinal mucin fraction is extracted by using a molecular weight of about 2 million or more as the standard in gel filtration chromatography. The purified product thus obtained, corresponding to the blood type derived from the human body, is designated as human type A intestinal mucin, human type B intestinal mucin, and human type 0 intestinal mucin. Hereinafter, in this human intestinal mucin (HCM), type A intestinal mucin is sometimes referred to as A-HCM, and type B intestinal mucin is referred to as B-HCM. The type of intestinal mucin is called Ο-HCM. For each of the obtained HCMs, the human blood group matrix antigenicity of the blood type was confirmed by the antigen-antibody reaction. The immobilization of each of the HCMs obtained as described above to the BIACORE sensing sheet was carried out in a biosensor BIACORE 1000 (BIACORE) by an amine-based coupling method. First, a sensor sheet CM5 (BIACORE) into which a carboxymethyl dextran group was introduced was introduced into a mixture of 75.0 mg/ml of N-ethyl-N'-(3-diaminopropyl)carbon. Amino acid hydrochloride (EDC) 50 μΐ and 11.5 mg/ml hydrazine-perylene adenine imine (NHS) 50 μΐ mixed reagent (EDC/NHS) to activate carboxyl groups in carboxymethyl dextran . Then, a mixed solution of 30 μΐ of any of the above purified HCM solutions (A-HCM, Β-HCM, Ο-HCM) was added, and it was poured into 120 μM of immobilized acetate buffer (10 mM, pH). In the value 4.0), HCM is covalently bonded to the carboxyl group on the sensor sheet by an amine coupling reaction. Further, 1 Μ ethanolamine hydrochloride-NaOH (pH 8.5) was used to block the residual survival group of the portion on the sensor sheet to which the probe was not bound. As the electrophoresis buffer, HBS-EP buffer was used. The amount of HCM immobilization shown by the difference between the report point after the introduction of EDC/NHS and the ethanolamine solution was set to 1 〇〇〇 to 2000 RU. The HCM immobilization sensor piece thus obtained in 119625.doc -34-200817508 was used in the above test of the analyte solution. 3. Analysis of surface plasma resonance spectrum Further, the amount of HCM on each HCM immobilization sensor and the amount of lactic acid bacteria in the above analyte solution was analyzed using a biosensor BIACORE 1000 (BIACORE). Hereinafter, the measurement conditions of BIACORE 1000 used for the analysis will be described. Electrophoresis buffer: 1 «84? buffer (? 11 value 7.4)

Addition amount of analyte solution (sample) · 20 μΐ Flow rate: 3 μΐ/min Reaction temperature: 25 °C Regeneration solution: 1 Μ胍 hydrochloride solution 5 μΐ In the analysis by BIOACORE system, analytes and probes The amount of bonding (interaction) of the needle is represented by a value in which a resonance unit (RU) is used. The so-called 1 resonance unit (RU) means that 1 pg of substance is bound per 1 mm 2 on the surface of the sensing sheet. That is, according to the binding/dissociation curve obtained in the analysis, the value of the RU value before the binding is subtracted from the combined RU value, which corresponds to each of the lactic acid bacteria as the analyte and each of the intestinal mucin as the probe. The amount of binding of mm2. This binding amount corresponds to the ability of the lactic acid bacteria to bind to each intestinal mucin. As a result of the analysis, a strain which showed a value of 10 0 RU or more as a binding ability to at least one intestinal mucin was selected from the lactic acid bacteria 194 strain. Among the strains screened, it was considered that the intestinal adhesion was enhanced compared with other average lactic acid strains (high intestinal adhesion). The strains of the selected strains 119625.doc -35 - 200817508 and their analytical results are shown in Figures 1 to 3. [Example 3] Synthesis and purification of ONPG1C-6P for measuring Ρ-β-glc activity Next, attempts were made to screen a strain having high intestinal adhesion and having high lactose-degrading enzyme activity. Therefore, in the examples, first, ONPG1C-6P for measuring its activity was prepared. 1· Chemical synthesis of ONPG1C-6P for the determination of acid-based β-glucose (Ρ-β-glc) activity of ortho-succinylbenzene-β-D glucoside-6-phosphate (o-Nitrophenyl-PD- Glucpyranoside 6-phosphate ; ONPGlc-6P), by Henststenberg and Morse (Hengstenberg, W. and Μ·L. Morse, Synthesis of o-Nitrophenyl-beta-D-galactopyranoside 6-phosphate; Carbohydr. Res, 10. The improved method of pp. 463-465 (1969)) for chemical synthesis. Specifically, first, 〇·9 g of o-nitrophenyl-β-D-glucoside (ONPGlc, Sigma, St. Louis, USA) is dissolved in pre-ice and cooled, and then mixed with 54 μM of distilled water and 840 μM of phosphonium trimethyl phosphate in 7.5 ml. Then, the mixture was stirred on ice for 5 hours while allowing to react. After the reaction, borneol (steamed water) was added, and concentrated aqueous ammonia was added dropwise thereto, and the solution was neutralized (pH 7.0) using a pH meter to stop the reaction. 2. Purification of ONPGlc_6P Using a rotary evaporator (Tokyo Science Machinery, Tokyo), the above-mentioned reaction solution was repeatedly concentrated and dried at 40 ° C to remove o-nitrobenzene (ONP) formed during the reaction. Then, the concentrated reaction solution was mixed with activated carbon (50 g) and allowed to stand at 4 ° C for 2 hours, whereby chemically synthesized 119625.doc -36-200817508 ONPGlc-6P and unreacted QNpGici were attached to the activity. After carbon, it was washed with 20 times of distilled water (2 L) to carry out desalting. The elution of ONPG1C-6P adsorbed on activated carbon was carried out using a pyridine:water:ethanol mixture (l:l:l (v/v) '6〇〇 mi). The eluate was concentrated in a rotary evaporator at 4 °C to remove pyridine. This was followed by filter paper chromatography (PPC). That is, 'Picture for PPC (Whatman, 3 MM, 46x57 cm,

On MaidSt (me, En§land), the above crude purified 〇NPGlc-6P was linearly coated with a 100 μΐ micropipette using butanol:pyridine:water (6:4:3〇/ν/ν) The developing solvent was subjected to single development by a rising method. After the completion of the development for 2 days, the filter paper was dried in a ventilated chamber, and the 0NPGlc-6P and 0NPGlc bands were confirmed using a UV light box (302 nm, Zhouyue, Tokyo), and only the ONPG1C-6P tape was cut. The cut filter paper was immersed in distilled water and stirred overnight to extract 〇NPGlc-6P. After the extraction, the filter paper was removed, and the aqueous layer was concentrated by a rotary evaporator, and then filtered by gel filtration using a TOYOPEARL HW40S column (2·6φχ90 cm, T〇s〇H, Tokyo) having distilled water as a mobile phase. A by-product of the reaction. After gel filtration, the 〇NPGlc-6P fraction in each of the eluted fractions was recovered using thin layer chromatography (TLC, Silicagel 60, iqx cm, Merck, Darmstadt, Germany). The developing solvent of TLC was subjected to single-stranding using butanol: 2-propanol··water (3:12:4 (v/v/v)). After the air was dried, a 5°/〇 (v/v) sulfuric acid-methanol solution was uniformly sprayed onto the thin plate, and heated in a desiccator (Yarnato, Tokyo) heated to 150 ° C for 3 minutes to carry out the test. The recovered 〇NPGlc-6P fraction was freeze-dried to prepare purified ONPG1c, 6P. The purity of the purified product was confirmed by TLC, and the structure was confirmed by H-NMR by 119625.doc -37 - 200817508. [Example 4] Measurement of P-gal, Ρ-β-gal, and Ρ-β-glc activity For the test lactic acid bacteria 104 strain prepared in Example 1, the Fisher method was used to measure β-gal, Ρ-β-gal. And lactose-degrading enzyme activity of Ρ-β-glc. • That is, for each strain, 0.5 mg of the freeze-dried cells were suspended in 1.0 ml of 0.05 Μ phosphate buffer (pH 6.8), and 50 μM of toluene-acetone mixture (1:9 (Wv)) was added and stirred vigorously. 3 minutes. In 25 μΐ of this suspension, Ρ added o-nitrobenzene-β-D-galactopyranoside (ONPGal), o-nitrophenyl-β-Ν-galactopyranoside-6-phosphate (〇 NPGal-6P) or the above-prepared ortho-succinylbenzene-β-D-glucose-6-acid acid S (ONPGlc-6P) 0.05 sputum acid buffer (pH 6.8) 100 μΐ at 37 ° C The reaction was carried out for 15 minutes. After the reaction, 125 μL of a 0.5% sodium carbonate solution was added to stop the reaction, and the cells were removed by centrifugation (6,000 x rpm, 10 minutes, 4.

The absorbance of the supernatant at 405 nm was determined by Multiskan MS-UV (Labsystems, Helsinki, Finland). The ortho-nitrophenol (/(〇ΝΡ)1 μηιοί疋 is 1 卓元 in 1 minute, and the unit is used as a unit to calculate the activity value per 1 mg of the cells and per 1 mg of the culture supernatant. The activity value of the contained protein. Further, the protein was quantified by using the Bicr〇bca Protein Assay Reagent Kit (Pierce® Rockford ^ Illinois ^ USA) based on the BCA method for 2 hours, and then the sputum was measured using Muhiskan Ms_uv. The absorbance in nm was measured, and the calibration curve was prepared by using bovine serum albumin (BSA), and the amount of protein was calculated here. In Fig. 3, there is shown a representative example of the strain selected in Example 2 The lactose-degrading enzyme was subjected to the activity of 119625.doc -38-200817508. [Example 5] Screening of lactic acid strain According to the measurement result of the adhesiveness of the material indicated by the above-mentioned _ towel, _(4) (4) The activity of the lytic enzyme was also high. The lactic acid strain, as a result, was 1: high, and for the strains which showed no significant higher activity in (4), which was called 纠·(4), the lactose fraction was not selected, and three milks were selected (OLL 2836 strain, OLL2948 strain, and 〇Ιχ2848 (4)~3, another aspect, most of the lactic acid strains with high lactose-degrading enzyme activity, low colonic adhesion (for type A, B, and sputum type - human intestinal mucin, RU value) As a result, the results were excluded. As shown in Figures 1 to 3, the OLL2836 strain represents the Ρ-β-gal activity of 46 576 units/〇^ protein, and the OLL2948 strain represents 50.194 units/mg of protein. The activity, OLL stage strain showed 107. 〇9〇 unit/mg protein mouth (iv) activity, but these activity values were very high values even compared with the potted lactic acid strain with higher intestinal adhesion. Moreover, these strains have a very high value for the expression of the intestinal adhesion. In particular, 'OLL2836 strain shows relative to human b-type intestinal mucin and human intestinal gut mucin. Particularly strong binding ability, and 〇ll2948 strain and OLL2828 strain show a particularly strong binding ability to human type A intestinal mucin. The above results show that for these lactic acid strains, it can be expected to be separately fixed in the intestine In the road, the effect of continuous decomposition of lactose In addition, the lactose decomposition effect of 119625.doc -39-200817508 can also be obtained in the test body of any blood type by using the 3 strains in combination. Further, the OLL2836 strain, the OLL2948 strain and the OLL2848 strain can be obtained. In the strain identification test, the OLL2836 strain and the OLL2948 strain were identified as strains belonging to Lactobacillus gasseri, and the OLL2848 strain was identified as a strain belonging to Lactobacillus mucosae (see the following examples). On June 9th, 2006 (original registration day), the three lactic acid strains were deposited with the Patent Microbiology Depository Center (2-5-8, Kishozu, Kisarazu, Chiba Prefecture, Japan). Apply and be accepted for deposit. The registration number of the deposit application is NITE AP-241 for OLL2836, NITE AP-242 for OLL2948, NITE AP-243 for OLL2848, and NITE P-241, NITE P-242, NITE for registration. P-243. These deposited strains were handed over to the Budapest Treaty-based deposit (international deposit) for safekeeping on March 27, 2007, and were accepted as the original deposit date on June 9, 2006. The international deposit numbers are: NLE BP-241 for the OLL2836 strain, NITE BP-242 for the OLL2948 strain, and NITE BP-243 for the OLL2848 strain. In addition, these lactic acid strains were also deposited with the Taiwan Food Industry Development Research Institute (FIRDI) on June 4, 2007. The registration number of the OLL2836 strain is BCRC 91035 1, the registration number of the OLL2948 strain is BCRC 910353, and the deposit of the OLL2848 strain. The number is BCRC 910352. The fungal properties of these strains were confirmed as described in Table 1 above. As described above, by using the screening method of the present invention, a lactic acid strain having high intestinal adhesion and high lactose-degrading enzyme activity can be obtained. Further, the activity of lactose-degrading enzyme was measured for β-gal, Ρ-β-gal, and Ρ-β-glc, and strains showing significantly higher activity against the three lactase-degrading enzymes were selected by 119625.doc -40-200817508, respectively. By combining the obtained strains, it is also possible to obtain a combination of very useful lactic acid bacteria which can be applied to a wide range of subjects. It is considered that the lactic acid strain obtained by the method of the present invention is a useful probiotic lactic acid strain which is effective in reducing the lactose in the intestinal tract, and is effective for improving lactose intolerance. [Example 6] Identification of strains Three strains of lactic acid strains OLL2836, OLL2948 and OLL2848 selected in Example 5 were identified for strain identification. The identification was carried out by analysis of the 16S rDNA base sequence based on amplification and sequence measurement of the V3 region. Hereinafter, an identification test performed in the OLL2848 strain and the OLL2948 strain is disclosed as an example, and the OLL2836 strain is also identified in substantially the same manner. 1. Detection of 16S rDNA fragment directly by the PCR method The OLL2848 strain and the OLL2948 strain were subcultured by MRS medium as described above, and then directly subjected to PCR. In a microcapillary tube of 0.6 ml volume, 4 μM of ultrapure water was injected into each tube, and the cells scraped by the sterilized toothpick were suspended therein. To the suspension, Taq DNA polymerase (TaKaRa, Kyoto), lOx PCR buffer, dNTP Mix (TaKaRa, Kyoto), and a set of primers were added to prepare a PCR reaction solution. As a set of primers, a general primer 27F (5'-GAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 1), and SlSRa-ATTACCGCGGCTGCTGGd1, SEQ ID NO: 2) were used. The PCR conditions were as follows: [95 ° C for 10 minutes, 55 ° C for 3 minutes, 72 ° C for 1 minute] as one cycle, [95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds] as 39 cycles, and [72 ° C 1 minute] was carried out as one cycle. 119625.doc -41 - 200817508 2. Agarose gel electrophoresis The DNA fragment amplified by the above PCR reaction was subjected to electrophoresis. The electrophoresis electrophoresis gel was carried out using a 2% agarose gel, and the electrophoresis buffer was carried out using a buffer of cesium (90 mM triboric acid, 2 mM EDTA, pH 8.0) at a constant voltage of 100 V. The swimming device used Mupid-2 (COSMOBI〇, Tokyo). The molecular weight marker used was a 100b DNA ladder (TaRaRa, Kyoto). Gel staining after electrophoresis was carried out by dipping for 10 minutes in an ethidium bromide (EB) solution (0.5 pg/ml). 3. The DNA extraction from the agar-gel gel was carried out by UV-UV light box TRANSILLMINATOR TM-20 (After-Korean Pharmaceutical Co., Ltd., Tokyo), and the gel was irradiated on the agarose gel electrophoresis and stained gel to confirm the target DNA fragment. Cut it out. DNA was extracted by purifying the set of MagExtractor (TOYOBO, large P counter) with a DNA fragment. Namely, after the cut agarose gel was transferred to a 1.5 ml-volume tube, 400 μM of the adsorption solution was added, and the mixture was heated to 55 ° C to be completely dissolved. Then, 15 μM of magnetic beads were added to the reaction solution, and the mixture was stirred at room temperature for 1 minute. After centrifugation (6,000 rpm, 5 sec, 4 ° C), the supernatant was removed, and 500 μM of a washing solution was added for mixing. Again, after separation of the heart (6,000 卬 111, 5 seconds, 4 ° ,, add 11111 of 75 ° / 〇 ethanol for stirring, centrifugation (15,000 rpm, 1 minute, 4 ° C), remove the supernatant After adding 1 5 μΐ of steamed water for stirring, the supernatant was recovered by centrifugation (1,500 rpm, 1 minute, 4 ° C) as a purified DNA solution. Decision and homology analysis 119625.doc -42- 200817508 Base sequence determination of purified DNA using primer 27F (5,-GAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 3) Base sequence determination by Operon Biotechnology In this way, the nucleotide sequence of the V3 region of 16S rDNA measured for the OLL2848 strain is represented by SEQ ID NO: 4, and the nucleotide sequence of the V3 region of 16S rDNA measured for the OLL2948 strain is represented by SEQ ID NO: 5. On the network of the Japanese DNA database (DDBJ), homology analysis was performed on these base sequences by the program BLASTN. As a result, the base sequence of the V3 region of the OLL2848 strain and Lactobacillus mucosae were obtained. Between the sequences of the V3 region 99.6% homology was shown, and on the other hand, the base sequence of the V3 region of the OLL2948 strain showed 99.2% homology with the sequence of the V3 region of Lactobacillus gasseri. It can be identified that the OLL2848 strain belongs to Lactobacillus faecalis lactic acid bacteria, and the OLL2948 strain belongs to lactic acid bacteria of Lactobacillus gasseri. [Industrial Applicability] The method of the present invention can be used for effective screening for improving lactose intolerance. Lactic acid strain. The lactic acid strain obtained by the method of the present invention can be simply administered to a patient, and can be advantageously used as an active ingredient for a drug or a functional food which can continuously improve lactose intolerance. [Sequence free content] The sequences of SEQ ID NOs: 1 to 3 are primers. The entire contents of all publications, patents, and patent applications cited in this specification are hereby incorporated herein by reference. 119625.doc -43 - ζυυδί [Simple description] The lanthanide series indicates the results of the activity test. The k adhesion is compared with the results of the intestinal tract adhesion-activity test in comparison with Fig. 2. The 3 line shows the results of the test for the adhesion test of the intestinal tract. The β-gal activity of the Ρ-β-gh strain of the Ρ-β-gal strain of the representative strain of 叼 is representative of 119625.doc 44- 200817508 Sequence List <110> Consortium Food Research Institute National University Corporation Northeastern University <120>Lactic acid bacteria that improve lactose intolerance

<130> PH-3099TW <140> 096123069 <141>2007-06-26 <150>JP 2006-175897 <151>2006-06-26 <160> 5 <170> Patentln Ver. 2.1 <210> 1 <211> 19 <212> DNA <213>Artificial sequence <220> 223>Artificial sequence type: Primer <220><223> Inventor: Saito, Tadao; Kitazawa, Haruki Inventor: Kawai, Yasushi; Itoh, Hiroyuki <400> 1 gagtttgatc ctggctcag <210> 2 <211> 17 <212> DNA <213>Artificial sequence <220><223> Sequence type: Primer <400> 2 attaccgcgg ctgctgg <210> 3 <211> 19 <212> DNA <213>Artificial sequence <220> 119625.doc 200817508 Artificial sequence type: Primer <400> 3 gagtttgatc ctggctcag < 210 > 4 < 211> 559 < 212 > DNA < 213> mucosa Lactobacillus < 400 > 4 tgggggaggg gggggggggg ggggccggcg gtgtgtctct atatacatgc aagtccgaac gcgttggccc aactgattga acgtgcttgc acggaacttg acgttggttt accagcgagt ggcggacggg tgagtaacac gtaggtaacc tgccccaaag cgggggataa catttggaaa cagatgctaa taccgcataa caatttgaat cgcatgattc aaatttaaaa gatggcttcg gctatcactt tgggatggac ctgcggcgca ttagcttgtt ggtagggtaa cggcctacca aggctgtgat gcgtagccga gttgagagac tgatcggcca caatggaact gagacacggt ccatactcct acgggaggca gcagtaggga atcttccaca atgggcgcaa gcctgatgga gcaacaccgc gtgagtgaag aagggtttcg gctcgtaaag ctctgttgtt agagaagaac gtgcgtgaga gcaactgttc acgcagtgac ggtatctaac cagaaagtca cggctaacta cgtgccagca gcccgcggt < 210 > 5 < 211> 524 < 212 > DNA < 213> gasseri Lactobacillus < 400 > 5 ggcgtgcctt atacatgcaa gccgagcgag cttgcctaga tgaattttgg tgcttgcacc acgatgaaac tagatacaag cgagcggcgg acgggtgagt aacacgtggg taacctgccc aagagactgg gataacacct ggaaacagat gctaataccg gataacaaca ctagacgcat gtctagagtt taaaagatgg ttctgctatc actcttggat ggacctgcgg tgcattagct agttggtaag gtaacggctt accaaggcaa tgatgcatag ccgagttgag agactgatcg gccacattgg gactgagaca cggcccaaac Tcctacggga ggcagcagta gggaatcttc cacaatggac gcaagtctga tggagcaacg ccgcgtgagt gaagaagggt ttcggctcgt aaagctctgt tggtagtgaa Gaaagataga ggtagtaact ggcctttatt tgacggtaat tacttagaaa gtcacggcta actacgtgcc agcagcccgc ggta 119625.doc

Claims (1)

200817508 X. The scope of application for patents: 1. The method of lactic acid bacteria, the special screening of intestinal adhesion and lactose is a screening. The improvement of lactose intolerance is caused by the lactic acid bacteria of Lactobacillus, and the enzyme activity is enhanced. The bacteria. 2. The method according to the method of determining the binding ability of the lactic acid bacteria to at least one of human type A intestinal mucin, human type B intestinal mucin, and human sputum intestinal mucin using surface electrical destruction resonance analysis. The bacteria having an RU value of 100 Ru or more indicating that the ability is sufficient are selected as bacteria having enhanced intestinal adhesion. 3. As requested! Or 2 method wherein the lactase-degrading enzyme activity is at least one of lactase-degrading enzyme activity of galactosidase, phosphate-β-galactosidase, and phosphate-P-glucosidase. 4. The method according to any one of the preceding claims, wherein the Lactobacillus gasseri, or Lactobacillus mycosae 〇5, a lactic acid bacterium, is as claimed in claim 1 The method of any one of the methods of 4, wherein the intestinal adhesion and the lactose-degrading enzyme activity are combined together. 6·----------------------------------------------------------------------------------------------- Any one of them. A lactose intolerance improving agent comprising at least one lactic acid bacterium as claimed in claim 5 or 6. 8. The lactose intolerance improving agent according to claim 7, which comprises at least lactic acid lactic acid θ 119625.doc 200817508 bacterium OLL2836 strain (registered number BCRC 9 1035 1), and Lactobacillus gasseri OLL2948 strain (registered number BCRC 91 0353 ), and Lactobacillus mulidis OLL2848 strain (registration number BCRC 9103 5 2) trilactic acid bacteria. A beverage food comprising at least one lactic acid bacterium according to claim 5 or 6. 1 . The beverage food according to claim 9 which comprises at least Lactobacillus kawaii OLL2836 strain (Accession No. BCRC 91035 1), Lactobacillus kawaii OLL2948 strain (Accession No. BCRC 91 0353), and Lactobacillus mucosa OLL2848 strain (registered No. BCRC 910352) Trilactic acid bacteria. 11. The beverage food according to claim 9 or 10, which is formed from baby food, baby food, breastfeeding women's food, aged food, patient food, health functional food, supplement, fermented milk and lactic acid bacteria beverage. Group of selectors 0 119625.doc