TWI383049B - Improved lactose intolerance of lactic acid - Google Patents

Description

Lactic acid bacteria that improve lactose intolerance

The present invention relates to a lactic acid bacterium useful for improving lactose intolerance and its use.

"Lactose intolerance" refers to a constitution in which digestive symptoms such as diarrhea or abdominal pain are easily caused by indigestion of lactose in the milk. It is generally believed that this lactose intolerance usually becomes significant with age, but it is also visible in early childhood. In order to alleviate the symptoms of lactose intolerance, 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 calcium, for example, it will increase the risk of osteoporosis in the elderly, and it will become larger in terms of nutrition. obstacle. Osteoporosis is one of the biggest diseases in Japan that is rapidly developing into an aging population. Therefore, it is particularly important to promote the active intake of dairy products in terms of reducing the risk of osteoporosis in the elderly.

Lactose is usually decomposed by lactose-degrading enzymes produced in cells of the inner layer of the small intestine and absorbed. On the other hand, various microorganisms such as lactic acid bacteria are known to produce lactase, and lactose is decomposed. The manufacturer believes that yoghurt 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 in the intestines. The slow proliferation in the tract makes the lactose decomposition ability in the intestinal tract increase, and as a result, the lactose intolerance itself is improved.

It has been known from the past that lactose metabolism of lactic acid bacteria has two lactose-degrading enzymes of β-galactosidase (β-gal) and phosphate-β-galactosidase (P-β-gal). Further, the inventors of the present invention also known that a third lactose-degrading enzyme of a phosphate-β-glucosidase (P-β-glc) contained in a lactic acid bacterium is also present (Non-Patent Document 1). The lactic acid bacteria having high activity of such lactose-degrading enzymes are considered to be effective in alleviating the symptoms of lactose intolerance. However, there is a higher lactose-decomposing activity in the parenteral lactic acid bacteria, but it is generally considered that it is also present in the human intestinal tract. Such a lactic acid bacterium does not continue to produce lactose-degrading enzyme in the intestinal tract of the human body, and it is considered that lactose-degrading enzyme activity of more than the amount of oral ingestion cannot be obtained (Non-Patent Document 2). Therefore, in order to improve lactose intolerance by oral administration of lactic acid bacteria, it is not only required to have a high lactose-degrading enzyme activity, but also a lactate-producing lactic acid bacterium in the human intestinal tract, but no significant progress has been made in the exploration of the lactic acid bacteria described above. .

However, it is known that bacteria having a high intestinal adhesion are present in the intestinal lactic acid bacteria. As a method for exploring such a bacteria having a high intestinal adhesion, for example, there is a method of screening a lactic acid bacterium having a high intestinal adhesion suitable for each blood type of a human body by measuring the binding ability to human intestinal mucin (patent) Document 1). The method is based on the following contents and reports: in order to make the lactic acid bacteria in the intestinal tract in the early stage of infection in the human body, it is indispensable to adhere to the surface of intestinal epithelial cells; the use of large intestinal mucin in Takahashi et al. Report on the screening of lactic acid bacteria (RCM) [using polystyrene beads coated with colorectal mucin (RCM) for the screening of Lactobacillus acidophilus lactic acid strains, strongly binding to the surface proteins of lactic acid strains on this RCM ( SLP) is also reported in the human mucous layer of the large intestine. Non-Patent Document 3, etc.; The chemical structure of the sugar chain constituting the human large intestinal mucin is reported differently according to the ABO type blood type (for example, Non-Patent Document 4). However, this screening method has not been applied yet It is suitable for use in applications other than the exploration of lactic acid bacteria of various blood types.

[Patent Document 1] Japanese Patent Laid-Open Publication No. 2004-101249

[Non-Patent Document 1] Discovery of a new way of lactose assimilation system in Lactobacillus gasseri (Gasiaria) originating from the intestines of the human body, Saito Tayoshi, Ito Katsuyuki, Yoshino Yoshio, Yuki Yamazaki, Bioinstitutional Society, (2001) , 79(6), pp.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. 1031-1037

[Non-Patent Document 3] Takahashi N, Saito T, Ohwada S, Ota H, Hashiba H, Itoh T, "A new screening method for the sclection of Lactobacillus acidophilus group lactic acid bacteria with high adhesion to human colonic mucosa.", Biosci Biotechnol Biochem, (1996), 60 (9), pp. 1434-1438

[Non-Patent Document 4] Blood type sugar chain structure on the mucin of the genus Amano and the digestive tract - Explanation of the mechanism of bacterial infection, Biochemistry, Society of Japanese Biochemical Society, (1999) 71(4), pp. 274~277

It is an object of the present invention to provide a lactic acid bacterium having the ability to improve lactose intolerance.

In order to solve the above problems, the inventors of the present invention have found that a part of Lactobacillus lactic acid bacteria has enhanced intestinal adhesion and lactose-degrading enzyme activity, and the present invention has been completed based on this knowledge.

That is, the present invention includes the following.

[1] A method for screening a lactic acid bacterium having an improvement ability of lactose intolerance, which is characterized in that bacteria which are mutually enhanced in intestinal adhesion and lactose-degrading enzyme activity are selected from a lactic acid bacterium of the genus Lactobacillus.

In the method, it is better to use the surface plasma resonance analysis to screen the bacteria, and to measure at least at least one of human type A intestinal mucin, human type B intestinal mucin, and human type O intestinal mucin. The binding ability of one is a bacteria having a RU value of 100 RU or more in combination with the binding ability, and is screened as a bacteria having enhanced intestinal adhesion.

In the method, the lactose-degrading enzyme activity in the selected bacteria is preferably lactose of at least one of β-galactosidase, phosphate-β-galactosidase, and phosphate-β-glucosidase. Decomposes enzyme activity.

In the method, the Lactobacillus faecalis which is screened is preferably Lactobacillus gasseri or Lactobacillus mucosae.

[2] A lactic acid bacterium which is enhanced by the intestinal adhesion and the lactose-degrading enzyme activity obtained by the method of the above [1].

[3] A lactic acid bacterium which is any one of Lactobacillus kawaii OLL2836 strain (Accession No. BCRC 910351), Lactobacillus kawaii OLL2948 strain (Accession No. BCRC 910353), and Lactobacillus mucosa OLL2848 strain (Accession No. BCRC 910352). .

[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 (Accession No. BCRC 910351), Lactobacillus kawaii OLL2948 strain (Accession No. BCRC 910353), and Lactobacillus mucosa OLL2848 strain (registered number). BCRC 910352) Three lactic acid bacteria.

[5] A food or drink comprising at least one lactic acid bacterium as described in [3] above.

More preferably, the beverage or food comprises at least Lactobacillus kawaii OLL2836 strain (Accession No. BCRC 910351), Lactobacillus kawaii OLL2948 strain (Accession No. BCRC 910353), and Lactobacillus mucosa OLL2848 strain (Accession No. BCRC 910352). Three kinds of lactic acid bacteria.

As the food and beverage, it is preferably a food for infants, a food for children, a food for breastfeeding women, a food for the elderly, a food for patients, a functional food for health care, a supplement, a lactated milk, or a lactic acid bacteria beverage.

The beverage food is particularly preferably used as a beverage food for improving lactose intolerance.

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, a medicament for improving lactose intolerance or various foods and drinks can be produced.

The present specification contains the contents disclosed in Japanese Patent Application No. 2006-175897, which is the priority of the present disclosure.

Hereinafter, the present invention will be described in detail.

1. Screening method for lactic acid bacteria having the ability to improve lactose intolerance

The present invention relates to a method for screening a lactic acid bacterium having an improvement ability of lactose intolerance by screening a bacterium of Lactobacillus lactic acid bacteria for screening for adhesion of intestinal adhesion and lactose-degrading enzyme activity.

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, including the previous morphological classification or physiology based on the classification of known Lactobacillus species. Classification of biochemical traits, but for example, for bacteria that have not been identified, the molecular phylogenetic classification based on the homology analysis using the 16S rDNA sequence, especially the base sequence of the V3 region, can be clearly performed. Classification is preferred. 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), and acidophilic lactic acid. Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus helvelicus , Lactococcus lactis, Lactobacillus crispatus, hemolytic lactic acid Bacillus (Lactobacillus johnsonii), Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus mucosae, etc., but are not limited thereto. In the method of the present invention, as the Lactobacillus lactic acid bacteria, intestinal lactic acid bacteria are preferred, and Lactobacillus gasseri and Lactobacillus mucosa are particularly preferred. In the method, the "Lactobacillus lactic acid bacterium" for screening 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 adhesion and the lactose-degrading enzyme activity 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 used to measure at least one of the human intestinal type A intestinal mucin, the human type B intestinal mucin, and the human type O 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 bacteria to the intestinal mucin can be carried out by, for example, a method of surface plasma resonance analysis disclosed in Patent Document 1 and International Patent Application No. WO 2006/067940. In the case of using surface plasmon resonance analysis, it is preferred to select bacteria having a RU value of 100 RU or more indicating the binding ability of lactic acid bacteria to intestinal mucin, and to select candidates for lactic acid strains of bacteria having enhanced intestinal adhesion.

Hereinafter, an analysis system for the binding ability of the intestinal mucin will be described in more detail. First, in order to prepare a blood type of human intestinal mucin, a surface layer is taken from the human intestinal tract of each blood type (type A, type B, and type O), and salt is used. The solubilizing agent such as samarium is subjected to gel filtration, and the target fraction is collected and purified by protein absorption (absorbance at 280 nm) and a high neutral sugar content. For a detailed description of the gel filtration purification, for example, see Purushothaman SSet al, "Adherence of Shigella dysenteriae 1 to Human Colonic Mucin." Curr. Miorobiol., 42 (6), p. 381-387 (2001) A method for purifying human large intestinal mucin. After purification, it is more preferable to use an anti-blood group antigen antibody to confirm that a blood group antigen is found 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 can be used. Blood Group A Trisaccaride-BSA (for example, Calbiochem) or glycoprotein. Blood Group B Trisaccaride-BSA (for example, Calbiochem), etc., as an antigen, can produce an anti-blood-type antigen antibody by various antibody production methods well known to the practitioner.

In the screening method of the present invention, human body A-type intestinal mucin, human B-type intestinal mucin, and human intestinal mucin of human O-type intestinal mucin are respectively used as probes for surface plasma resonance spectrum analysis. Thereby, the ability of the lactic acid bacteria to bind to human intestinal mucin can be determined. For the surface plasma resonance spectrum analysis, a BIACORE system (BIACORE), such as BIACORE 1000, can be used as a biosensor (biological intermolecular interaction analysis device).

The BIACORE system is based on the principle of surface plasma resonance spectroscopy (SPR), which can simultaneously monitor the interaction (bonding and dissociation) of biological molecules without using markers. Specifically, the intermolecular interaction between the ligand (hereinafter, also referred to as a probe) and the analyte is measured. In the present invention, lactic acid bacteria are used as the analyte system, and human colorectal mucin (type A, type B, and type O) is used as a ligand system. A detailed description of the principles of surface plasma resonance has been published. If the principle used in the device is briefly described, the substance is bound/dissociated on the gold film, and the change in the refractive index of the reflected light accompanying the mass change on the gold film caused by the bonding/dissociation is measured, thereby calculating the gold. The amount of material bound to the membrane. More specifically, in the apparatus, a glass substrate with a gold film called a sensor sheet is provided on the way of flowing the sample or the reagent, and a sample or the like is flowed into the passage. Inside, on the sensing sheet, the 760 nm polarized light is condensed into a wedge shape by using a crucible or the like, and is reflected on the gold film, and the refractive index of the reflected light is monitored, and the angle of the surface plasma resonance spectrum is changed. It is shown to be proportional to the change in the amount of binding of the substance on the gold film. Therefore, in the BIACORE system, first, the probe is flowed into the channel, the probe is fixed on the gold film of the sensing sheet in advance, and then the portion to which the probe is not bound is blocked, so that the combination to be inspected is to be inspected/ The substance (analyte) of the dissociation reaction flows into the passage, and the refractive index of the reflected light is monitored over time, whereby the amount of binding of the probe to the analyte can be calculated from the amount of change. In this system, the change of the angle of the surface plasma resonance spectrum of 0.1° is defined as 1000 resonance units (RU), and the change in the refractive index of the reflected light is expressed by the value of the resonance unit (RU value). Here, 1 resonance unit (RU) corresponds to a mass change of 1 pg/1 mm ² on the surface of the sensor sheet. That is, the amount of binding of the substance to the gold film on the sensor sheet can be calculated from the difference between the time before the combination of the substance (before the addition) and the RU value at the end of the combination. If the BIACORE system is used, the probe (in the present invention, each human intestinal mucin) and the analyte (in the present invention) can be obtained as the mass change (pg) per 1 mm ^{2 of the} sensor sheet, that is, the Ru value. , the binding amount of lactic acid bacteria, the binding amount (RU value) is equivalent to the binding ability of the analyte and the probe.

Furthermore, among the sensor sheets usable in the BIACORE system, there are several types of substances (probes), immobilization methods, and purpose of use that are fixed to the sensor sheet, for example, except for the most standard CM5 (glass surface). There is a gold film (typically 50 nm) with a carboxymethyl dextran layer (typically 100 nm) on the surface. The carboxyl group and coordination of the carboxymethyl dextran layer In addition to the amine group, sulfhydryl group, aldehyde group or carboxyl group of the group, and the ligand (probe) is immobilized on the sensor sheet, CM4, CM3, C1, SA, NTA, L1, HPA and the like are also present.

In the present invention, in order to immobilize intestinal mucin as a probe to the sensing sheet, for example, it can also be carried out according to the manufacturer's instruction manual of 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 (BIACORE) is used as the sensor sheet, first, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride is mixed at 1:1. A mixed reagent (EDC/NHS) of a salt (EDC) and N-hydroxysuccinimide (NHS) flows into the channel to activate a carboxyl group in the carboxymethyl dextran on the sensor sheet, and is then prepared. A mixed solution of a human intestinal mucin solution (each using A-HCM, B-HCM, and O-HCM) is added to an acetate buffer (pH 4.0) for immobilization, and further, it is allowed to flow into the passage, and By the amine coupling reaction, the human intestinal mucin is covalently bonded to the carboxyl group on the sensing sheet. Further, it is also preferred to use ethanolamine hydrochloride-NaOH (pH 8.5) to block the sensing sheet on which the probe is not bound. The carboxyl group is made into an intestinal mucin (HCM) immobilization sensor. In the present invention, the amount of HCM immobilization in the sensor sheet is not particularly limited, but is preferably 1000 to 2000 RU.

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 BIACORE 1000 which can be used for the analysis is shown below.

Electrophoresis buffer: HBS-EP buffer (pH 7.4)

Addition amount of analyte solution (sample): 20 μl

Flow rate: 3 μl/min

Reaction temperature: 25 ° C

Regeneration solution: 1 M of hydrazine hydrochloride solution 5 μl

According to the binding/dissociation curve obtained based on such an assay, the RU value obtained by combining the analytes minus the RU value of the RU value before the analyte binding can be used as the lactic acid bacteria as the analyte and the intestinal tract as the probe. The binding amount (pg) per 1 mm ² of mucin, which is a value indicating the binding ability of the lactic acid bacteria to intestinal mucin. In the present invention, when at least one of the human type A intestinal mucin, the type B intestinal mucin, and the type O intestinal mucin exhibits a value of 100 RU or more, the combination of the lactic acid bacteria and the intestinal mucin The ability can be judged as "increased intestinal adhesion" or "high intestinal adhesion".

Furthermore, the RU value can vary depending on the measurement conditions in the BIACORE system. The temperature conditions in the method are, for example, 20 to 40 ° C, preferably 20 to 30 ° C, more preferably 23 to 28 ° C. Further, the analyte solution in the method The concentration of (sample) is preferably 0.1 to 0.5 mg/mL, and the flow rate in the method is preferably 3 to 10 μl/min. If it is in the above range, the RU value does not change even if the above various conditions are changed. Therefore, it is possible to judge whether or not the intestinal adhesion is high by using "100 RU" as a standard. In addition, when the conditions such as the concentration of the analyte solution are outside the above range, the intestinal adhesion of the lactic acid bacteria is exhibited when the RU value in the above condition is converted to the same binding ability as 100 RU. Something has been enhanced. Further, in the screening method of the present invention, it is considered that the higher the RU value of the bacteria, the higher the intestinal adhesion, that is, the intestinal adhesion is enhanced as compared with the conventional lactic acid bacteria. In the present invention, it is preferred that the binding ability to at least one human intestinal mucin indicates a lactic acid bacteria having a RU value of 100 RU or more, more preferably 300 RU or more, particularly preferably 1000 RU or more, screening. It is a bacteria with enhanced intestinal adhesion.

Further, in the screening method of the present invention, β-galactosidase (β-gal), phosphate-β-galactosidase (P-β-gal), and phosphate-β-glucose are measured for the Lactobacillus lactic acid bacterium. A lactose-degrading enzyme activity (lactolytic enzyme activity) of at least one lactase-degrading enzyme in the glucosidase (P-β-glc), and screening the strain having enhanced activity.

Specifically, according to the Fisher method, the freeze-dried cells are suspended in a 0.05 M phosphate buffer, and a toluene-acetone mixed solution (1:9 (v/v)) is added thereto and vigorously stirred, if it is β-gal For the determination of lactose-degrading enzyme activity, a phosphate buffer containing o-nitrophenyl-β-D-galactopyranoside (ONPGal) is added to the suspension; if it is a lactose-degrading enzyme activity of P-β-gal For the determination, o-nitrobenzene-β-D-galiprazole is added to the suspension. a phosphate buffer of glucoside-6-phosphate (ONPGal-6P); or, if it is a lactose-degrading enzyme activity of P-β-glc, adding o-nitrobenzene-β-D to the suspension A phosphate buffer of glucoside-6-phosphate (ONPGlc-6P) was reacted at 37 ° C for 15 minutes. After the reaction, a 0.5 M sodium carbonate solution was added to stop the reaction, and the bacterial components were removed by centrifugation (6,000 × rpm, 10 minutes, 4 ° C) using an absorbance photometer (for example, Multiskan MS-UV (Labsystems) , Helsinki, Finland)), the absorbance of the supernatant at 405 nm was determined. 1 μmol of o-nitrophenol (ONP) which was free in 1 minute was defined as 1 unit, and was converted into an activity value. From the activity values, 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 were calculated.

Further, when the protein is quantified, for example, a BCA method based Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, Illinois, USA) can be used to react for 2 hours, and an absorbance photometer (for example, Multiskan MS-) is used. UV) The absorbance at 540 nm was measured. 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-gal, P-β-gal, and P-β-glc calculated in the above manner, and the activity value per 1 mg of the protein contained in the culture supernatant, A lactic acid bacterium having a particularly high activity of lactase-degrading enzyme (the lactose-degrading enzyme activity is enhanced compared with ordinary lactic acid bacteria).

Although not limited, for example, regarding lactose-degrading enzyme activity of β-gal (also referred to as β-gal activity in the present invention), it is particularly preferred to select 15 units or more per 1 mg of protein in the culture supernatant. Good is more than 100 units The strain. Further, regarding the lactose-degrading enzyme activity of P-β-gal (also referred to as P-β-gal activity in the present invention), it is particularly preferable that the amount of 1 mg of protein in the culture supernatant is 15 units or more. It is good to show strains of more than 45 units. Further, regarding the lactose-degrading enzyme activity of P-β-glc (also referred to as P-β-glc activity in the present invention), it is particularly preferred that the screening culture supernatant exhibits 25 units or more per 1 mg of protein, preferably It is a strain showing more than 50 units.

In the method of the present invention, the above-mentioned Lactobacillus lactic acid bacteria (more preferably Lactobacillus, Gallibacterium or Lactobacillus. Mucosa) are measured for intestinal adhesion and lactose-degrading enzyme activity, and the result is It can effectively screen lactic acid strains with enhanced intestinal adhesion and lactose-degrading enzyme activity. The invention also relates to a lactic acid strain thus screened. The Lactobacillus lactic acid bacterium obtained in this manner is excellent in proliferative and fixed in the intestinal tract and maintains a very effective ability to break down lactose, and therefore is very useful for improving lactose intolerance. In the present invention, the term "improvement of lactose intolerance" means reducing the symptoms of digestive system (abdominal pain, diarrhea, hunger of the abdomen, etc.) of lactose intolerance when ingesting a beverage product (dairy product) containing lactose. The frequency of occurrence, either completely preventing the onset, or reducing the severity of the symptoms. Moreover, "the ability to improve lactose intolerance" 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 in which intestinal adhesion and lactose-degrading enzyme activity are remarkably enhanced. These three lactic acid bacteria are Lactobacillus gasseri OLL2836 strain (later, also known as OLL2836 strain) and Lactobacillus gasseri OLL2948 strain. Later, also known as OLL2948 strain), and Lactobacillus mucosa OLL2848 strain (later, also known as OLL2848 strain). These lactic acid strains are deposited in the patent microbial deposit center of the independent administrative legal person product evaluation technology infrastructure. The information relating to such deposits is as follows. (1) The name of the depository institution: the patent administrative microbial deposit center of the independent administrative legal person product evaluation technology base organization (2) The liaison office of the depository agency: 2-5-8 (3) registration date of the Kishozu City, Chiba Prefecture, Japan (3) Date): June 9, 2006 (4) Deposit 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: registration number NITE BP-243 (original Registered collection number NITE AP-243).

These lactic acid strains were further deposited with the Taiwan Food Industry Development Research Institute (FIRDI) on June 4, 2007 under the following registration number.

‧ Lactobacillus kawaii OLL2836 strain: registration number BCRC 910351

‧ Lactobacillus kawaii OLL2948 strain: registration number BCRC 910353

‧ Lactobacillus mucosa OLL2848 strain: registration number BCRC 910352

The deposited OLL2836 strain, OLL2948 strain and OLL2848 strain, It has the properties shown in Table 1 below.

Lactobacilli MRS Broth in Table 1 (MRS medium; for example, Difco, Ref. No. 288160), is a medium which can be preferably used for culturing such lactic acid strains. Typical compositions of MRS medium are shown in Table 2.

The three strains shown in Table 1 are considered to have particularly high improvement ability of lactose intolerance, and can be particularly advantageously used in the present invention.

2. A lactose intolerance improving agent containing the lactic acid bacteria of the present invention

The lactic acid bacteria of the present invention exerts potent lactose-degrading enzyme activity by reaching the intestinal tract in a state of survival after oral administration, and as a result, lactose intolerance can be improved. Further, in the mash produced by the lactic acid bacteria of the present invention, the decomposition rate of lactose 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 lactose-degrading enzyme activity obtained by the above screening method. The lactose intolerance improving agent of the present invention has an effect of improving lactose intolerance by administering a test substance exhibiting lactose intolerance or suspected lactose intolerance, that is, reducing lactose intolerance The frequency of discomfort of digestive system symptoms (abdominal pain, diarrhea, abdominal hunger, etc.), or complete prevention of the disease, 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 (1 strain or more) of the lactic acid bacteria of the present invention. The lactose intolerance improving agent of the present invention preferably comprises, as a lactic acid bacterium of the present invention, a strain of Lactobacillus kawaii OLL2836 (Accession No. BCRC 910351), Lactobacillus kawaii OLL2948 strain (Accession No. BCRC 910353), and a mucosa. At least one selected from the group consisting of Lactobacillus OLL2848 strain (Accession No. BCRC 910352). 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 kawaii OLL2948 strain (Accession No. BCRC 910353), and Lactobacillus mucosa OLL2848 strain. Three lactic acid bacteria (registration number BCRC 910352). The lactose intolerance improving agent containing the three lactic acid bacteria can be combined with the intestinal mucin containing any of the blood group antigens of type A, type B, and type O, 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 of the present invention, or may be a yeast produced by using the lactic acid bacteria. , a culture, or a composition comprising such concentrates. The lactic acid bacteria of the present invention contained in the lactose intolerance improving agent of the present invention may be a living cell or a dead cell, and may be a moist bacteria or a dry bacteria. However, in a more preferred embodiment, the lactose intolerance improving agent of the present invention comprises a composition of the lactic acid bacteria of the present invention in a living state. The lactose intolerance improving agent of the present invention is not limited, and may be in any form such as a liquid, a gel, a powder, a granule, a solid, a capsule or a sheet.

By adding an appropriate amount of the lactose intolerance improving agent of the present invention, it is possible to impart a lactose intolerance improving effect to a beverage or food composition. The lactose intolerance improving agent of the present invention may contain a carrier or an additive which is acceptable for a pharmaceutical preparation, in addition to the lactic acid bacteria of the present invention or a culture thereof, a mash, or a concentrate thereof as an active ingredient. Examples of the carrier and the additive include organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, and carboxylic acid. Methyl starch sodium, pectin, sanxian gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, nectar Other than the sugar alcohol, sorbitol, carboxymethyl cellulose, a surfactant which can be used as a pharmaceutical additive, and the like, an artificial cell constituent such as a liposome or the like can be 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 (for example, intragastric administration or intragut administration), and it is particularly preferred to administer it orally. The lactose intolerance improving agent of the present invention administered orally may be a solid preparation such as a tablet, a granule, a powder, a pill or a capsule, a gel, or a liquid agent, and a suspension A dosage form such as a liquid preparation such as a float or a syrup. When it is used as a liquid preparation, it may be supplied, and it is intended to be a dried product which is re-dissolved when the lactose intolerance improving agent of the present invention is used.

In the above dosage form, the oral solid preparation may further contain an additive such as a pharmaceutically acceptable binder, an excipient, a lubricant, a disintegrator, a wetting agent or the like. Further, the oral liquid preparation may further contain an additive such as a stabilizer, a buffer, a flavoring agent, a preservative, a fragrance, a coloring agent or the like which is generally used in pharmaceutically.

The administration amount of the lactose intolerance improving agent of the present invention varies depending on the age and weight of the subject, the administration route, and the number of administrations, and the manufacturer can change it in a wider range by discretion. For example, in the case of oral administration, the administration amount of the lactic acid bacteria of the present invention contained in the lactose intolerance improving agent of the present invention is suitably 1 to 1000 mg/kg/day. The lactose intolerance improving agent of the present invention can be administered in a single administration, and can be repeatedly administered at intervals of 6 to 8 hours.

The subject to which the lactose intolerance improving agent of the present invention is administered is not limited, and is preferably a mammal including a human, a domestic animal, a pet, an experimental (test) animal, or the like. Further, as the subject of the present invention, it is preferred that the lactose intolerance or the lactose intolerance is a mammal in the early childhood, adulthood, and old age, and the lactose of the present invention is not administered as a drug. The subject of the resistance-improving agent is preferably a mammal having a decreased lactose-decomposing ability due to an increase in age or disease, or a mammal having a genetic cause or an environmental cause of lactose intolerance. The lactose intolerance improving agent of the present invention has less concern for side effects, and therefore can be used very effectively in terms of continuous use.

3. Beverage food containing the lactic acid bacteria of the present invention

The present invention also relates to a beverage food comprising a Lactobacillus lactic acid bacterium which is significantly enhanced by the intestinal adhesion and lactose-degrading enzyme activity obtained by the above screening method. 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 completely prevent the onset, or reduce the severity of the symptoms of lactose intolerance.

In the present invention, the "beverage food" is not limited, and the type of the beverage food containing the lactic acid bacteria of the present invention including beverages, foods, and functional foods is not particularly limited. For example, the beverage containing the lactic acid bacteria of the present invention may be exemplified by fermented milk (such as yoghurt), lactic acid bacteria beverage, milk beverage (coffee milk, fruit milk, etc.), tea beverage (green tea, black tea, oolong tea, etc.), and fruit. Vegetable-based beverages (beverages containing oranges, apples, grapes, etc., or vegetable juices such as tomatoes and carrots), alcoholic beverages (beer, sparkling wine, wine, etc.), carbonated drinks, and soft drinks. For the production methods of various beverages, reference may be made to existing reference books such as "Latest. Soft Drinks" (2003) (Guanglin Co., Ltd.) and the like.

The food containing the lactic acid bacteria of the present invention is not particularly limited, and may be a fresh food or a processed food. As a particularly good food, a lactic acid or a lactic acid bacteria beverage which allows the lactic acid bacteria of the present invention to reach the intestinal tract in a living state can be mentioned. The food containing the lactic acid bacteria of the present invention may be a dairy product such as yoghurt or cheese. Yeast for the manufacture of fermented milk.

Further, as a food and beverage containing the lactic acid bacteria of the present invention, it is particularly preferable Functional food. The "functional food" of the present invention means a food having a specific function for a living body, and includes, for example, a health-care functional food containing a specific health food (including a conditional special protection [specific health food]) and a nutritious functional food. So-called health foods such as special-purpose foods, nutritional supplements, health supplements, supplements (for example, tablets, coated tablets, sugar-coated tablets, capsules, and liquids) and beauty foods (for example, diet foods) All. The functional food of the present invention further includes a health food to which a health claim based on a food specification of Codex (FAO/WHO Contract Food Specification Committee) is applied.

As a more specific example of the functional food of the present invention, there are food for patients, pregnant women. Special-purpose foods for breastfeeding women, such as powdered milk, infant formula, and food for the elderly. The lactic acid bacteria of the invention slowly improve the intestinal environment by mild action, thereby improving the lactose decomposition ability in the intestinal tract, improving the lactose intolerance and reducing the symptoms, and therefore it is particularly good for treating weak physical strength. The use of milk powder or liquid-prepared milk for lactose intolerance in infants and young children (for example, it can be added to raw milk-derived milk raw materials), or for pregnant women or lactating women such as breast-feeding women Foods, and the use of foods for the elderly and foods for patients to treat lactose intolerance in elderly or patients with reduced physical strength.

As a preferred functional food of the present invention, a supplement for breast milk for treating a subject exhibiting lactose intolerance due to congenital or acquired causes, maternal maternal. Supplements for breastfeeding women, supplements for the elderly and supplements for patients.

As a preferred example of the functional food containing the lactic acid bacteria of the present invention, Health foods can be cited. The health-care function food system is designed not only for ordinary foods but also for foods in the form of tablets, capsules, and the like, in accordance with the domestic and international trends and the integration with the previous specific health food system. Under this system, health-care functional foods include two types of specific health foods (individually licensed) and nutritious functional foods (standard types). Further, it also includes novel types such as special conditions (special health foods).

In the functional food of the present invention, it is preferred that the lactic acid bacteria of the present invention are introduced into the intestine to fix the lactose-decomposing ability in the intestinal tract (preferably, continuous improvement), and as a result, lactose is not obtained. Tolerance improvement effect. The functional food of the present invention may be a first object for use in applications other than the improvement of lactose intolerance. The functional food of the present invention (preferably a specific health food or a conditional special protection [special health food]) may also reveal or show an increase in lactose decomposition ability in the intestinal tract, thereby improving lactose intolerance. The effect of the disease or its symptoms. Such disclosure or display may also recognize the display for the provisions set forth in the health-care food system. For example, in the functional food of the present invention, "suitable for a person who wants to adjust the abdomen", "a good condition for maintaining the abdomen", "adjusting the environment in the intestine", "suitable for a person who is not suitable for drinking milk", etc., may be considered, but Not limited to these.

The functional food of the present invention may be a solid preparation such as a tablet, a granule, a powder, a pill or a capsule, a liquid preparation such as a liquid preparation, a suspension preparation or a syrup preparation, or a preparation form such as a gel preparation, or may be a general beverage. The shape of the food (for example, fermented milk, beverages, powdered tea, snacks, etc.).

The amount of the lactic acid bacteria to be added of the present invention in the food or drink is not particularly limited, and It should be based on the situation. The specific addition amount can be appropriately determined after the manufacturer considers the type or the taste or taste of the beverage food. However, it is generally preferred that the total amount of the lactic acid bacteria of the present invention is 0.001 to 100% by weight, and particularly preferably 0.1 to 100% by weight.

The lactic acid bacteria of the present invention can be contained in a food or drink by any appropriate method available to the manufacturer. For example, the lactic acid bacteria of the present invention can be directly mixed into a raw material of a beverage or food. The lactic acid bacteria of the present invention may also be coated, covered, impregnated or sprayed onto beverages and foods. The lactic acid bacteria of the present invention can be uniformly dispersed into beverages and foods, and can also be unevenly dispersed in beverages and foods. A capsule or the like to which the lactic acid bacteria of the present invention are added may be prepared. The lactic acid bacteria of the present invention may also be coated with an edible film or an edible coating agent or the like. Further, in the lactic acid bacteria of the present invention, after adding an appropriate excipient or the like, it may be formed into a shape such as a tablet. The food or drink containing the lactic acid bacteria of the present invention may be further processed, and the above-mentioned processed products are also included in the scope of the present invention. Further, particularly preferred is a food or drink containing a chylomicron produced using the lactic acid bacteria of the present invention. In short, the beverage food 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 conventionally used in foods and drinks can also be used. The additive is not limited, and examples thereof include a color developer (such as sodium nitrite), a coloring material (such as xanthine pigment and red 102), a fragrance (such as orange flavor), and a sweetener (stevia and aspartame). Etc.), preservation materials (sodium acetate, sorbic acid, etc.), emulsifiers (sodium chondroitin sulfate, propylene glycol fatty acid esters, etc.), antioxidants (disodium edetate, vitamin C, etc.), pH adjusters ( Citric acid, etc., chemical seasoning (sodium inosine, etc.), tackifier (Sanxianjiao) Etc., a swelling agent (calcium carbonate or the like), an antifoaming agent (calcium phosphate), an adhesive (such as sodium polyphosphate), a nutrient enhancer (calcium enhancer, vitamin A, etc.), and the like. Further, functional raw materials such as Asian cockroach extract, Siberian cockroach extract, eucalyptus extract, and Eucommia tea extract may be further added.

The beverage food of the present invention comprises at least one (1 strain or more) of the lactic acid bacteria of the present invention. The beverage food of the present invention preferably contains, as the lactic acid bacteria of the present invention, Lactobacillus kawaii OLL2836 strain (Accession No. BCRC 910351), Lactobacillus kawaii OLL2948 strain (Accession No. BCRC 910353), and Lactobacillus mucosa OLL2848. At least one selected from the strain (registration number BCRC 910352). In a particularly preferred embodiment, the beverage food of the present invention comprises at least Lactobacillus kawaii OLL2836 strain (Accession No. BCRC 910351), Lactobacillus kawaii OLL2948 strain (Accession No. BCRC 910353), and Lactobacillus mucosa OLL2848 strain (registered number). BCRC 910352) three lactic acid bacteria. The beverage food containing the three lactic acid bacteria can be combined with intestinal mucin containing any blood type antigen of type A, B type, and O type, and thus is effective for a wide range of subjects.

The subject to be ingested or administered the beverage food of the present invention is not limited, but preferably includes humans, livestock (pig, horse, etc.), pets (dogs, cats, etc.), experimental (test) animals (mouse, large A mammal such as a rodent or a rabbit such as a rat. Further, as the subject of the present invention, it is preferred whether the lactose intolerance or the suspected lactose intolerance is a mammal in the early childhood, adulthood, or old age, and the present invention is ingested or administered. The test subject of beverages and foods is more preferably a mammal whose lactose decomposition can be decreased due to an increase in age or disease, or a genetic cause or environmental cause of lactose intolerance. mammal.

[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, the human-derived lactic acid strain provided by Meiji Dairy Co., Ltd., from the American American Type Culture Collection (ATCC), the Institute of Physical and Chemical Research, the Microbial System Preservation Facility (JCM), the Ministry of Agriculture, Forestry and Fisheries The lactic acid strain purchased from the field (NIAI) and the National Collections of Food Bacteria (NCFB) in the United Kingdom, and the lactic acid strain isolated from the child's stool by the inventors of the present invention using a modified LBS medium, totaled 104 strains. 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 OLL or MEP in the strain name indicates a strain retained by Meiji Dairy Co., Ltd.

The cells of each of the tested lactic acid bacteria were dispersed in a sterilized 10% (w/v) skim milk medium (Snow Printing Dairy Co., Ltd., Sapporo), and stored in a refrigerator at -80 ° C until use.

When cultured, each strain was cultured by MRS (Difco, Detroit, MI, USA) Subculture (37 ° C, 24 hours) 3 times, subculture (37 ° C, 24 hours) 2 times by MRSL medium (MRS medium in which 2% glucose was replaced by lactose). Then, the obtained bacterial cell culture solution was inoculated to 1% of the reconstituted MRSL medium, followed by incubation at 37 ° C for 18 hours. The thus prepared culture solution (6,000 × g, 20 minutes, 4 ° C) was centrifuged, and the cells were collected and washed with 0.05 M phosphate buffer (pH 6.8), and then suspended in distilled water, followed by Freeze drying.

[Example 2] Screening of intestinal adhesion strains

From the above-mentioned test lactic acid strain, a strain having a high intestinal adhesion (RU value of 100 or more) was screened using an analysis system developed by the inventors of the present invention for examining the binding ability to intestinal mucin. The analysis system used is also disclosed in detail in the international application WO2006/067940.

Specifically, biosensors BIACORE 1000 (BIACORE) was used for surface plasma resonance spectroscopy to determine lactic acid bacteria for human type A intestinal mucin, human B type intestinal mucin, and human O-type intestinal adhesion. Protein binding ability.

1. Preparation of analyte solution

First, each strain was subcultured three times by MRS medium after inoculation, and after incubation at 37 ° C for 12 hours, 500 μl was dispensed into a 1.5 ml volume tube. The culture solution was centrifuged (6,000 rpm, 4 ° C, 10 minutes), and the cells obtained by removing the supernatant were washed twice with PBS buffer (pH 7.2), and then suspended in distilled water. Freeze-dried. The cells were conditioned to 0.1 using HBS-EP buffer (0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20). The concentration of mg/ml was used to prepare a solution solution.

2. Preparation of human intestinal mucin (HCM) and preparation of HCM immobilization sensor

The human intestinal mucin is prepared and immobilized into a sensor sheet. Human type A intestine (from the large intestine of blood type A human body), human type B intestine (from the large intestine of blood type B human body), and human O type intestine (from the large intestine of blood type O type human body) From the Graduate School of Medicine, Graduate School of Tohoku University, Japan, samples were purchased as specimens. Furthermore, the sample was taken by an ethics committee of the Graduate School of Medicine, Tohoku University, Japan, and patient consent was obtained. In the normal part of the large intestine of these intestines, the mucin mucin layer was collected by surface scraping. The mucin mucin layer is obtained by Folch solvent (chloroform-methanol mixture (2:1 (v/v)); J. Folch et al.,: J. Biol. Chem. (1957) 226, p. 497-500) After degreasing with diethyl ether, it was dried and extracted with a 4 M guanidine hydrochloride solution at 37 ° C for 2 hours. The extract was then purified by gel filtration. Gel filtration purification based on human colorectal mucin revealed in Purushothaman SSet al, "Adherence of Shigella dysenteriae 1 to Human Colonic Mucin." Curr. Miorobiol., 42 (6), p. 381-387 (2001) The purification method is carried out. The moving layer was a 4 M guanidine hydrochloride solution, and the column for gel filtration chromatography was Toyopearl HW-65F (90 cm × 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 result was protein absorption (absorbance at 280 nm), and , choose the highest peak of neutral sugar content, and then, to condense The molecular weight of the gel filtration chromatography is about 2 million or more as a standard, and the large intestinal mucin fraction is separately extracted. The purified product thus obtained corresponds to the blood type derived from the human body, and is respectively designated as human type A intestinal mucin, human type B intestinal mucin, and human type O intestinal mucin. Hereinafter, in this human colon mucin (HCM), type A intestinal mucin is sometimes referred to as A-HCM, and type B intestinal mucin is referred to as B-HCM, and type O is used. The intestinal mucin is called O-HCM. For each of the obtained HCMs, the human blood group matrix antigenicity of the blood type was confirmed by an 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-dimethylaminopropyl) carbon. 50 μl of diimine hydrochloride (EDC) and 11.5 mg/ml of N-hydroxysuccinimide (NHS) 50 μl mixed reagent (EDC/NHS) to activate carboxyl groups in carboxymethyl dextran . Then, a mixed solution containing 30 μl of any of the above-purified purified HCM solutions (A-HCM, B-HCM, O-HCM) was prepared and poured into 120 μl of an 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 M ethanolamine hydrochloride-NaOH (pH 8.5) was used to block the residual survival group of the site 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 points after the introduction of EDC/NHS and the ethanolamine solution was set to 1000 to 2000 RU. The HCM immobilization sensor thus obtained is then used to dissolve the above analyte In the test of the liquid.

3. Surface plasma resonance spectrum analysis

Furthermore, the amount of binding of HCM on each HCM immobilization sensor to the lactic acid bacteria in the above analyte solution was analyzed using a biosensor BIACORE 1000 (BIACORE). Hereinafter, the measurement conditions of the BIACORE 1000 used for the analysis are shown.

Electrophoresis buffer: HBS-EP buffer (pH 7.4)

Addition amount of analyte solution (sample): 20 μl

Flow rate: 3 μl/min

Reaction temperature: 25 ° C

Regeneration solution: 1 μM hydrochloride solution 5 μl

In the analysis by the BIOACORE system, the amount of binding (interaction) between the analyte and the probe is expressed by a unit of resonance unit (RU). The so-called 1 resonance unit (RU) means that 1 pg of substance is bound per 1 mm ² on the surface of the sensor 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 mm ² . The amount of binding is equivalent to the ability of the lactic acid bacteria to bind to each intestinal mucin.

As a result of the analysis, among the 104 strains of the lactic acid bacteria to be tested, strains which showed a value of 100 RU or more as a binding ability to at least one intestinal mucin were selected. Among the selected strains, intestinal adhesion was considered to be enhanced compared with other average lactic acid strains (high intestinal adhesion). Representative examples of the strains to be screened and their analysis results are shown in Figs.

[Example 3] Synthesis and purification of ONPGlc-6P for measuring P-β-glc activity

Hereinafter, attempts are made to screen strains having high intestinal adhesion and having high lactase-degrading enzyme activity. Therefore, in the examples, first, ONPGlc-6P for measuring its activity was prepared.

1. Chemical synthesis of ONPGlc-6P

o-Nitrophenyl-β-D-glucopyranoside 6-phosphate; ONPGlc for the determination of phospho-β-glucosidase (P-β-glc) activity -6P), by the synthesis method of Hengstenberg and Morse (Hengstenberg, W. and MLMorse, Synthesis of o-Nitrophenyl-beta-D-galactopyranoside 6-phosphate; Carbohydr. Res, 10, pp. 463-465 (1969) )) The improved method for chemical synthesis. Specifically, first, 0.9 g of o-nitrophenyl-β-D-glucoside (ONPGlc, Sigma, St. Louis, USA) was dissolved in pre-ice and cooled, and then mixed with 54 μl of distilled water and 840 μl. Phosphorus chloroformate in trimethyl phosphate 7.5 ml. Then, the mixture was stirred on ice for 5 hours while allowing to react. After the reaction, borneol (distilled water) was added, and concentrated ammonia water 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

The above-mentioned reaction solution was concentrated and dried at 40 ° C using a rotary evaporator (Tokyo Science, Tokyo) 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 the chemically synthesized ONPGlc-6P and the unreacted ONPGlc were adsorbed to the activated carbon, and then 20 times. The distilled water (2 L) was washed and desalted. The elution of ONPGlc-6P adsorbed on activated carbon was carried out using a pyridine:water:ethanol mixture (1:1:1 (v/v), 600 ml). The eluate was repeatedly concentrated in a rotary evaporator at 40 ° C to remove pyridine. This was followed by filter paper chromatography (PPC). That is, the crude purified ONPGlc-6P was linearly coated on a PPC filter paper (Whatman, 3 MM, 46×57 cm, Maidstone, England) using a 100 μl micropipette, using butanol: pyridine: water (6). : 4:3 (v/v/v)) As a developing solvent, single development was carried out by a rising method. After the completion of the development for 2 days, the filter paper was dried in a ventilated chamber, and the ONPGlc-6P and ONPGlc tapes were confirmed using a UV ultraviolet light box (302 nm, Zhouyue, Tokyo), and only the ONPGlc-6P tape was cut. The cut filter paper was immersed in distilled water and stirred at 4 ° C overnight to extract ONPGlc-6P. After the extraction, the filter paper was removed, and the aqueous layer was concentrated by a rotary evaporator, and then the reaction was removed by gel filtration using a TOYOPEARL HW40S column (2.6 Φ × 90 cm, TOSOH, Tokyo) having distilled water as a mobile phase. by-product. After gel filtration, the ONPGlc-6P fraction in each of the eluted fractions was recovered using thin layer chromatography (TLC, Silicagel 60, 10 x 10 cm, Merck, Darmstadt, Germany). The developing solvent for TLC was subjected to single-stranding using butanol: 2-propanol: water (3:12:4 (v/v/v)). After air drying, a 5% (v/v) sulfuric acid-methanol solution was uniformly sprayed on a thin plate and heated in a desiccator (Yamato, Tokyo) heated to 150 ° C for 3 minutes for detection. The recovered ONPGlc-6P fraction was freeze-dried to prepare purified ONPGlc-6P. The purity of the purified product was confirmed by TLC, and the structure was confirmed by ¹ H-NMR.

[Example 4] Determination of β-gal, P-β-gal and P-β-glc activities

With respect to 104 strains of the test lactic acid bacteria prepared in Example 1, the lactase activity of β-gal, P-β-gal, and P-β-glc was measured using the Fisher method. That is, for each strain, 0.5 mg of the freeze-dried cells were suspended in 1.0 ml of 0.05 M phosphate buffer (pH 6.8), and 50 μl of a toluene-acetone mixture (1:9 (v/v)) was added, which was intense. Stir for 3 minutes. In 25 μl of this suspension, o-nitrophenyl-β-D-galactopyranoside (ONPGal), o-nitrophenyl-β-N-galactopyranoside-6-phosphate (ONPGal-) was added. 6P) or 100 μl of 0.05 M phosphate buffer (pH 6.8) of o-nitrobenzene-β-D-glucoside-6-phosphate (ONPGlc-6P) prepared above, and reacted at 37 ° C for 15 minutes. After the reaction, 125 μl of a 0.5 M sodium carbonate solution was added to stop the reaction, and the bacterial components were removed by centrifugation (6,000 × rpm, 10 minutes, 4 ° C) using Multiskan MS-UV (Labsystems, Helsinki, Finland). The absorbance of the supernatant at 405 nm was measured. 1 μmol of free o-nitrophenol (ONP) in 1 minute was defined as 1 unit, and the activity value per 1 mg of the cells and the protein contained in 1 mg of the culture supernatant were calculated as the unit. Activity value.

Further, the protein was quantified by using a BCA method based Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, Illinois, USA), and after 2 hours of reaction, the absorbance at 540 nm was measured using Multiskan MS-UV. . The calibration curve was prepared using bovine serum albumin (BSA), and the amount of protein was calculated here. Fig. 1 to Fig. 3 show the results of measuring the activity of the lactose-degrading enzyme of the representative example of the strain selected in Example 2.

[Example 5] Screening of lactic acid strain

According to the results of determination of intestinal adhesion and β-gal, P-β-gal and P-β-glc activities indicated in the above examples, screening of lactic acid strains having high intestinal adhesion and high lactose-degrading enzyme activity . As a result, three strains of lactic acid bacteria were selected as strains having high intestinal adhesion and showing significantly higher activity against any of the lactose-degrading enzymes of β-gal, P-β-gal, and P-β-glc. Strains (OLL 2836 strain, OLL 2948 strain, and OLL 2848 strain) (Figs. 1 to 3). On the other hand, most of the lactic acid strains with high lactose-degrading enzyme activity have low intestinal adhesion (for the human intestinal mucin of type A, B, and O, the RU value is less than 100 RU). ), therefore, the results were excluded.

As shown in Figures 1-3, the OLL2836 strain represents P-β-gal activity of 46.576 units/mg protein, the OLL2948 strain represents P-β-glc activity of 50.194 units/mg protein, and the OLL2848 strain represents 107.090 units/mg of protein β. -gal activity, but these activity values are very high values even compared to other lactic acid strains having high intestinal adhesion. Moreover, these strains also showed very high values with respect to the RU value indicating intestinal adhesion. Specifically, OLL2836 strain showed a particularly strong binding ability to human B-type intestinal mucin and human O-type intestinal mucin, and OLL2948 strain and OLL2828 strain showed particularly strong human A-type intestinal mucin. Binding ability.

From the above results, it is shown that, in addition to the effect of separately decomposing lactose in the intestinal tract, it is also possible to use the 3 strains in combination with the lactic acid strains in the test body of any blood type. A higher lactose decomposition effect can also be obtained.

Furthermore, strain identification tests were carried out on OLL2836 strain, OLL2948 strain and OLL2848 strain, and OLL2836 strain and OLL2948 strain were identified as strains belonging to Lactobacillus gasseri, and Lactobacillus mucosae was identified as a strain of LOLtobacillus mucosae. Strain (see the examples below). With regard to the three kinds of lactic acid strains, on June 9, 2006 (original storage day), the application was filed with the Patent Microbiology Depository Center of the Independent Evaluation Corporation's Product Evaluation Technology Foundation (2-5-8, Kishozu City, Chiba Prefecture, Japan) And was accepted for registration. 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 registration numbers are: OLY 2836 is NITE BP-241, OLL 2948 is NITE BP-242, and OLL 2848 is NITE BP-243. 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 was BCRC 910351, the registration number of the OLL2948 strain was BCRC 910353, and the registration number of the OLL2848 strain. For 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 a high lactose-degrading enzyme activity can be obtained. Further, the activity of lactose-degrading enzyme was measured for β-gal, P-β-gal, and P-β-glc, and the activities of the three lactose-degrading enzymes were significantly higher. The strain, by combining the obtained strains, can also 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 lactose in the intestinal tract, and is effective for improving lactose intolerance.

[Example 6] Identification of strains

The strains of the three lactic acid strains OLL2836, OLL2948 and OLL2848 screened in Example 5 were identified. Identification is based on amplification based on the V3 region. The sequence analysis of the 16S rDNA base sequence was carried out. Hereinafter, as an example, an identification test performed in the OLL2848 strain and the OLL2948 strain was revealed, and the OLL2836 strain was also identified in substantially the same manner.

1. Detection of 16S rDNA fragments directly by PCR method

The OLL2848 strain and the OLL2948 strain were subjected to subculture by MRS medium as described above, and then PCR was directly performed. In a microcapillary tube of 0.6 ml volume, 4 μl 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), 10× PCR buffer, dNTP Mix (TaKaRa, Kyoto), and a set of primers were added to prepare a PCR reaction solution. As a set of primers, universal primers 27F (5'-GAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 1), and 518R (5'-ATTACCGCGGCTGCTGG-3', 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 ° C1 Minutes] are performed as one cycle.

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 1×TBE buffer (90 mM triboric acid, 2 mM EDTA, pH 8.0) at a constant voltage of 100 V. The swimming device used Mupid-2 (COSMOBIO, Tokyo). The molecular weight marker was 100b DNA ladder (TaKaRa, Kyoto). Gel staining after electrophoresis was carried out by immersion in an ethidium bromide (EB) solution (0.5 μg/ml) for 10 minutes.

3. DNA extraction from agarose gel

The gel was electrophoresed on the agarose gel electrophoresis and the gel after staining with a UV-UV light box TRANSILLMINATOR TM-20 (Australia Pharmaceutical Co., Ltd., Tokyo), and the target DNA fragment was confirmed and cut. The DNA was extracted by a DNA fragment purification kit MagExtractor (TOYOBO, Osaka). Namely, after the cut agarose gel was transferred to a 1.5 ml volume tube, 400 μl of the adsorption solution was added, and the mixture was heated to 55 ° C to completely dissolve. Then, 15 μl of magnetic beads were added to the reaction mixture, and the mixture was allowed to stand at room temperature for 1 minute. After centrifugation (6,000 rpm, 5 seconds, 4 ° C), the supernatant was removed, and 500 μl of the washing solution was added and stirred. After centrifugation (6,000 rpm, 5 seconds, 4 ° C), 1 ml of 75% ethanol was added and stirred, and centrifugation (15,000 rpm, 1 minute, 4 ° C) was carried out, and then the supernatant was removed. After 15 μl of distilled water was added and stirred, the supernatant was recovered by centrifugation (15,000 rpm, 1 minute, 4 ° C) as a purified DNA solution.

4. DNA sequence determination and homology analysis

Use primer 27F (5'-GAGTTTGATCCTGGCTCAG-3', sequence No. 3), the base sequence of the purified DNA was determined. Base sequence determination was performed by Operon Biotechnology Co., Ltd. Thus, 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 the base sequences by the program BLASTN.

As a result, the base sequence of the V3 region of the OLL2848 strain showed 99.6% homology with the sequence of the V3 region of Lactobacillus mucosae, and on the other hand, the base sequence of the V3 region of the OLL2948 strain. It showed 99.2% homology with the sequence of the V3 region of Lactobacillus gasseri. From this situation, it can be identified that the OLL2848 strain belongs to the lactic acid bacteria of Lactobacillus mucinae, and the OLL2948 strain belongs to the lactic acid bacteria of Lactobacillus gasseri.

[Industrial availability]

The method of the present invention can be used to effectively screen lactic acid strains useful for improving lactose intolerance. 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 or the like which can continuously improve lactose intolerance.

[sequence table free content]

The sequences of SEQ ID NOs: 1 to 3 are primers.

The entire disclosures of all publications, patents and patent applications cited in this specification are hereby incorporated by reference.

<110> Food Research Society of the National Corporation Corporation 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> PatentIn 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

<210> 2

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial Sequence Type: Primer

<400> 2

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial Sequence Type: Primer

<400> 3

<210> 4

<211> 559

<212> DNA

<213> Lactobacillus mucosa

<400> 4

<210> 5

<211> 524

<212> DNA

<213> Lactobacillus

<400> 5

Fig. 1 shows the results of a P-β-gal activity test for a representative strain having a high intestinal adhesion.

Fig. 2 shows the results of a P-β-glc activity test for a representative strain having a high intestinal adhesion.

Fig. 3 shows the results of a β-gal activity test for a representative strain having a high intestinal adhesion.

(no component symbol description)

Claims (6)

A lactic acid bacterium which is enhanced by intestinal adhesion and lactose-degrading enzyme activity, which is Lactobacillus gasseri OLL2836 strain (registered number BCRC 910351), Lactobacillus kawaii OLL2948 strain (registered number BCRC 910353), and Lactobacillus mucosa OLL2848 strain ( Any of the registration numbers BCRC 910352). A lactose intolerance improving agent comprising at least one lactic acid bacterium according to claim 1. The lactose intolerance improving agent according to claim 2, which comprises Lactobacillus kawaii OLL2836 strain (registered number BCRC 910351), Lactobacillus kawaii OLL2948 strain (registered number BCRC 910353), and Lactobacillus mucosa OLL2848 strain (registered number BCRC) 910352) three kinds of lactic acid bacteria. A beverage food comprising at least one lactic acid bacterium according to claim 1. The beverage food of claim 4, which comprises at least Lactobacillus kawaii OLL2836 strain (Accession No. BCRC 910351), Lactobacillus kawaii OLL2948 strain (Accession No. BCRC 910353), and Lactobacillus mucosa OLL2848 strain (Accession No. BCRC 910352). Three lactic acid bacteria. The beverage food according to claim 4 or 5, which is in the group consisting of baby food, baby food, breastfeeding women's food, aged food, patient food, health functional food, supplement, fermented milk and lactic acid bacteria beverage. Selector.