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WO2006067940A1 - Bacteries de l’ acide lactique se liant a des types sanguins abo humains - Google Patents

Bacteries de l’ acide lactique se liant a des types sanguins abo humains Download PDF

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Publication number
WO2006067940A1
WO2006067940A1 PCT/JP2005/022096 JP2005022096W WO2006067940A1 WO 2006067940 A1 WO2006067940 A1 WO 2006067940A1 JP 2005022096 W JP2005022096 W JP 2005022096W WO 2006067940 A1 WO2006067940 A1 WO 2006067940A1
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Prior art keywords
lactic acid
type
human
acid bacteria
blood group
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PCT/JP2005/022096
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English (en)
Japanese (ja)
Inventor
Tadao Saito
Yasushi Kawai
Hideaki Uchida
Katsunori Kimura
Kakuhei Isawa
Keisuke Furuichi
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Meiji Dairies Corp
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Meiji Dairies Corp
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Priority claimed from PCT/JP2005/011043 external-priority patent/WO2006059408A1/fr
Application filed by Meiji Dairies Corp filed Critical Meiji Dairies Corp
Priority to CA2588940A priority Critical patent/CA2588940C/fr
Priority to JP2006548758A priority patent/JP4738348B2/ja
Priority to US11/720,462 priority patent/US7897374B2/en
Publication of WO2006067940A1 publication Critical patent/WO2006067940A1/fr
Anticipated expiration legal-status Critical
Priority to US12/987,518 priority patent/US8465933B2/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to lactic acid bacteria and screening of lactic acid bacteria.
  • the human intestinal tract is inhabited by approximately 200 types of intestinal bacteria (intestinal resident microorganisms).
  • Microorganisms called probiotics improve the balance between useful and harmful bacteria in the human intestine and contribute to the health of the host.
  • the binding mechanism of lactic acid bacteria in the human intestine has not yet been elucidated. From previous studies on intestinal lactic acid bacteria, it has been confirmed that L. casei has binding properties to glycolipid sugar chains, and L. reuteri and L. cri spatus have binding properties to collagen. Furthermore, a lectin-like protein that binds to the intestinal lactobacilli has been identified.
  • Intestinal mucin is a mucous high molecular weight glycoprotein having an infinite number of mucin-type sugar chains linked to a polypeptide (core protein, apomucin) through a 0-glycoside bond.
  • intestinal settled lactic acid bacteria acquire intestinal connectivity by binding to the sugar chains of intestinal mucin through lectin-like proteins on the surface of the bacteria, and are considered to achieve stable growth. available.
  • Human ABO blood groups are distinguished by the type of antigenic substance expressed on the surface of erythrocytes.
  • the antigenic site of this ABO blood group substance is a sugar chain having a specific chemical structure (ABO blood group antigen).
  • ABO blood group antigen Both type A and type B antigens are molecules composed of three sugars, and a -N-acetylethyl latatosamine is bound to a basic structure called a type H antigen composed of two sugars in a specific binding mode. Is type A antigen and ⁇ -galactose is bound to type B antigen.
  • Type IV blood Humans of type IV blood express the type A antigen
  • humans of type B blood express the type B antigen
  • humans of type AB blood express both the type A antigen and the type B antigen on the erythrocyte surface.
  • type 0 blood humans express H-type antigens of the basic structure.
  • This method is an epoch-making method in which the adsorptive power between lactic acid bacteria and ABO blood group antigens is detected using surface plasmon resonance spectrum (SPR), and lactic acid bacteria that are compatible with the blood type are selected. Specifically, when ABO blood group antigen or intestinal mucin is used as a ligand and lactic acid bacteria are brought into contact with the ligand immobilized on the sensor chip, the binding between the lactic acid bacteria and the ligand is accompanied by the binding. This is a method to detect the mass change that occurs on the sensor chip as a surface plasmon resonance (SPR) signal.
  • SPR surface plasmon resonance
  • the present inventors have carried out the above method and confirmed that the Lactobacillus crispatus JCM8778 strain and the Lact obacillus acidophilus OLL2769 strain are A-type antigen recognizable (Patent Document Non-Patent Document 5).
  • probiotic lactic acid bacteria including yogurt Because of the expected increase in demand for foods that use blood, we have been waiting for the acquisition of blood group-binding lactic acid bacteria with even better binding properties!
  • Patent Document 1 JP 2004-101249 A
  • Non-Patent Document 1 Junko Amano, Biochemistry, Japan Biochemical Society, 1999, No. 71, p.274 -277
  • Patent Document 3 Holgersson, J., Jovall, PA, and Breimer, ME, Glycosphingolipids of human large intenstine: detailed structural characterization with special reference to blood group compounds and bacterial receptor structures.J. Biochem, (Tokyo), 110, 120 -131 (1991).
  • Non-Patent Document 4 Vanak, J., Ehrmann, J., Drimalova, D., Nemec, M., monoclonal antibo dies in the detection of blood group antigens A and B in the mucosa of the large inte stine.Cas Lek Cesk , 18, 364-367 (1988).
  • Non-Patent Document 5 Uchida, H. et al, Biosci. Biotechnol. Biochem., 68 (5), 1004-1010 (20 04).
  • Patent Document 6 Holmes, S. D. et al, Studies on the interaction of Staphylococcus aure us and Staphylococcus epidermidis with fibronectin using surface plasmon resonance (BIACORE). J. Microbiological Methods, 28, 77-84 (1997).
  • Non-Patent Document 7 Fratamico, P. M. et al, Detection of Escherichia coli 0157: H7 using a surface plasmon resonance biosensor. Biotechnol. Techniques., 7, 571-576 (1998). Disclosure of the Invention
  • the present invention has been made in view of such circumstances, and the problem to be solved by the present invention is to find a novel intestinal-binding lactic acid bacterium suitable for human ABO blood group.
  • Means for solving the problem [0009]
  • the inventors of the present invention who have solved the above-mentioned problems have implemented the lactic acid bacteria screening method based on the surface plasmon resonance spectrum described above. Although the above method has already been established as a screening method for lactic acid bacteria, an attempt has been made by the present inventors to set a “selection reference value” in the screening method for lactic acid bacteria in order to make it suitable for large-scale screening.
  • Non-patent document 6 Examples of detection using protein A or protein G immobilized on a chip via an antibody (Non-Patent Document 7) are known.
  • the RU value indicating the binding of viable cells is about 100 It is 1000R U.
  • examples of lactic acid bacteria studied by surface plasmon resonance spectrum are not known except for reports by the present inventors (Patent Document 1, Non-Patent Document 5).
  • the present inventors established a screening method for lactic acid bacteria compatible with the ABO blood group by using 100 RU as a criterion for bacterial binding under certain conditions.
  • the present inventors conducted the screening method described above for 238 strains of lactic acid bacteria of human intestinal origin isolated from the human intestinal tract, and further conducted a test for judging suitability for yogurt production. Finally, We specifically found strains compatible with blood group A and type 0. That is, the present invention relates to a lactic acid bacterium suitable for blood group compatible yogurt and a method for screening the lactic acid bacterium, and specifically provides the following.
  • Lactobacillus gasseri lactic acid bacteria capable of binding to the human ABO blood group antigen represented by any one of the following formulas (a) to (c)
  • RU value that indicates the ability to bind human ABO blood group antigen of type A is 100RU or more
  • the RU value indicating the ability to bind to human ABO blood group antigens of type B and Z or H is 100RU or less
  • the RU value indicating the ability to bind to human type A intestinal mucin is 100 RU or more
  • the RU value that indicates the ability to bind B-type human ABO blood group antigen is 100RU or more
  • the RU value indicating the ability to bind to human ABO blood group antigens of type A and Z or H is 100RU or less
  • the RU value that indicates the ability to bind human B-type intestinal mucin is 100 RU or more
  • the RU value that indicates the ability to bind H-type human ABO blood group antigen is 100RU or more
  • the RU value indicating the ability to bind human type 0 intestinal mucin must be 100 RU or more.
  • the RU value that indicates the ability to bind B-type human ABO blood group antigen is 100RU or more
  • the RU value which indicates the ability to bind human ABO blood group antigens of type A and H, is 100RU or less
  • the RU value indicating the ability to bind to human type B intestinal mucin must be greater than the RU value indicating the ability to bind to human type A intestinal mucin and the RU value indicating the ability to bind to human type 0 intestinal mucin.
  • Fig. 1 is a diagram showing the chemical structure of a Pioti-Louis polymer (BP) probe having a sugar chain antigen site of human ABO blood group.
  • BP Pioti-Louis polymer
  • FIG. 2 Diagram showing the sugar chain part of the A-type antigen BP-probe (top), the B-type antigen BP-probe sugar chain part (middle), and the type 0 antigen BP-probe sugar chain part (bottom) is there.
  • the present invention comprises (a) [GalNAc a 1-3 (Fuc a 1-2) Gal-], (b) [Gal a 1-3 (Fuc a 1-2) Gal-], (c) An intestinal-binding Lactobacillus gasseri lactic acid bacterium having binding ability to a human ABO blood group antigen represented by any formula of [Fucal-2Ga a] is provided.
  • lactic acid bacteria are a general term for a group of bacteria that produce glucose strength lactic acid in a molar ratio of 50% or more.
  • O Lactobacillus genus, Lactococcus rot, Streptococcus moth, Leuconostoc moth are representative genus of lactic acid bacteria. is there.
  • the genus Bifidobacterium is also included in the lactic acid bacteria in this specification.
  • the genus Lactobacillus is further classified into species. As a typical example of the species of the genus Lactobacillus, Lactobacillus delbruekn suosp. Bulgancus (and bulgaricus), Lactobacillus de lbruekn subsp.
  • Delbruekii (L. Delbruekii), Lactobacillus acidophilus Group ⁇ ⁇ flop lactic acid bacteria (L. acidophilus Gunore 1 ⁇ ⁇ -flops), Lactobacillus casei (L. casei), Lactobacillus plantarum (L. plantarum.), Lactobacillus brevis (L. brevis), Lactobacillus buchneri (L. buchneri), Lactobacillus fermentum (L. fermentum), Lactobacillus helveticus (L. helveticus) Can be mentioned. L.
  • Lactobacillus acidophilus group lactic acid bacteria are classified into Lactobacillus acidophilus (A-1), Lactobacillus cnspatus (A-2), Lactobacillus amylovorus ( ⁇ -3), Lactobacillus gallinarum (A-4) according to the results of DNA-DNA homology and cell wall composition analysis. ), L actobacillus gasseri (B-1), and Lactobacillus johnsonii (B-2).
  • Lactobacillus gasseri (hereinafter abbreviated as "L. gasserij”) lactic acid bacteria of the present invention is an intestinal tract Lactobacillus gasseri characterized by having a binding ability to a human ABO blood group antigen.
  • ABO human blood group antigen is a sugar chain that determines blood group Specifically, type A sugar chain (type A antigen): [GalNAc a 1-3 (Fuc a 1-2) Gal-], type B sugar chain (type B antigen): [Gal a 1-3 ( Fuc a 1-2) Gal-], O-type sugar chain (hereinafter referred to as a sugar chain that determines blood group 0, also referred to as H-type antigen, 0-type antigen): [Fuc a to 2Ga].
  • type A sugar chain type A antigen
  • type B sugar chain type B antigen
  • O-type sugar chain hereinafter referred to as a sugar chain that determines blood group 0, also referred to as H-type antigen, 0-type antigen
  • intestinal mucins present on the surface of the intestinal tract have different sugar chains depending on the ABO blood group. The present inventors confirmed that there is L.
  • gasseri lactic acid bacteria that bind to type A sugar chains which are antigens that determine human blood group A, among lactic acid bacteria that bind to mucin prepared for human intestinal force of type A did. It was also confirmed that type A sugar chains were expressed in the mucin. Therefore, in the L. gasseri lactic acid bacterium of the present invention, the lactic acid bacterium that binds to the A-type sugar chain and does not adsorb to other types of sugar chains binds to the A-type sugar chain on the intestinal mucin, whereby the ABO blood group It has acquired intestinal connectivity for type A humans.
  • the L. gasseri lactic acid bacteria that binds to type A sugar chains which are antigens that determine human blood group A, among lactic acid bacteria that bind to mucin prepared for human intestinal force of type A did. It was also confirmed that type A sugar chains were expressed in the mucin. Therefore, in the L. gasseri lactic acid bacterium of the present invention, the lactic acid bacterium
  • gasseri lactic acid bacterium is considered to stably bind to and proliferate in the intestinal tract of blood group A humans and exert a probiotic function. That is, it can be said that the L. gasseri lactic acid bacteria can contribute to the health of blood group A humans and is suitable for blood group A humans.
  • the L. gasseri lactic acid bacterium of the present invention that binds to the B-type sugar chain and does not adsorb to other types of sugar chains is compatible with ABO blood group B humans.
  • the L. gasseri lactic acid bacterium of the present invention that binds to the type 0 sugar chain and does not adsorb to other types of sugar chains is suitable for ABO blood group 0 type humans.
  • the L. gasseri lactic acid bacteria of the present invention (hereinafter sometimes referred to as "blood group-specific L. gasseri lactic acid bacteria”) can be separated from human feces. If it is L. gasseri lactic acid bacteria (hereinafter abbreviated as "A-type compatible lactic acid bacteria”) compatible with blood group A humans, it is compatible with humans of blood group B from ABO blood group A human feces L. gasseri lactic acid bacteria (hereinafter abbreviated as “B-type compatible lactic acid bacteria”) from ABO blood type B human feces to L.
  • A-type compatible lactic acid bacteria L. gasseri lactic acid bacteria
  • gasseri lactobacilli compatible with blood type 0 humans (Hereinafter abbreviated as “type 0 compatible lactic acid bacteria”)
  • type 0 compatible lactic acid bacteria the properties of L. gasseri lactic acid bacteria well known to those skilled in the art can be used as a guide.
  • gonococcus, homo-fermentation, aerobic growth, no gas generation, etc. can be used as a guide.
  • any medium suitable for culturing Lactobacillus is generally used.
  • a medium containing a nitrogen source such as yeast extratate, casein, or whey protein, and inorganic nutrients such as magnesium sulfate, iron sulfate, or manganese sulfate can be used.
  • a medium containing a nitrogen source such as yeast extratate, casein, or whey protein, and inorganic nutrients such as magnesium sulfate, iron sulfate, or manganese sulfate can be used.
  • Lactobacilli MRS broth Disifco, Re
  • the culture conditions are not particularly limited as long as the intestinal lactic acid bacteria can grow.
  • Preferred conditions are, for example, pH 5.0-pH 8.0, temperature 20 ° C-45 ° C, and more preferred conditions are anaerobic, pH 5.0-pH 7.0, temperature 30 ° C-40. ° C.
  • L. gasseri separated and cultured as described above has intestinal tract-binding properties and blood group antigens by blood group is determined by binding to human intestinal mucin or blood group antigens. It can be known by judging the presence or absence of sex.
  • a cell surface protein SLP
  • SLP cell surface protein
  • detection may be performed by a hybridization method after electrophoresis of SLP of a test bacterium using labeled intestinal mucin or labeled blood group antigen.
  • a surface plasmon analyzer for example, BIACORE
  • the surface part of the human intestinal force of a specific blood type is also collected and subjected to gel filtration using a solubilizing agent such as guanidine hydrochloride to reduce protein absorption.
  • Purification can be carried out based on the high neutral sugar content. For example, the method described in Purushothaman, b. B. Et al, Adherence of Shigella dysentenae 1 to Human colonic Mucin. Curr. Ic robiol, 42 (6), 381-387 (2001). It is more preferable to confirm that the blood group antigen is expressed in the prepared human intestinal mucin using an anti-blood group antigen antibody. Specific examples include the methods described in the examples.
  • blood group antigens can be synthesized on the basis of the sugar chain sequence shown in the formula below or in FIG. 2 (eg, commercially available sugar chain probes (eg, manufactured by Seikagaku Corporation) or neoglycoprotein 'Blood Group A Trisaccaride- BSA (eg Calbiochem) or neoglycoprotein 'Blood Group B Trisaccaride- BSA (eg Calbiochem) may be used.
  • commercially available sugar chain probes eg, manufactured by Seikagaku Corporation
  • neoglycoprotein 'Blood Group A Trisaccaride- BSA eg Calbiochem
  • neoglycoprotein 'Blood Group B Trisaccaride- BSA eg Calbiochem
  • accession number: NITE BP- Lactic acid bacteria identified by 26 and accession number: NITE BP-27 are type A compatible lactic acid bacteria
  • lactic acid bacteria specified by accession number: NITE BP-25 and accession number: NITE BP-28 are type 0 compatible lactic acid bacteria.
  • These strains are lactic acid bacteria that have been confirmed by the present inventors to be compatible with the above-mentioned blood types.
  • L. gasseri lactic acid bacteria according to blood group of the present invention include Lactobacillus gasser i specified by accession number: NITE BP-145 and accession number: NITE BP-146.
  • the lactic acid bacteria specified by the accession number: NITE BP-145 and the accession number: NITE BP-146 are B-type compatible lactic acid bacteria. These strains were confirmed by the present inventors to be compatible with blood group B.
  • the present inventors deposited these strains with the Patent Microorganism Depositary Center, National Institute of Product Evaluation Technology, based on the Budapest Treaty concerning the international recognition of the deposit of microorganisms in the patent procedure. The contents specifying the deposit are described below.
  • Lactobacillus gasseri OLL2915 strain (Accession number NITE BP-25)
  • Lactobacillus gasseri OLL2804 strain (Accession number NITE BP-26)
  • Lactobacillus gasseri OLL2818 strain (Accession number NITE BP-27)
  • Lactobacillus gasseri OLL2827 strain (Accession number NITE BP-28)
  • Accession numbers NITE BP-145 and 146 are as follows.
  • Lactobacillus gasseri OLL2877 strain (Accession number MTE BP-145)
  • Lactobacillus gasseri OL 2901 strain (Accession number MTE BP-146)
  • the gasseri lactic acid bacterium according to the blood type of the present invention can be used for the production of foods and drinks suitable for each blood type.
  • the foods and beverages produced by using gasseri lactic acid bacteria according to the present invention may be functional foods, specific health foods, health foods, and nursing foods that are not restricted in categories or types. It may be a dairy product such as cheese or yogurt, a seasoning or the like. There are no restrictions on the shape of the food or drink, and any form of food or drink that can normally be distributed can be used, such as solid, liquid, liquid food, jelly, tablet, granule, and capsule. Manufacture of the said food-drinks can be performed by those skilled in the art.
  • the gasseri lactic acid bacterium according to the blood type of the present invention can be mixed with general foods and drinks, and can be used as a starter for producing dairy products and fermented milk such as yogurt and cheese. .
  • a starter as long as there is no hindrance to growth and dairy product production, the presence of L. gasseri? Good.
  • it may be mixed with Lacto bacillus delbruek subsp.bulgancus, Streptococcus thermophilus, Lactobacillus aci dophilus, etc. It can be mixed to make a starter.
  • Yogurt production using the above starter can be carried out according to a conventional method.
  • plain milk dart can be produced by mixing the above starter with milk or dairy products cooled after warming 'mixing' and 'homogenizing' and sterilizing, followed by fermentation and cooling.
  • the food and drink produced by using the gasseri lactic acid bacterium according to the present invention according to blood type contains the same bacterium in the food and drink.
  • this food or drink is ingested by a human who is compatible with the AB0 blood group, the gasseri lactic acid bacteria bind and grow in the intestinal tract according to the blood type of the present invention, and the intestinal balance is adjusted and maintained. Early take-out function is expected. Therefore, the
  • the foods and drinks are useful not only as general foods and drinks but also as functional foods for the purpose of promoting health, such as “health and nutrition foods”, “care foods” and “health foods”.
  • the present invention also provides a method for screening lactic acid bacteria by surface plasmon resonance spectrum.
  • the screening method of the present invention is a method for selecting lactic acid bacteria suitable for each blood group.
  • the screening method of the present invention comprises screening by measuring the binding of lactic acid bacteria to blood type human intestinal mucin and ABO blood group antigens by surface plasmon resonance spectrum, and (i) specific blood (Ii) ABO blood group antigens other than the specific blood group in (i) above and a certain amount or more of adsorbing ability. (Iii) Selection is made using as an index the ability to bind to a specific blood group of human intestinal mucin in (i) above.
  • human type A intestinal mucin refers to type A human intestinal mucin of the ABO blood group.
  • human type B intestinal mucin is a blood group B human-derived intestinal mucin
  • human type 0 intestinal mucin is a blood group 0 human intestinal mucin.
  • surface plasmon resonance spectrum analysis is performed using ABO blood group antigen and human intestinal mucin as a probe.
  • ABO blood group antigen and human intestinal mucin can be used as a probe.
  • BIACORE Biacore Co., Ltd.
  • the preparation method of ABO blood group antigen and human intestinal mucin is as described above.
  • immobilizing the probe it can be performed by a known immobilization method.
  • the immobilization method may be a physical adsorption method or a covalent bond adsorption.
  • the chip is coated with streptavidin and the probe is piotinated, it can be easily fixed by a piotine-avidin bond. You can also use a commercially available streptavidinized chip (BIACORE)! /.
  • the “binding ability” is represented by a resonance unit (RU). 1RU is, to 1mm 2 of lpg substance binding child Represents.
  • the “binding ability of a certain amount or more” is determined based on “100RU”. That is, it is determined that a certain amount or more of lactic acid bacteria binds to the probe based on a measured value of “100RU” or more. For example, if the measurement result using a blood group A human-derived intestinal mucin as a probe is 100 RU or more, the lactic acid bacterium is a lactobacillus that binds to the intestinal mucin.
  • the RU value can vary depending on the measurement conditions.
  • the temperature condition in this method is, for example, 20-40 ° C, preferably 20-30 ° C, more preferably 23-28 ° C.
  • the preferred sample concentration in this method is 0.1-0.5 mg / mL, and the preferred flow rate in this method is 3-1 ⁇ / zl / min. If it is within the above range, the RU value will not change even if the above conditions are changed. Furthermore, even when various conditions such as sample concentration are changed outside the above range, they are included in “100RU” in this method if they are substantially equivalent to 100RU under the above conditions.
  • selection may be made by a value other than “100RU” for the purpose of making the selection criteria stricter.
  • a bacterium having a higher RU value means a bacterium having a stronger ability to bind to a probe (ligand). Therefore, for the purpose of finding bacteria with stronger binding power, it is possible to screen for bacteria that bind to ABO blood group antigen probes with RU values higher than 100! For example, screening may be performed based on 2000 RU as in the examples described later, which may be 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 RU. Conversely, for recognition of blood types that are not intended, screening may be based on numerical values that are stricter than "100RU or less". For example, 90,80,70,60,50RU may be used as a standard.
  • the blood group-binding lactic acid bacteria were measured when the ABO blood group probe was used as the measured value (RU) when using the blood type human intestinal mucin as a probe according to the screening method of the present invention. Expected to be lower than the value (RU). This is because blood group antigens (sugar chains) bound to human intestinal mucins when human intestinal mucins are immobilized on a chip are compared only with blood group antigens (sugar chains) on a chip unit area. This is considered to be the force that is reduced when fixed.
  • the measured value when using a specific ABO blood group probe is sufficiently high compared to the measured value when using another ABO blood group probe, for lactic acid bacteria!
  • the blood group-specific binding lactic acid bacteria of the present invention can be obtained.
  • the RU value indicating the ability to bind to human ABO blood type antigen of type A is 100RU or more
  • human ABO blood of type B and H RU value indicating ability to bind to type A antigen is 100 RU or less
  • RU value indicating ability to bind to human type A intestinal mucin RU value and human ability to bind to human type B intestinal mucin It can be screened using as an indicator that it is larger than the RU value indicating the ability to bind to type 0 intestinal mucin.
  • the RU value indicating the binding ability with human ABO blood group antigen of type B is 100RU or more
  • RU value indicating the ability to bind to human type B intestinal mucin is the RU value indicating the ability to bind to human type A intestinal mucin and human type 0 It is possible to screen for an index that is greater than the RU value indicating the ability to bind to intestinal mucin.
  • the RU value that indicates the ability to bind to H-type human ABO blood group antigens is 100 RU or more, and (ii) the human ABO blood group antigens of type A and B RU value showing binding ability is 100RU or less, (iii) RU value showing binding ability with human type 0 intestinal mucin Force RU value showing binding ability with human type A intestinal mucin and human type B intestinal mucin It is possible to screen using an index that is larger than the RU value indicating the binding ability of.
  • the test bacterium may cause bacterial aggregation due to non-specific adsorption, and the RU value may become abnormally high. Therefore, adsorption with human-derived intestinal mucin and ABO blood group antigen is not correctly reflected in the measurement results. Therefore, if the RU value is abnormally high, it is suspected that nonspecific adsorption (bacterial aggregation) has occurred, and the lactic acid bacteria can be excluded from the screening.
  • the subject of the screening method of the present invention is not particularly limited as long as it is a lactic acid bacterium. Despite being listed as an object, Lactobacillus acidopnilus, Lactobacillus crispatus, Lactooaci llus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus johns onii, Lactobacillus caselus, Lactobacillus caselus Lactobacillus delbruekn subsp.
  • Suitable for use with actis Lactobacillus fermentum, Lactobacillus murinus, Bifidobacterium animalis, Bifi dobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacteriu m longum, Bifidobacterium pseudolongum, Enterococcus faecium, Enterococcus feca lis, Streptococcus thermophilus.
  • a more suitable subject example is the genus Lactobacillus, and the most preferred subject example is: Lactobacillus acidophilus group lactic acid bacteria containing L. gasseri.
  • the lactic acid bacteria screened by the method of the present invention is a lactic acid bacterium that easily binds to the human intestinal tract of a specific ABO blood group.
  • the human of the specific blood group can improve health due to the effect of improving the intestinal balance in a short period of time. Therefore, the lactic acid bacteria screened by the method of the present invention can be applied to foods and drinks for specific blood groups, functional foods, foods for specific health use, dairy products, lactic acid bacteria drinks, and the like.
  • lactic acid bacteria used for yogurt production do not produce gas. This is because Japanese laws require that the product be marketed in a sealed container, and also to prevent product defects and rupture due to container expansion associated with gas production. Therefore, a gas production test was conducted for 238 strains.
  • Lactic acid bacteria were activated and cultured twice (37 ° C, 18h) with MRS Broth (DIFCO).
  • a Durham tube and MRS Broth (5 ml) were put together in a test tube with an aluminum cap and sterilized (121 ° C, 15 minutes).
  • the Durham tube has an opening facing down, and bubbles are removed from the Durham tube during sterilization.
  • the sterilized test tube with an aluminum cap was inoculated with 10 ⁇ L of about 10 9 cfo / mL lactic acid bacteria solution, and anaerobically cultured at 37 ° C. for 24 hours. After completion of the culture, the presence or absence of gas accumulated in the Durham tube was visually observed. Gas production occurs when clear bubbles are observed (+ (Table 1).
  • lactic acid bacteria that recognize and bind to blood group antigens were selected by surface plasmon resonance spectrum measurement.
  • Prepare 238 strains belonging to Lactobacillus acidophilus group (L. gasseri, L. plant arum, L. crispatus, L. amylovorus, L. casei, L. salivarius, L. brevis, L. fermentum, etc.) as test bacteria did.
  • BIACORE1000 was used as the surface plasmon resonance spectrum analyzer.
  • the antigen structure of human ABO blood group substance was used as a probe for selection of bacteria. Specifically, the following trisaccharide sugar chain polymer probe (manufactured by Seikagaku Corporation) (hereinafter also referred to as “BP-probe”) was used.
  • a antigen probe [GalNAc a 1-3 (Fuc a 1-2) Gal-]
  • H antigen probe [Fuc a l-2Gal-]
  • FIG. 1 The chemical structure of the BP-probe is shown in FIG. 1 and the sugar chain structure is shown in FIG.
  • the blood antigen BP-probe was prepared to O.lmg / lOml with HBS-EP buffer (pH 7.4).
  • Sensor chip SA is a BIACORE dedicated chip on which streptavidin is immobilized.
  • the immobilized amount of the A antigen probe was 750 RU, and the immobilized amount of the B antigen probe was 850 RU.
  • the 0 antigen probe was immobilized as follows.
  • a antigen sugar chain probe (Seikagaku Corporation) ⁇ - ⁇ -Acetylgalactosaminidase (EC3.2.1) .97 Streptococcus pneumoniae derived, SIGMA, JAPAN) 0.5U was used to react in acetate buffer pH 4.5 at 55 ° C for 20 hours to remove terminal GalNAc. (It was confirmed that 80% or more of GalNAc was removed by measuring the removal rate with the A antigen antibody!)
  • This biotinylated H-type antigen sugar chain probe was treated in the same manner as the A-type antigen sugar chain probe. Then, it was immobilized on a sensor chip to which avidin was bound. The amount of binding at this time was about 1000 RU.
  • Human large intestine mucin was prepared as another ligand used for selection of bacteria.
  • Human type A intestinal tract large intestine
  • Human type B intestinal tract large intestine
  • human type 0 intestinal tract large intestine
  • the mucous mucin layer was collected from the normal colon by the surface layer removal method.
  • the mucin layer was degreased with Folch's solvent and jetyl ether, dried, and extracted with 4M guanidine hydrochloride solution at 37 ° C for 2 hours.
  • Purified human A type, human B type, or human type 0 intestinal mucin (human colon mucin HCM, sometimes referred to as A-HCM type B intestinal mucin, type B-HCM type 0) Intestinal mucin was abbreviated as 0-HCM) and used for the test.
  • Gel filtration purification is a purification method for human colon mucin described in Purushothaman, SS et al, Adherence of Shigella dysentenae 1 to Human colonic Mucin. Curr.icrobio 1., 42 (6), 381-387 (2001). Based on.
  • the moving bed was 4M guanidine hydrochloride solution, and the column was Toyopearl HW-65F (90 cm X 2.6 cm, To soh. Tokyo. Japan).
  • neutral sugars were measured by the phenol sulfate method (490 nm) and proteins were measured at 280.
  • the peak with the highest protein absorption and neutral sugar content was selected, and a fraction with a molecular weight of about 2 million or more was collected as a standard to obtain human large intestine mucin (HCM).
  • HCM human large intestine mucin
  • Immobilization of the HCM to the BIACORE chip was performed by the amine coupling method.
  • a mixed reagent in which 50 ⁇ l of (NHS) was mixed was flowed to activate the carboxyl group introduced at the dextran end.
  • Running buffer HBS-EP buffer (pH7.4)
  • Regeneration solution 1M guazine hydrochloride solution 5 ⁇ 1
  • Lactic acid bacteria were selected from the results so far. First, selection was performed from the BIACORE measurement result and the gas production test result of Example 1.
  • Blood type A yogurt lactic acid bacteria were selected from the viewpoints of (i) A antigen recognition and (ii) gas production. The selection criteria are
  • a antigen recognizability As measured by BIACORE, the result of A antigen probe was 2000 RU or more (upper 35%)
  • Lactobacillus 238 was selected to 43 strains.
  • yogurt lactic acid bacteria for blood group 0 were selected by (i) H antigen recognition and (ii) gas production. The selection criteria are
  • H antigen recognizability As measured by BIACORE, the result of H antigen probe is 700RU or higher (upper 13%)
  • Lactobacillus 238 strain was narrowed down to 14 strains.
  • yogurt lactic acid bacteria for blood group B were selected by (i) B antigen recognition and (ii) gas production. The selection criteria are
  • Lactobacillus 238 strain was narrowed down to 25 strains.
  • B antigen recognition When measured with BIACORE, the result of B antigen probe is 100RU or more. under,
  • H antigen recognition 100 RU or less with H antigen probe as measured by BIACORE (ii) Specific recognition of A antigen
  • a antigen recognition / B antigen recognition ratio is 70 or more, A antigen recognition / H antigen recognition ratio is 100 or more
  • yogurt lactic acid bacteria candidates for blood group 0 were also selected. The selection criteria are
  • a antigen recognition In BIACORE measurement, the result of A antigen probe is 100RU or less.
  • B antigen recognition In BIACORE measurement, B antigen probe results are less than 100RU,
  • H antigen recognition / A antigen recognition ratio is 800 or more
  • H antigen recognition / B antigen recognition ratio is 20 or more
  • yogurt candidate strain for blood group B was also selected.
  • the selection criteria are
  • a antigen recognition In BIACORE measurement, the result of A antigen probe is 100RU or less.
  • H antigen recognition In BIACORE measurement, the result with H antigen probe is 100RU or less.
  • the ratio of B antigen recognition / A antigen recognition was 50 or more, and the ratio of B antigen recognition / H antigen recognition was 50 or more. Based on these, 6 strains were selected from 25 strains.
  • the strains selected above were further selected based on blood type human colon mucin (HCM) recognition.
  • HCM blood type human colon mucin
  • the selection criteria were (i) A-HCM recognizability of 100RU or higher and (ii) O-HCM recognizability of 100RU or lower in BIACORE measurement results by human HCM.
  • MEP165511 strain and MEP165530 strain were excluded because A-HCM recognizability was 1000 RU or more and bacterial aggregation was suspected. From the above selection, 2 strains (L. gasseri OLL 2804, L. gasseri OLL 2818) were selected from 13 strains.
  • yogurt lactic acid bacteria candidates for blood group 0 are selected from (i) those that recognize 0-HCM, and (ii) those with relatively high recognition of A-HCM. Was excluded.
  • the selection criteria were (i) O-HCM recognizability of 100RU or higher and (ii) A-HCM recognizability of 1000RU or lower in the BIACORE measurement results by human HCM. From the above selection, 2 strains (L. gasseri OLL 2827, L. gasseri OLL 2915) were selected from 5 strains.
  • the selection criteria for blood type B yogurt candidate strains were that B-HCM recognition was higher than A-HCM and H-HCM recognition. From the above selection, 2 strains (L. gasseri OLL2877, L. gasseri OLL2901) were selected from 6 strains.
  • Selection criteria vary depending on the type of bacteria Since all of the bacterial species selected this time are Lactobacillus gas seri, they were set to 0.50 or more. Blood group type A yogurt lactic acid bacteria (L.gasseri OLL 280 4, L.gasseri OLL 2818), blood group type yoghurt (L.gasseri OLL 2827, L.gasseri O LL 2915) and blood group B yogurt lactic acid bacteria (L.gasseri OLL2877, L.gasseri OLL2901) both met the above criteria (Table 1).
  • Lactobacilli MRS broth (DIFCO) 2 times of activation culture (37 ° C, 18 hours) Lactobacilli MRS broth (DIFCO) containing 5% of Lactobacilli MRS broth (DIFCO) Cultured at 37 ° C. After 18 hours of culture, the turbidity (OD) of the medium was measured.
  • the selection criteria were 0.08 or more for blood type A yogurt lactic acid bacteria and blood group 0 yogurt lactic acid bacteria, which differ depending on the type of bacteria. Both blood type A yogurt lactic acid bacteria (L.gasseri OLL 2804, L.gasseri OLL 2818) and blood type O yogurt lactic acid bacteria (Lg asseri OLL 2827, L.gasseri OLL 2915) met the above criteria. (table 1). Among blood type B yogurt lactic acid bacteria, L. gasseri OLL2901 had a bile acid tolerance of 0.031, but it is considered that there is no particular problem with intestinal retention. Table 2 shows the scientific characteristics of the above six strains.
  • Plain yogurt was prepared using L. gasseri OLL 2804 strain.
  • Yogurt mix (SNF: 9.5%, FAT: 3.0%) heated at 95 ° C for 5 minutes, 1% each of starter of L. bulgar icus JCM 1002 ⁇ and S. thermophilus ATCC 19258, L. gasseri OLL 2804 strain starter was inoculated 5% and fermented at 43 ° C for 4 hours.
  • thermophilus ATCC 19258 was 9.60 X 10 7 CFU / mL, 7.50 X 10 7 CFU / mL, 11.0 X 10 7 CFU / mL, and the survival rate of the L. gasseri OLL 2804 strain was 77% on the first day of storage, with a slight decrease in the number of viable bacteria.
  • the preserved product has good flavor and physical properties.
  • Plain yogurt was prepared using L. gasseri OLL 2818 strain. First, 1% each of L. gasseri OLL2818 strain, L. bulgaricus JCM 1002 T and S. thermophilus ATCC19258 was inoculated into 10% nonfat dry milk medium, and cultured at 37 ° C for 15 hours to prepare a Balta starter .
  • Yogurt mix (SNF: 9.5%, FAT: 3.0%) heated at 95 ° C for 5 minutes, 1% each of L. bulga ricus JCM 1002T and S. thermophilus ATCC 19258 starter, L. gasser i OLL 2818 starters were inoculated with 5% and fermented at 43 ° C for 4 hours.
  • the viable cell counts of L. gasseri OLL 2818, L. bulgaricus JCM 1002 T and S. thermophilus ATCC 19258 were 14.0 X 10 7 CFU / mL, 20.0 X 10 7 CFU / mL, 11.4 X 10 8 CFU / mL. The flavor and physical properties were both good.
  • the viable count of gasseri OLL 2818, L. bulgaricus JCM1002T and S. thermophilus ATCC 19258 was 11.4 X 10 7 CFU / mL, 7.00 X
  • the survival rate of L. gasseri OLL 2818 was 82% on the first day of storage, and the viable cell count decreased only slightly, 10 7 CFU / mL, 10.0 X 10 7 CFU / mL.
  • the preserved product has good flavor and physical properties.
  • a lactic acid bacterium suitable for human ABO blood group and a screening method thereof are provided.
  • the lactic acid bacterium of the present invention or the lactate strain obtained by the screening method of the present invention is a lactic acid bacterium having a high intestinal binding property for each ABO blood type, and functional yogurt classified by blood type is obtained by applying the lactic acid bacterium to food and beverage production. New probiotic foods and drinks can be provided.

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Abstract

Les bactéries de l’acide lactique sont criblées par spectroscopie par résonance plasmonique de surface, la mucine intestinale humaine et des antigènes des types sanguins étant utilisés en tant que sondes. Afin de s’adapter à un criblage de grande échelle, on s’efforce de définir des normes de sélection pour le criblage des bactéries de l’acide lactique tel que décrit ci-dessus. Il s’avère par conséquent que les bactéries de l’acide lactique correspondant respectivement aux types sanguins ABO peuvent être criblées au moyen de 100RU à des conditions spécifiques en tant que norme de liaison des bactéries. 238 souches de bactéries de l’acide lactique sont soumises à ce criblage. Ensuite, un test destiné à déterminer l’aptitude à la production de yaourt est réalisé. Enfin, les souches correspondant aux types sanguins individuels A, B et O sont clarifiées en pratique.
PCT/JP2005/022096 2004-12-01 2005-12-01 Bacteries de l’ acide lactique se liant a des types sanguins abo humains Ceased WO2006067940A1 (fr)

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CA2588940A CA2588940C (fr) 2004-12-01 2005-12-01 Lactobacilles de liaison par les groupes sanguins humains a, b et o
JP2006548758A JP4738348B2 (ja) 2004-12-01 2005-12-01 ヒトａｂｏ式血液型結合性乳酸菌
US11/720,462 US7897374B2 (en) 2004-12-01 2005-12-01 Human ABO blood group-binding lactobacilli
US12/987,518 US8465933B2 (en) 2004-12-01 2011-01-10 Method for screening human ABO blood group-binding lactobacilli

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JP2008179630A (ja) * 2006-12-28 2008-08-07 Glico Dairy Products Co Ltd 免疫賦活作用を有する乳酸菌，免疫賦活作用を有する製品及びその製造方法。
WO2009069704A1 (fr) * 2007-11-30 2009-06-04 Meiji Dairies Corporation Bactérie de l'acide lactique ayant l'effet d'abaisser le niveau sanguin d'acide urique
US8460918B2 (en) 2007-11-29 2013-06-11 Meiji Co., Ltd. Lactic acid bacteria having action of lowering blood uric acid level
JP2015073483A (ja) * 2013-10-09 2015-04-20 独立行政法人農業・食品産業技術総合研究機構 消化管粘膜付着性を有する乳酸菌
JP2019528738A (ja) * 2016-09-16 2019-10-17 デュポン ニュートリション バイオサイエンシーズ エーピーエス 細菌
WO2022102753A1 (fr) * 2020-11-13 2022-05-19 味の素株式会社 Agent microbien viable contenant lactococcus lactis, microorganisme appartenant au genre lactobacillus ou mélange associé, et son procédé de production

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008179630A (ja) * 2006-12-28 2008-08-07 Glico Dairy Products Co Ltd 免疫賦活作用を有する乳酸菌，免疫賦活作用を有する製品及びその製造方法。
KR101450511B1 (ko) 2007-11-29 2014-10-15 가부시키가이샤 메이지 혈중 요산치 저감작용을 갖는 유산균
US8460918B2 (en) 2007-11-29 2013-06-11 Meiji Co., Ltd. Lactic acid bacteria having action of lowering blood uric acid level
KR101385864B1 (ko) 2007-11-29 2014-04-17 가부시키가이샤 메이지 혈중 요산치 저감작용을 갖는 유산균
US8541223B2 (en) 2007-11-29 2013-09-24 Meiji Co., Ltd. Lactic acid bacteria having action of lowering blood uric acid level
US8389266B2 (en) 2007-11-30 2013-03-05 Meiji Co., Ltd. Lactic acid bacterium having effect of lowering blood uric acid level
KR20100109911A (ko) * 2007-11-30 2010-10-11 메이지 데어리즈 코포레이션 혈중 요산치 저감작용을 갖는 유산균
CN102016004B (zh) * 2007-11-30 2012-11-28 株式会社明治 具有降低血中尿酸值作用的乳酸菌
JPWO2009069704A1 (ja) * 2007-11-30 2011-04-14 明治乳業株式会社 血中尿酸値低減作用を有する乳酸菌
WO2009069704A1 (fr) * 2007-11-30 2009-06-04 Meiji Dairies Corporation Bactérie de l'acide lactique ayant l'effet d'abaisser le niveau sanguin d'acide urique
KR101584380B1 (ko) 2007-11-30 2016-01-21 가부시키가이샤 메이지 혈중 요산치 저감작용을 갖는 유산균
JP2015073483A (ja) * 2013-10-09 2015-04-20 独立行政法人農業・食品産業技術総合研究機構 消化管粘膜付着性を有する乳酸菌
JP2019528738A (ja) * 2016-09-16 2019-10-17 デュポン ニュートリション バイオサイエンシーズ エーピーエス 細菌
JP7111700B2 (ja) 2016-09-16 2022-08-02 デュポン ニュートリション バイオサイエンシーズ エーピーエス 細菌
WO2022102753A1 (fr) * 2020-11-13 2022-05-19 味の素株式会社 Agent microbien viable contenant lactococcus lactis, microorganisme appartenant au genre lactobacillus ou mélange associé, et son procédé de production

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