WO2008001676A1 - Bactérie d'acide lactique utilisée pour améliorer l'intolérance au lactose - Google Patents

Bactérie d'acide lactique utilisée pour améliorer l'intolérance au lactose Download PDF

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Publication number
WO2008001676A1
WO2008001676A1 PCT/JP2007/062522 JP2007062522W WO2008001676A1 WO 2008001676 A1 WO2008001676 A1 WO 2008001676A1 JP 2007062522 W JP2007062522 W JP 2007062522W WO 2008001676 A1 WO2008001676 A1 WO 2008001676A1
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Prior art keywords
lactic acid
strain
food
lactobacillus
acid bacteria
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PCT/JP2007/062522
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English (en)
Japanese (ja)
Inventor
Tadao Saitou
Haruki Kitazawa
Yasushi Kawai
Hiroyuki Itou
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The Food Science Institute Foundation
Tohoku University
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Priority to JP2008522516A priority Critical patent/JP5054687B2/ja
Priority to CN2007800237640A priority patent/CN101479382B/zh
Publication of WO2008001676A1 publication Critical patent/WO2008001676A1/fr
Priority to HK09112106.7A priority patent/HK1134937A1/xx

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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
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to a lactic acid bacterium useful for improving lactose intolerance and use thereof.
  • Lactose intolerance is a constitution that easily causes uncomfortable digestive symptoms such as diarrhea and abdominal pain due to indigestion of ratatoses (lactose) in ingested milk. This lactose intolerance is generally considered to become more prominent with aging, but is also observed in infancy. Lactose To reduce symptoms of intolerance, it is necessary to avoid consumption of products containing latatoses. The inability to take dairy products, which are a good source of calcium, can reduce the risk of osteoporosis in older people, for example. It is a great obstacle for nutrition. Osteoporosis is one of the biggest diseases that threatens our country, which is rapidly aging, and it is particularly important to promote the active intake of dairy products in order to reduce the risk of osteoporosis in the elderly.
  • Ratatoose is usually decomposed and absorbed by latatolytic enzymes produced by cells in the lining of the small intestine.
  • various microorganisms such as lactic acid bacteria produce latatose-degrading enzymes and degrade latatoses.
  • Liver lactic acid bacteria and yeast-containing yogurt and lactobacillus beverages are thought to be relatively less likely to cause lactose intolerance because some of the ratatoses are degraded and are not maintained.
  • these bacteria gradually grow in the intestine and increase the ability of extra-intestinal lath in the intestine. As a result, lactose intolerance itself is improved.
  • Non-patent Document 2 Non-patent Document 2
  • a lactic acid bacterium capable of degrading latatose continuously in the human intestine as well as having a high latatase activity is necessary. The search has not progressed much.
  • intestinal lactic acid bacteria include bacteria with high intestinal adhesion.
  • a method for searching for such a highly intestinal adherent for example, a method for screening a lactic acid bacterium having a high intestinal adherence suitable for each human blood type by measuring the binding ability to human intestinal mucin. Yes (Patent Document 1). This method is considered to be indispensable to adhere to the surface of intestinal epithelial cells in order for lactic acid bacteria to infect humans and establish in the intestine, using Takahashi et al.'S colonic mucin (RCM).
  • Non-patent document 3 Screening of Lactobacillus acidophilus gnolepe lactic acid bacteria using polystyrene beads coated with large intestine mucin (RCM) revealed that surface protein (SLP) derived from lactic acid bacteria that strongly bound to the RCM was Reported that it was also bound to the mucus layer.
  • SLP surface protein derived from lactic acid bacteria that strongly bound to the RCM was Reported that it was also bound to the mucus layer.
  • Non-patent document 3 etc.] and the chemical structure of the sugar chain constituting human large intestine mucin differed depending on the ABO blood group (for example, non-patent document 4).
  • the screening method has not been applied to applications other than the search for lactic acid bacteria suitable for each blood group.
  • Patent Document 1 Japanese Patent Laid-Open No. 2004-101249
  • Non-Patent Document 1 Tadao Saito, Toshitoshi Ito, Yoshio Kanno, Yukiko Yamazaki, Discovery of a new pathway of ratatoses utilization system in Lactob acillus gasseri (Gaseri fungus) of human intestinal tract, Journal of Biotechnology, (2001), 79 ( 6), pp.172-173
  • Non-Patent Document 2 Marteau P, Minekus M, Have thigh r R, Huis in't Veld JH, Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: validation a nd 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 selection of Lactobacillus acidophilus group lactic acid bac teria with high adhesion to human colonic mucosa., Biosci Biotechnol Biochem, (19 96), 60 (9), pp.1434--1438
  • Non-Patent Document 4 Junko Amano, Blood-type glycan structure on gastrointestinal mucin Toward elucidation of the mechanism of bacterial infection, Biochemistry, Japan Biochemical Society, (1999) 71 (4), pp.274-277 Disclosure of the invention
  • An object of the present invention is to provide a lactic acid bacterium having an ability to improve lactose intolerance.
  • the present invention includes the following.
  • a lactic acid bacterium having an ability to ameliorate lactose intolerance characterized by selecting a bacterium having enhanced intestinal adhesion and exonuclease activity from Lactobacillus lactic acid bacteria. How to Leeung.
  • surface plasmon resonance analysis is used to select the ability of lactic acid bacteria to bind to at least one of human type A intestinal mucin, human type B intestinal mucin, and human type 0 intestinal mucin. It is more preferable to select a bacterium having an RU value of 100 RU or more indicating the binding ability as a bacterium having enhanced intestinal adhesion.
  • the latatolytic enzyme activity enhanced by the selected bacterium is preferably at least one latatase activity of ⁇ -galatatosidase, phospho- ⁇ -galatatosidase, and phospho- ⁇ -glucosidase.
  • the Lactobacillus lactic acid bacterium to be screened is Lactobacillus gasseri or Lactobacillus mucosae.
  • [0013] A lactic acid bacterium obtained by the method of the above [1], wherein both intestinal tract adhesion and exonuclease activity are enhanced.
  • Lactobacillus gasseri OLL2836 strain (Accession No. BP-241), Lactobacillus' Gas A lactic acid bacterium which is one of Seri OLL2948 strain (Accession No. NITE BP-242) and Lactobacillus mucosae OLL284 8 strain (Accession No. MTE BP-243).
  • a lactose intolerance improving agent comprising at least one lactic acid bacterium according to [3] above.
  • Lactose intolerance improving agents include Lactobacillus gasseri OLL2836 strain (Accession number NIT E BP-241), Lactobacillus gasseri OLL2948 strain (Accession number NITE BP-242), and Ratatobacillus mucosae OLL2848. More preferably, the strain (accession number NITE BP-243) contains at least three lactic acid bacteria.
  • the foods and drinks include Lactobacillus 'Gasseri OLL2836 strain (Accession number NITE BP_241), Lactobacillus' Gasseri OLL2948 strain (Accession number NITE BP-242), and Lactobacillus mucosa OLL2848 strain (Accession number NITE BP- Those containing at least the three lactic acid bacteria of 243) are more preferred.
  • the food or drink is preferably an infant food, infant food, nursing food, elderly food, sick food, health functional food, supplement, fermented milk, or lactic acid bacteria beverage.
  • This food / beverage product is particularly suitable as a food / beverage product for improving lactose intolerance.
  • lactic acid strains useful for improving lactose intolerance can be efficiently selected. Moreover, if the lactic acid strain of this invention is used, the medicine useful for improvement of lactose intolerance and various food-drinks can be manufactured.
  • Fig. 1 shows the results of a P- ⁇ -gal activity test for a representative strain having high intestinal adhesion.
  • FIG. 2 shows the results of a P- ⁇ -glc activity test for a representative strain having high intestinal adhesion.
  • FIG. 3 shows the results of a ⁇ -gal activity test for a representative strain having high intestinal adherence.
  • the present invention relates to a method for screening a lactic acid bacterium having an ability to improve lactose intolerance by selecting a bacterium having enhanced intestinal adhesion and latatolytic enzyme activity from Lactobacillus lactic acid bacteria.
  • Lactobacillus lactic acid bacteria to be used for screening in the method of the present invention refers to bacteria classified as Lactobacillus or any bacterial species belonging to the genus Lactobacillus according to a normal taxonomic technique. Means.
  • the taxonomic method is not particularly limited, and includes conventional morphological taxonomy and taxonomy based on physiological 'biochemical properties' that have been used for the classification of known extra-L.
  • molecular phylogenetic classification based on homology analysis using 16S rDNA allotment lj, especially its V3 base sequence.
  • Lactobacillus lactic acid bacteria include Lactobacillus bulgaricus (7 Lactobacillus delbrueckii subsp. Bulgaricus), Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus acidopmlus, Lactobacillus casei, Lactobacill us plantarum, Lactobacillus brevis, Lactobacillus brevis (Lactobacillus brevis), Lactobacillus brevis (Lactobacillus brevis) buchneri), Fukutono, Chinores 'Fa 1- Mentum (Lactobacillus fermentum), Lactobacillus helveticus, Ratatococcus' Lactococcus lactis, La ⁇ Powers that are limited to Lactobacillus johnsonii, Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus mucosae, etc.
  • enteric lactic acid bacteria are preferred as Lactobacillus genus Lactobacillus, particularly Lactobacillus gasseri and Lactobacillus * mucosae.
  • Lactobacillus genus Lactobacillus particularly Lactobacillus gasseri and Lactobacillus * mucosae.
  • Lactobacillus refers to multiple types of Lactobacillus milk It may be a cell population or cell mixture containing acid bacteria cells, or may be a collection of individual strains of Lactobacillus lactic acid bacteria.
  • the intestinal adhesion and lactose-degrading enzyme activity of the lactic acid bacteria belonging to the genus Lactobacillus as described above are measured, and the bacteria in which both are enhanced are selected.
  • intestinal adherence refers to the ability of lactic acid bacteria to bind to the intestinal epithelial cell surface of a subject.
  • lactic acid bacteria against at least one intestinal mucin of human type A intestinal mucin, human type B intestinal mucin, and human type O intestinal mucin are used.
  • the binding ability is measured, and the obtained measurement value can be used as a value indicating intestinal adhesion.
  • the binding ability of lactic acid bacteria to intestinal mucin can be measured by a method using surface plasmon resonance analysis described in, for example, Patent Document 1 and International Application WO2006 / 067940.
  • a bacterium with an RU value of 100 RU or more indicating the ability of lactic acid bacteria to bind to intestinal mucin may be selected as a lactic acid strain candidate as a bacterium with enhanced intestinal adhesion. I like it.
  • Blood group antigens include commercially available sugar chain probes (for example, manufactured by Seikagaku Corporation) or neoglycoprotein 'Blood Group A Trisaccari de- BSA (for example, Calbiochem) or neoglycoprotein' Blood Groop B Trisaccaride e_BSA ( For example, Calbiochem) can be used, and anti-blood group antigen antibodies can be prepared by using various antibody production methods known to those skilled in the art using the antigen as an antigen.
  • sugar chain probes for example, manufactured by Seikagaku Corporation
  • neoglycoprotein 'Blood Group A Trisaccari de- BSA for example, Calbiochem
  • neoglycoprotein' Blood Groop B Trisaccaride e_BSA for example, Calbiochem
  • anti-blood group antigen antibodies can be prepared by using various antibody production methods known to those skilled in the art using the antigen as an antigen.
  • human type A intestinal mucin human type B intestinal mucin, human type 0
  • BIACORE a biosensor (biological intermolecular analysis device)
  • BIACORE1000 a biosensor (biological intermolecular analysis device)
  • the BIACORE system is an analyzer that monitors the interaction (binding and dissociation) of biological molecules in real time without using labels, based on the principle of surface plasmon resonance spectrum (SPR). Specifically, the intermolecular interaction between the ligand (hereinafter also referred to as probe) and the analyte is measured.
  • probe the ligand
  • lactic acid bacteria were used as the analyte
  • human colon mucin A type, B type, ⁇ type
  • a simple explanation of the principle used in this device is that by bonding and dissociating substances on the gold film and measuring the change in the refractive index of the reflected light due to the mass change on the gold film due to the bond / dissociation, The amount of material bound on the gold film is calculated. More specifically, in the apparatus, a glass substrate on which a gold film called a sensor chip is affixed is placed in the middle of a flow path for flowing a sample or a reagent so as to be exposed to the flow. While the sample is flowing through the flow path, the polarized light of 760 nm is condensed into a wedge shape with a prism etc. on the sensor chip, reflected on the gold film, and the refractive index of the reflected light is monitored.
  • the change in the angle of the surface plasmon resonance spectrum is shown in proportion to the change in the amount of binding. Therefore, in the BIACORE system, the probe is first flowed through the flow path, the probe is immobilized on the gold film of the sensor chip in advance, and then the site where the probe is not bound is blocked, and then the binding / dissociation reaction with the probe is performed. By monitoring the refractive index of the reflected light over time while flowing the substance to be examined (analyte) in the flow path, the force S can be calculated from the amount of change.
  • a change of 0.1 ° in the surface plasmon resonance spectrum is defined as 1000 resonance units (RU), and the change in the refractive index of reflected light is expressed based on a value (RU value) in units of resonance units.
  • one resonance unit (RU) corresponds to a mass change of lpg / lmm 2 on the surface of the sensor chip.
  • the amount of substance binding to the gold film on the sensor chip is the time before the substance binding (before addition) and when the binding is complete. It can be calculated from the difference in RU values.
  • the amount of binding between the probe (each human intestinal mucin in the present invention) and the analyte (lactic acid bacteria in the present invention) can be determined as the mass change (p g ) per sensor chip lmm 2, that is, the RU value.
  • the amount of binding (RU value) corresponds to the ability of the analyte to bind to the probe.
  • CM5 Gold film (typically 50 nm) is applied to the glass surface, and a carboxymethyl dextran layer (typically lOOnm) is provided on the surface.
  • carboxy group of the carboxymethyl dextran layer and In addition to the ligand (probe) immobilized on the sensor chip via the amino group, thiol group, aldehyde group or carboxyl group of the ligand), CM4, CM3, Cl, SA, NTA, Ll, HPA, etc. is there.
  • intestinal mucin may be immobilized on the sensor chip as a probe, for example, according to the manufacturer's instructions for the BIACORE system.
  • the immobilization method may be, for example, a physical adsorption method or a covalent bond adsorption.
  • CM5 BIACORE
  • EDC N-ethynole-N '-(3-dimethylaminopropyl-carbodiimide hydrochloride
  • a mixed reagent EDC / NHS
  • PHC a mixed reagent
  • PHC / NHS N-hydroxysuccinimide
  • the human intestinal mucin is covalently bound to the carboxyl group on the sensor chip, and ethanolamine hydrochloride-NaOH (pH 8.5) is used, and the probe binds to the carboxyl group on the sensor chip. Blocking the base site, In also preferred.
  • the present invention can be a tube mucin (HC M) immobilized sensor chip, HCM immobilization of the sensor chip specifically limited such les, but preferably a 1000 ⁇ 2000RU.
  • a sample (analyte solution) containing lactic acid bacterial cells is flowed through the flow path, and the HCM (probe) on each HCM-immobilized sensor chip and the lactic acid bacterial cells (analyte) in the analyte solution.
  • the amount of binding is calculated.
  • an example of the measurement conditions of BIACORE1000 that can be used for this analysis is shown below.
  • Running buffer HBS-EP buffer (pH 7.4)
  • Regeneration solution 1M guanidine hydrochloride solution 5 ⁇ 1
  • the RU value obtained by subtracting the RU value before the analyte binding from the RU value after the analyte binding is calculated with the lactic acid bacteria as the analyte and each intestinal mucin as the probe. It can be used as a binding amount (pg) per lmm 2 , that is, a value indicating the binding ability of lactic acid bacteria to intestinal mucin.
  • Judgment when the ability of lactic acid bacteria to bind to intestinal mucin shows a value of 100 RU or more for at least one of human type A intestinal mucin, type B intestinal mucin, and type 0 intestinal mucin, Judgment is “enhanced” or “high intestinal adherence”.
  • the RU value may vary depending on the measurement conditions in the BIACORE system.
  • the temperature condition in this method is, for example, 20 to 40 ° C, preferably 20 to 30 ° C, more preferably 23 to 28 ° C.
  • the concentration of a preferable analyte solution (sample) in the present method is 0.1 to 0.5 mg / mL, and a preferable flow rate in the present method is 3 to 10 / il / min.
  • the RU value does not change even when the above conditions are changed, and therefore whether or not the intestinal tract adherence is high can be determined based on “100 RU”.
  • the lactic acid bacteria Intestinal adherence is enhanced. Furthermore, in the screening method of the present invention, it can be said that the intestinal adherence is higher as the RU value is higher, that is, the intestinal adherence is enhanced than that of normal lactic acid bacteria.
  • a lactic acid bacterium having a RU value of 100 RU or more, more preferably 300 RU or more, particularly preferably 1000 RU or more as a binding ability to at least one human intestinal mucin is used as a bacterium having enhanced intestinal adhesion. It ’s better to select.
  • Ratatase activity (latatose degradation) of at least one latatolytic enzyme among ⁇ -galatatosidase ( ⁇ -gal), phospho- ⁇ -galatatosidase ( ⁇ - ⁇ -gal), and phospho- ⁇ -dalcosidase ( ⁇ - ⁇ -glc) (Enzyme activity) is measured, and a strain having the enhanced activity is selected.
  • lyophilized cells were suspended in 0.05M phosphate buffer, and the mixture was stirred vigorously with a toluene-acetone mixture (1: 9 (v / v)).
  • a toluene-acetone mixture (1: 9 (v / v)
  • 0-nitrophenyl- / 3-D-galactopyranoside ONPGal
  • P- / 3-gal is used for measuring latatase activity.
  • 0_Nitrophenyl- ⁇ _D-galactopyranoside_6_phosphate ONPGa ⁇ 6P
  • ⁇ NPGlc_6P 0-nitrophenyl- ⁇ -D-darcobilanoside-6-phosphate
  • O_Nitrophenol ( ⁇ ) ⁇ ⁇ ⁇ released per minute is defined as 1 unit and converted to activity value. From the activity value, the activity value per lmg of cells and the activity value per lmg of protein contained in the culture supernatant are calculated.
  • Lactic acid bacteria having particularly high lactase activity in which latatose-degrading enzyme activity is enhanced as compared with normal lactic acid bacteria can be selected.
  • ⁇ -gal has a lactatase activity (also referred to as / 3-gal activity in the present invention) of 15 units or more, preferably 100 units per mg of protein in the culture supernatant. It is particularly preferred to select strains that are greater than.
  • a strain showing 15 units or more, preferably 45 units or more per mg of protein in the culture supernatant can be selected. Particularly preferred.
  • ⁇ - ⁇ _glc activity in the present invention
  • intestinal adherence and exolytic activity of the lactic acid bacteria of the genus Lactobacillus are obtained as described above.
  • Lactobacillus 'Gaseri' or Lactobacillus 'Mukosae' are obtained as described above.
  • the present invention also relates to a lactic acid strain thus selected. Lactobacillus lactic acid bacteria obtained in this way grow and settle well in the intestinal tract, and retain the ability to break down lacese (lactose) very efficiently, which is very useful for improving lactose intolerance. It is.
  • “improvement of lactose intolerance” refers to an unpleasant digestive symptom of lactose intolerance (abdominal pain, diarrhea, tummy, etc.) It means to reduce the frequency of appearance of the sensation (such as a feeling of ruffles) or to completely prevent the onset, or to reduce the severity of symptoms.
  • “Lactose intolerance improvement ability” refers to the ability of lactic acid bacteria to improve lactose intolerance in this way.
  • Lactobacillus genus Lactobacillus belonging to the genus Lactobacillus genus Lactobacillus that have both intestinal adhesion and lactose-degrading enzyme activity significantly enhanced are actually obtained. I was able to.
  • These three lactic acid bacteria are Lactobacillus gasseri OLL2836 strain (hereinafter referred to as OLL2836 strain), Lactobacillus gasseri OLL2948 strain (hereinafter referred to as OLL2948 strain), and Lactobacillus mucosae OLL2848 strain (hereinafter referred to as OLL2836 strain).
  • OLL2836 strain Lactobacillus gasseri OLL2836 strain
  • OLL2948 strain Lactobacillus gasseri OLL2948 strain
  • OLL2836 strain Lactobacillus mucosae OLL2848 strain
  • the deposited OLL2836, OLL2948, and OLL2848 strains have the properties shown in Table 1 below.
  • Lactobacilli MRS Broth (MRS medium; for example, Difco, Ref. No. 288160) in Table 1 is a medium that can be suitably used for culturing these lactic acid strains.
  • Table 2 shows the typical composition of MRS medium.
  • Lithium phosphate 2.20 g
  • the lactic acid bacterium according to the present invention can reach the intestine in an alive state by oral administration, can settle in the intestine, and exhibits a strong exonuclease activity, thereby improving lactose intolerance. be able to. Moreover, in the fermented product manufactured using the lactic acid bacteria according to the present invention, the degradation rate of latatoose (lactose) is improved due to the high activity and latatolytic activity.
  • the present invention contains a Lactobacillus lactic acid bacterium (a lactobacillus according to the present invention) obtained by using the above screening method and having significantly enhanced both intestinal adherence and rasogenic enzyme activity.
  • the present invention also relates to a lactose intolerance improving agent.
  • This lactose intolerance improving agent of the present invention is effective for improving lactose intolerance by being administered to a subject who has or is suspected of having lactose intolerance, That is, the ability to reduce the frequency of occurrence of uncomfortable digestive symptoms of lactose intolerance (such as abdominal pain, diarrhea, or tummy sensation of tummy) or the onset of symptoms completely, or the severity of symptoms of lactose intolerance Can be reduced.
  • 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 contains at least one lactic acid bacterium according to the present invention (one or more strains).
  • the lactose intolerance improving agent of the present invention comprises, as lactic acid bacteria according to the present invention, Lactobacillus gasseri OLL2836 strain (Accession No. NITE BP-241), Lactobacillus gasseri OLL2948 strain (Accession No. MTE BP-242), and lactose It preferably contains at least one selected from Bacillus mucosae OLL2848 strain (Accession No. NIT E BP-243).
  • the lactose intolerance improving agent of the present invention contains at least Lactobacillus gasseri OLL28.
  • 36 strains accesion No. MTE BP-241
  • Lactobacillus gasseri OLL2948 accesion No. ⁇ BP-242
  • Lactobacillus mucosae OLL2848 accesion No. MTE BP-243. contains. Lactose intolerance improving agents containing a combination of these three lactic acid bacteria can bind to intestinal mucins with blood group antigens of type A, B, and O, and are effective for a wide range of subjects. It is.
  • the lactose intolerance improving agent of the present invention may be a composition containing the purified lactic acid bacteria according to the present invention, and a fermented product, a culture, or a product produced using the lactic acid bacteria.
  • a composition containing a concentrate of The lactic acid bacterium according to the present invention contained in the lactose intolerance improving agent of the present invention may be a living cell, a dead cell, a wet cell or a dry cell.
  • the lactose intolerance improving agent of the present invention is a composition containing the lactic acid bacteria according to the present invention in a living state.
  • the lactose intolerance improving agent of the present invention is not limited, but may be in any form such as liquid, gel, powder, granule, solid, capsule or tablet.
  • the lactose intolerance improving agent of the present invention is a pharmaceutically acceptable carrier or additive in addition to the lactic acid bacterium according to the present invention as an active ingredient or a culture, fermentation product, or concentrate thereof. May be contained.
  • carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polybutyl alcohol, polybulur pyrrolidone, carboxybule polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch.
  • artificial cell structures such as ribosomes can be mentioned.
  • the additives used are selected appropriately 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, enteral administration, etc.), and it is particularly preferable to administer it orally.
  • the lactose intolerance improving agent of the present invention to be administered is a solid preparation such as a tablet, granule, powder, pill, or capsule, a liquid preparation such as a liquid preparation, a suspension, or a syrup. Or the like. When used as a liquid preparation, it may be supplied as a dry product intended to be redissolved when the lactose intolerance improving agent of the present invention is used.
  • the oral solid preparation may contain additives such as binders, excipients, lubricants, disintegrants, wetting agents and the like generally used in pharmacy.
  • Oral liquid preparations may contain additives such as stabilizers, buffers, taste-masking agents, preservatives, fragrances, and coloring agents that are commonly used in pharmacology.
  • the dose of the lactose intolerance improving agent of the present invention varies depending on the age and weight of the administration subject, the administration route, and the number of administrations, and can be varied widely within the discretion of those skilled in the art.
  • the dose of the lactic acid bacterium according to 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 may be administered once or repeatedly at intervals of 6 to 8 hours.
  • Subjects to which the lactose intolerance improving agent of the present invention is administered are preferably, but not limited to, mammals including humans, domestic animals, pets, experimental (test) animals and the like. Furthermore, infants, adults, and elderly mammals that are suspected of being lactose intolerant are also preferred as subjects of the present invention, and mammals that have reduced ratatoses due to aging or disease, etc. A mammal having a genetic predisposition to lactose intolerance or an environmental predisposition is more preferable as a subject to which the lactose intolerance improving agent of the present invention is administered. Since the lactose intolerance improving agent of the present invention is less likely to cause side effects, it can be used very effectively for continuous use.
  • the present invention also relates to a food or drink containing the lactic acid bacteria belonging to the genus Bacillus which has both the intestinal tract adhesion and the activity of exonuclease degrading enzyme, both of which are remarkably enhanced, obtained using the above screening method.
  • the food or drink of the present invention is not particularly limited, but is particularly preferably a food and drink for improving lactose intolerance.
  • lactose intolerance improvement '' refers to subjects with an indication of lactose intolerance, strength, or suspicion of it, and unpleasant digestive symptoms of lactose intolerance (abdominal pain) , Diarrhea, tummy feeling, etc.) Is intended to completely prevent or reduce the severity of symptoms of lactose intolerance.
  • “food and drink” is not limited, but the type of food and drink containing the lactic acid bacteria according to the present invention including drinks, foods and functional foods is not particularly limited.
  • beverages containing lactic acid bacteria according to the present invention fermented milk (such as yogurt), lactic acid bacteria beverages, milk beverages (such as coffee milk and fruit milk), tea-based beverages (such as green tea, tea and oolong tea), fruit and vegetable-based beverages (Beverages containing fruit juices such as oranges, apples and grapes, and vegetable juices such as tomatoes and carrots), alcoholic beverages (beer, sparkling wine, wine, etc.), carbonated beverages and soft drinks .
  • existing reference books such as “Latest 'Soft Drinks” (2003) (Kokai Co., Ltd.) can be referred to.
  • the food containing the lactic acid bacterium according to the present invention is not particularly limited and may be a fresh food or a cache food, but the lactic acid bacterium according to the present invention is particularly suitable as a food. Examples include fermented milk and lactic acid bacteria beverages that can be delivered to the intestines alive.
  • the food containing lactic acid bacteria according to the present invention may be a dairy product such as yogurt or cheese, or a starter for producing fermented milk.
  • the “functional food” of the present invention means a food having a certain functionality for a living body, and includes, for example, food for specified health use (including conditional tokuho [food for specified health use]) and food with nutritional function.
  • Functional foods, special-purpose foods, dietary supplements, health supplements, supplements (for example, in various dosage forms such as tablets, coated tablets, dragees, capsules and liquids) and beauty foods (for example, diet foods) Includes so-called health foods in general.
  • the functional food of the present invention also includes health food to which a health claim based on the food standard of Codex (FAO / WHO Joint Food Standards Committee) is applied.
  • More specific examples preferable as the functional food of the present invention include special-purpose foods such as foods for patients, pregnant women's milk powder, infant formulas, and elderly foods.
  • the lactic acid bacteria according to the present invention can gradually improve the intestinal environment with mild action, improve the resolution of ratatoses in the intestine, improve lactose intolerance, and reduce the symptoms.
  • infant formula or liquid formula for the treatment of lactose intolerance in weak infants for example, it can be added to the normal infant formula
  • infants such as infant formula
  • Suitable functional foods of the present invention further include supplements for infants, subjects for maternal / lactating women, and supplements for elderly people for subjects exhibiting lactose intolerance due to innate or acquired factors. And sick supplements.
  • Preferable examples of functional foods containing lactic acid bacteria according to the present invention include health functional foods.
  • the health functional food system was established not only for normal foods but also for foods in the form of tablets, capsules, etc., based on domestic and external trends and consistency with conventional food systems for specified health use.
  • functional health foods consist of two types: foods for specified health use (individual permission type) and functional foods for nutrition (standard type).
  • new types such as conditional tokuho [food for specified health use] are included.
  • the functional food of the present invention introduces and establishes the lactic acid bacteria according to the present invention in the intestine, and improves (preferably, continuously) the intestinal leuth resolution, resulting in lactose intolerance. It is preferable to have an effect of improving the symptoms.
  • the functional food of the present invention may be intended primarily for uses other than the improvement of lactose intolerance.
  • the functional food of the present invention (preferably, food for specified health use or conditional tokuho [food for specified health use]) improves the intestinal extra-cellular resolution, thereby improving lactose intolerance or its symptoms. It may be a description or display of the effect to be performed. Such description or labeling may have been approved for labeling based on the provisions established in the Health Functional Food System.
  • the functional food of the present invention is in the form of solid preparations such as tablets, granules, powders, pills and capsules, liquid preparations such as liquids, suspensions and syrups, or preparations such as gels.
  • solid preparations such as tablets, granules, powders, pills and capsules
  • liquid preparations such as liquids, suspensions and syrups
  • preparations such as gels.
  • normal food and drink for example, fermented milk, beverages, powdered tea leaves, confectionery, etc. It may be.
  • the blending amount of the lactic acid bacteria according to the present invention in the food or drink is not particularly limited, and may vary depending on the case.
  • the specific blending amount can be appropriately determined by those skilled in the art in consideration of the type of food and drink, the desired taste and texture.
  • a blending amount such that the total amount of lactic acid bacteria according to the present invention is 0.001 to 100% by weight, particularly 0.1 to 100% by weight, is appropriate.
  • the lactic acid bacteria according to the present invention may be contained in the food and drink by any appropriate method available to those skilled in the art.
  • the lactic acid bacteria according to the present invention may be directly mixed into a raw material for food and drink.
  • the lactic acid bacteria according to the present invention may be applied, coated, permeated or sprayed on food and drink.
  • the lactic acid bacteria according to the present invention may be uniformly dispersed in food or drink, or may be unevenly distributed. You may prepare the capsule etc. which put the lactic acid bacteria based on this invention.
  • the lactic acid bacteria according to the present invention may be wrapped with an edible film or an edible coating agent. Moreover, after adding an appropriate excipient
  • the lactic acid bacteria based on this invention you may shape
  • Such processed products that may be further processed by the food and drink containing the lactic acid bacteria according to the present invention are also included in the scope of the present invention.
  • the food / beverage products containing the milk fermented product manufactured using the lactic acid bacteria based on this invention are especially preferable.
  • the food or drink of the present invention is not particularly limited, but it is particularly preferable to contain the lactic acid bacteria according to the present invention in a living state.
  • Additives include, but are not limited to, color formers (sodium nitrite, etc.), coloring agents (gardenia pigment, red 102, etc.), flavorings (orange flavors, etc.), sweeteners (stevia, astel palm, etc.) , Preservatives (sodium acetate, sorbic acid, etc.), emulsifiers (sodium chondroitin sulfate, propylene glycol fatty acid esters, etc.), antioxidants (EDTA disodium, vitamin C, etc.), pH regulators (taenoic acid, etc.), chemical Seasonings (such as sodium inosinate), thickeners (such as xanthan gum), swelling agents (such as calcium carbonate), antifoaming agents (such as phosphate phosphate), binders (such as sodium polyphosphate), nutrition enhancement Agents (calcium fortifier,
  • the food or drink of the present invention contains at least one lactic acid bacterium according to the present invention (one or more strains).
  • the lactic acid bacteria according to the present invention include Lactobacillus gasseri OLL2836 strain (Accession number MTE BP-241), Lactobacillus gasseri OLL2948 strain (Accession number BP BP-242), and Lactobacillus mucosa. It preferably contains at least one selected from the OLL2848 strain (Accession No. MTE BP-243).
  • the food and drink of the present invention comprises at least Lactobacillus gasseri OLL2836 strain (Accession No.
  • MTE BP_241 Ratobacillus gasseri OLL2948 strain (Accession No. MTE BP-242), and Lactobacillus mucosa.
  • the subject who ingests or administers the food or drink of the present invention is not limited, but is preferably a human, a domestic animal (pig, horse, etc.), a pet animal (Inu, cat, etc.), an experiment.
  • (Test) Mammals including animals (rodents such as mice and rats, and rabbits).
  • rodents such as mice and rats, and rabbits.
  • infants, adults, and elderly mammals that are suspected of being lactose intolerant are also preferred as subjects of the present invention, and mammals that have reduced latatose resolution due to aging or disease, etc.
  • Mammals having a genetic or environmental predisposition to lactose intolerance are also preferred as subjects for ingesting or administering the food or drink of the present invention.
  • test lactic acid bacteria used in the following are human-derived lactic acid strains provided by Meiji Dairies Co., Ltd., American Type Culture Collection (ATCC), RIKEN Microbiology Storage Facility (JCM), MAFF Livestock Experiment Station (NIAI) ), And a lactic acid strain obtained from the National Collections of Food Bacteria (NCFB) in the UK, and a total of 104 lactic acid strains isolated from infant feces by a modified LBS agar by the present inventors.
  • ATCC American Type Culture Collection
  • JCM RIKEN Microbiology Storage Facility
  • MAFF Livestock Experiment Station a lactic acid strain obtained from the National Collections of Food Bacteria (NCFB) in the UK, and a total of 104 lactic acid strains isolated from infant feces by a modified LBS agar by the present inventors.
  • NCFB National Collections of Food Bacteria
  • lactic acid bacteria include Lactobacillus bulgaricus, Lattobacillus noles' Lactobacillus acidophilus, Lactobacillus acidophilus, us lactis), Lactobacillus gasseri, Lattobacillus crispatus, Lactobacillus crispatus, Fuchno Chinores 1 ⁇ (Lactobacillus johnsonii), and various strains of Lactobacillus mucosae
  • strains with unidentified strains were included.
  • the strain described as OLL or MEP in the strain name indicates a strain possessed by Meiji Dairies Co., Ltd.
  • each lactic acid bacterium was dispersed in a sterilized 10% (w / v) skim milk medium (Snow Brand Milk Products Co., Ltd., Sapporo) and stored at _80 ° C in a deep freezer until use. .
  • each strain was subcultured 3 times in MRS medium (Difco, Detroit, MI, USA).
  • MRSL medium in which 2% glucose was replaced with ratatose
  • the obtained bacterial cell culture solution was inoculated to a newly prepared MRSL medium at 1%, and then cultured at 37 ° C for 18 hours.
  • the cells collected by centrifugation of the main culture thus prepared (6,000 X g, 20 minutes, 4 ° C) are washed with 0.05 M phosphate buffer (pH 6.8), and then suspended in distilled water. It became cloudy and then freeze-dried.
  • strains with high intestinal adhesion were selected using the Atsey system developed by the present inventors for examining the binding ability to intestinal mucin.
  • the Atsy system used is also described in detail in international application WO2006 / 067940.
  • each strain was inoculated and then subcultured 3 times in MRS medium. After culturing at 37 ° C for 12 hours, 500 ⁇ ⁇ was dispensed into a 1.5 ml tube. This culture solution is centrifuged (6,000 mm, 4 ° C, 10 minutes), and the cells obtained by removing the supernatant are washed twice in PBS buffer (pH 7.2) and suspended in distilled water. Freeze-dried. The bacterial cells were prepared to a concentration of 0.1 mg / ml using HBS-EP buffer (0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20) to prepare an analyte solution. [0078] 2. Preparation of human intestinal mucin (HCM) and production of HCM-immobilized sensor chip
  • Human intestinal mucin was prepared and immobilized on a sensor chip.
  • Human type A intestinal tract (blood type A human-derived large intestine), human type B intestine (blood type B human-derived large intestine), and human type O intestinal tract (blood type 0 human-derived large intestine) Received a sample collection from the graduate School of Medicine, Tohoku University. This sample was collected through the Ethics Committee of the graduate School of Medicine, Tohoku University, and with the consent of the patient.
  • a mucus mucin layer was collected from the normal large intestine portion of the intestinal tract by a surface layer removal method.
  • the mucous mucin layer consists of Folch solvent (mixed form-methanol (2: l (v / v)); J.
  • the moving bed was 4M guanidine hydrochloride solution and the column for gel filtration chromatography was Toyopearl HW_65F (90cm X 2.6cm, Tosoh. Tokyo. Japan). Measure the absorbance of protein at 280 ⁇ m by phenol sulfate method (measurement of absorbance at 490 nm after reaction). As a result, protein has absorption (280 nm absorbance) and the highest peak of neutral sugar content.
  • the mucin fraction of the large intestine was fractionated with a molecular weight of about 2 million or more in gel filtration chromatography as a guideline, and the purified product thus obtained was used for human A according to the blood type of the human.
  • HCM human colon mucin
  • B-type intestinal mucin B-.
  • O_H M M O type intestinal mucin
  • Immobilization of each HCM obtained as described above to the sensor chip for BIACORE was performed by a amine coupling method in a nano sensor BIACORE1000 (BIACORE).
  • BIACORE1000 a nano sensor BIACORE1000
  • 75.0 mg / ml N-ethyl _N '-(3-dimethylaminopropyl-carbodi) was applied to sensor chip CM5 (BIAC ORE) into which carboxymethyldextran group had been introduced in advance.
  • a mixed reagent (EDC / NHS) mixed with 50 ⁇ 1 of midhydrochloride (EDC) and 50 ⁇ 1 of 11.5 mg / ml ⁇ -hydroxysuccinimide (NHS) was used to activate the carboxy group in carboxymethyldextran. It was.
  • the HCM immobilization amount shown as the difference between report points before EDC / NHS introduction and after ethanolamine solution addition was 1000-2000 RU.
  • the HCM-immobilized sensor chip thus obtained was then used for testing the analyte solution.
  • BIACORE1000 biosensor BIACORE1000
  • Running buffer HBS-EP buffer (pH 7.4)
  • Regeneration solution 1M guanidine hydrochloride solution 5 ⁇ 1
  • the amount of binding (interaction) between the analyte and the probe is expressed as a value in units of resonance units (RU).
  • 1 Resonance unit (RU) means that the substance lpg is bound per lmm 2 on the surface of the sensor chip. That is, based on the binding / dissociation curve obtained in this analysis, the value obtained by subtracting the RU value before binding from the RU value after binding is the lmm 2 between each lactic acid bacterium as an analyte and each intestinal mucin as a probe. It corresponds to the amount of binding per hit. This amount of binding corresponds to the binding ability between the lactic acid bacteria and each intestinal mucin.
  • the binding ability to at least one intestinal mucin was 100 RU or more.
  • the strains that showed values were selected from 104 lactic acid bacteria. In the selected strain, it can be said that the intestinal adhesion is enhanced (higher intestinal adhesion) than other average lactic acid strains. Representative examples of the selected strains and the analysis results are shown in FIGS.
  • the solution in which the reaction was stopped was repeatedly concentrated and dried at 40 ° C. using a rotary evaporator (Tokyo Science Machine, Tokyo) to remove 0-nitrophenyl (ONP) generated in the reaction.
  • the concentrated reaction mixture was mixed with activated carbon (50 g) and allowed to stand at 4 ° C for 2 hours to adsorb chemically synthesized ONPGlc_6P and unreacted ONPGlc to activated charcoal. It was washed with double volume of distilled water (2 L) and desalted.
  • the elution of ⁇ NPGlc_6P adsorbed on activated carbon was performed using a pyridine: water: ethanol mixture (1: 1: 1 (v / v), 600 ml).
  • the eluate was repeatedly concentrated on a rotary evaporator at 40 ° C to remove pyridine. Subsequently, it was subjected to preparative paper chromatography (PPC). That is, 100 PPC filter paper (Whatman, 3MM, 46 X 57cm, Maidstone, England) Using a ⁇ 1 micropipette, apply the above-mentioned roughly purified ONPGlc-6P in a straight line, and simply use the ascending method using butanol: pyridine: water (6: 4: 3 ( ⁇ / ⁇ / ⁇ )) as the developing solvent. Expanded.
  • PPC preparative paper chromatography
  • ONPG1C-6P was extracted by immersing the cut paper in distilled water and stirring at 4 ° C. After extraction, the filter paper is removed, the aqueous layer is concentrated on a rotary evaporator, and the mixed reaction by-product is a TOYOPEARL HW40S column using distilled water as the mobile phase (2.6 ⁇ X 90 cm, Tosoichi, Tokyo) It was removed by gel filtration using.
  • the ONPGlc-6P fraction was collected from each eluted fraction using thin layer chromatography (TLC, Silicatel 60, 10 ⁇ 10 cm, Merck, Darmstadt, Germany).
  • TLC thin layer chromatography
  • the developing solvent for TLC was simply developed using butanol: 2-propanol: water (3: 12: 4 (v / v / v)).
  • a 5% (v / v) sulfuric acid-methanol solution was sprayed evenly onto a thin layer plate, and detected by heating for 3 minutes in a dryer (Yamato, Tokyo) heated to 150 ° C.
  • the collected ONPGlc-6P fraction was lyophilized to obtain purified ONPGlc-6P.
  • the purity was confirmed by TLC and the structure was confirmed by ⁇ -NMR.
  • the 104 lactic acid bacteria strains prepared in Example 1 were measured for the -gal, P- ⁇ -gal, and P_i3-glc ratatase activities using the Fisher method. That is, for each strain, 0.5 mg of freeze-dried cells were suspended in 1.0 ml of 0.05M phosphate buffer (pH 6.8), and toluene-acetone mixture (1: 9 (v / v)) 50 ⁇ 1 was added to the solution. Stir vigorously for 3 minutes.
  • Lactic acid strains with high intestinal adherence and high ratatase activity based on the measurement results of intestinal adherence and ⁇ -gal, ⁇ - ⁇ -gal and ⁇ - ⁇ -glc activities shown in the above examples were selected.
  • three strains of lactic acid OLL2836, OLL2948, OLL2836, strains with high intestinal adherence and markedly high activity for any one of ⁇ _gal, ⁇ - ⁇ -gal, and ⁇ _ ⁇ _glc latatase activity.
  • OLL2848 strain were selected ( Figures 1-3).
  • many lactic acid bacterial strains with relatively high latatase activity are excluded because of their low intestinal adherence (RU values of less than 100 RU for human intestinal mucins of type A, B, and O). It became the result.
  • OLL2836 strain showed P- ⁇ -gal activity of 46.576 units / mg protein
  • 0948 strain showed P-glc activity of 50.194 units / mg protein
  • the strain OLL2848 showed ⁇ -gal activity of 107.090 units / mg protein. These activities were considerably higher than those of other lactic acid strains with high intestinal adherence. In addition, these strains also showed very high values for RU values indicating intestinal adhesion.
  • OLL2836 strain is particularly strong and capable of binding to human type B intestinal mucin and human type O intestinal mucin
  • OLL2948 and OLL2848 are particularly strong in binding to human type A intestinal mucin. Indicated.
  • each of these lactic acid strains alone can be expected to have an effect of colonizing in the intestinal tract and continuously degrading ratatose, and in addition, by using the three strains in combination, It has been shown that subjects with different blood types will be able to obtain an effect of erosion, even high, and latatoses.
  • OLL2836, OLL2948 and OLL2848 strains further bacterial species identification tests were conducted.
  • L., OLL2836 and OLL2948 were identified as bacteria belonging to Lactobacillus gasseri and OLL2848 as Lactobacillus mucosae (see Examples below).
  • These three lactobacillus strains were obtained from the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center (2-5-8 Kisarazu Kazusa, Chiba, Japan) on June 9, 2006 (original deposit date) The application for deposit was made and received.
  • the receipt numbers for this deposit application are NITE AP_241 for OLL2836, NITE AP-242 for OLL2948, and NITE AP-243 for OLL2848, and the deposit numbers are NITE P-241, NITE P-242, and NITE P, respectively. -243.
  • These deposited strains were transferred to the deposit under the Budapest Treaty (international deposit) on March 27, 2007, and were deposited on June 9, 2006 as the original deposit date.
  • These international deposit numbers are NITE BP_241 for OLL2836, NITE BP_242 for OLL2948, and SNITE BP-243 for OLL2848. The mycological properties of these strains were confirmed as shown in Table 1 above.
  • a lactic acid strain having high intestinal adhesion and high ratatase activity can be obtained. Furthermore, iL-gal P- ⁇ -gal and ⁇ - ⁇ _glc were assayed for ratatase activity, and those strains that showed markedly high activity for each of the three latatolytic enzymes were selected. In combination, we were able to obtain a very useful combination of lactic acid bacteria that can be applied to a wide range of subjects.
  • the lactic acid strain obtained by the method of the present invention can be effectively used to improve lactose intolerance as a useful probiotic lactic acid strain that can settle in the intestinal tract and continuously reduce latatoses. Conceivable.
  • Bacterial species were identified for the three lactic acid strains OLL2836, OLL2948 and OLL2848 selected in Example 5. Identification was performed by 16S rDNA nucleotide sequence analysis based on amplification and sequencing of the V3 region. In the following, the identification tests performed on OLL2848 and OLL2948 strains are described as examples. However, the OLL2836 strain was identified in the same manner.
  • universal primers 27F 5, -GAGTTTGATCCTGGCTCAG-3 ', SEQ ID NO: 1) and 518R (5, _ATTAC CGCGGCTGCTGG-3', SEQ ID NO: 2) were used.
  • PCR conditions are 95 cycles at 95 ° C for 10 minutes, 55 ° C for 3 minutes, 72 ° C for 1 minute, 39 cycles at 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, and 72 ° C for 1 minute] One cycle was performed.
  • the DNA fragment amplified by the above PCR reaction was then subjected to electrophoresis. Electrophoresis was performed under a constant voltage of 100 V using 2% agarose gel as the electrophoresis gel and 1 X TBE buffer (90 mM Tris-boric acid, 2 mM EDTA, pH 8.0) as the electrophoresis buffer. Mupid-2 (Cosmo Bio, Tokyo) was used as the electrophoresis apparatus. As a molecular weight marker, a 100b DNA ladder (TaKaRa, Kyoto) was used. After electrophoresis, the gel was stained by immersing it in ethidium bromide (EB) solution (0.5 Aig / ml) for 10 minutes.
  • EB ethidium bromide
  • the gel after agarose gel electrophoresis and staining was irradiated with UV light using a UV transilluminator TRANS-20 (Funakoshi Pharmaceutical Co., Ltd., Tokyo), and the target DNA fragment was confirmed and cut out.
  • DNA was extracted using the DNA fragment purification kit MagExtractor (TOYOBO, Osaka). That is, after the excised agarose gel was transferred to a 1.5 ml tube, 400 / l adsorbed liquid was added and heated to 55 ° C. to completely dissolve it. Next, 15 ⁇ l of magnetic beads were added to the reaction solution and stirred, and then allowed to stand at room temperature for 1 minute.
  • the nucleotide sequence of the V3 region of OLL2848 strain showed 99.6% homology with the sequence of the V3 region of Lactobacillus mucosae.
  • the nucleotide sequence of the V3 region of OLL2948 strain showed 99.2% homology with the sequence of the V3 region of Lactobacillus gasseri. Therefore, OLL2848 was identified as a lactic acid bacterium belonging to Lactobacillus mucosae and OLL2948 as a lactic acid bacterium belonging to Lactobacillus gasseri.
  • the method of the present invention can be used to efficiently select lactic acid strains useful for improving lactose intolerance.
  • the lactic acid strain obtained by the method of the present invention can be conveniently used as an active ingredient for drugs, functional foods and the like that can be easily administered to patients and can continuously improve lactose intolerance.
  • sequences of SEQ ID Nos: 1 to 3 are primers.

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Abstract

La présente invention concerne une bactérie d'acide lactique utile pour l'amélioration de l'intolérance au lactose. Plus spécifiquement, la présente invention concerne un procédé destiné à cribler une bactérie d'acide lactique capable d'améliorer l'intolérance au lactose, l'invention étant caractérisée par la sélection d'une bactérie qui présente une propriété d'entéroadhérence accrue et une activité enzymatique dégradant le lactose accrue de bactéries d'acide lactique appartenant au genre Lactobacillus. L'invention concerne également une bactérie d'acide lactique générée par le procédé.
PCT/JP2007/062522 2006-06-26 2007-06-21 Bactérie d'acide lactique utilisée pour améliorer l'intolérance au lactose WO2008001676A1 (fr)

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WO2013168438A1 (fr) * 2012-05-10 2013-11-14 国立大学法人岩手大学 Protéine ayant une activité lactase, gène codant pour ladite protéine, vecteur recombinant portant ledit gène, transformant, procédé de fabrication de ladite protéine, et utilisation de ladite protéine
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
WO2015099617A1 (fr) 2013-12-23 2015-07-02 Medis, D.O.O. Nouvelles souches du gène lactobacillus et utilisation de celles-ci
JP2017511696A (ja) * 2014-03-05 2017-04-27 ディーエスエム アイピー アセッツ ビー．ブイ． 液状ラクターゼ組成物
CN109730156A (zh) * 2018-12-26 2019-05-10 李卫平 一种小儿止泻营养包及其制备方法
WO2021206106A1 (fr) 2020-04-08 2021-10-14 雪印メグミルク株式会社 Composition pour améliorer la flore bactérienne intestinale
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