CN101479382B - Lactic acid bacterium for amelioration of lactose intolerance - Google Patents

Lactic acid bacterium for amelioration of lactose intolerance Download PDF

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Publication number
CN101479382B
CN101479382B CN2007800237640A CN200780023764A CN101479382B CN 101479382 B CN101479382 B CN 101479382B CN 2007800237640 A CN2007800237640 A CN 2007800237640A CN 200780023764 A CN200780023764 A CN 200780023764A CN 101479382 B CN101479382 B CN 101479382B
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lactobacillus
strain
milk
acid bacteria
nite
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CN101479382A (en
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斋藤忠夫
北泽春树
川井泰
伊藤裕之
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Tohoku University NUC
Food Science Institute Foundation
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Food Science Institute Foundation
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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

Abstract

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

Description

Be used to improve the milk-acid bacteria of lactose intolerance
Technical field
The present invention relates to improving effective milk-acid bacteria of lactose intolerance and application thereof.
Background technology
" lactose intolerance " is owing to the lactose maldigestion in the emulsion of picked-up, and the physique of the symptom of digestive tract of discomforts such as diarrhoea or stomachache takes place easily.This lactose intolerance becomes significantly usually with advancing age, but also visible at infantile period.In order to alleviate the symptom of lactose intolerance, the picked-up of the goods of lactose must be avoided containing, but milk-product can't be absorbed as good calcium source, this can improve the risk of the elderly's osteoporosis, aspect nutrition, becomes very big problem.Osteoporosis is to threaten one of maximum disease along with the booming Japan of aging, sees from the angle that reduces senile osteoporosis disease risk, promotes the positive picked-up of milk-product to be even more important.
Lactose decomposes, absorbs through the lactose decomposing enzyme that in the cell of small intestine internal layer, produces usually.Various microorganisms lactose decomposing enzymes such as known milk-acid bacteria reduce lactose.Contain in live lactobacillus or zymic yogourt or the lactobacillus drink; The part lactose is decomposed, therefore difficult symptom with the generation lactose intolerance, and; Through orally ingestible constantly; Interior these bacterium of intestines are slowly bred, and the lactose resolving power strengthens in the intestines, and the possibility of result improves lactose intolerance itself.
Known in the past; In the metabolism of the lactose of milk-acid bacteria; Have beta-galactosidase enzymes (β-gal) and phosphoric acid-beta-galactosidase enzymes (and P-β-gal) two kinds of lactose decomposing enzymes are participated in, and the inventor etc. clear and definite phosphoric acid-beta-glucosidase enzyme that the third lactose decomposing enzyme-milk-acid bacteria had (the existing of P-β-glc) (non-patent literature 1).The active high milk-acid bacteria of these lactose decomposing enzymes is effective for the symptom that alleviates lactose intolerance.But we recognize: the milk-acid bacteria with high lactose degrading activity is also arranged in non-intestinal tract milk-acid bacteria, but can't in people's enteron aisle, survive usually.For above-mentioned milk-acid bacteria, can't in people's enteron aisle, continue to produce Sumylact L, can't have great expectations of the lactase activity higher (non-patent literature 2) than orally ingestible.Therefore, improve lactose intolerance for administered through oral gives milk-acid bacteria, just lactase activity is high, also must have in intestines, to continue the milk-acid bacteria that reduces lactose, but the research of above-mentioned milk-acid bacteria does not have bigger progress as yet.
Yet, have the high bacterium of enteron aisle adhesivity in the known intestinal tract milk-acid bacteria.Method as the high bacterium of the above-mentioned enteron aisle adhesivity of research for example has: through measuring and the mucinous binding ability of people's enteron aisle the method (patent documentation 1) of the milk-acid bacteria that the enteron aisle adhesivity of the suitable people's of screening various blood types is high.This method, is absolutely necessary with the intestinal epithelial cell surface adhesion in order people's initial stage to be infected and to be settled in the enteron aisle according to milk-acid bacteria; And the report of the milk-acid bacteria screening carried out of people's such as high bridge use large intestine Saliva Orthana (RCM) [when use scribbles the polystyrene bead screening Lactobacterium acidophilum group lactobacillus strain of large intestine Saliva Orthana (RCM), from the surface layer protein (SLP) of the strong bonded lactobacillus strain of this RCM also with National People's Congress's casing slime layer bonded report.Non-patent literature 3 etc.]; The chemical structure that constitutes the mucinous sugar chain of people's large intestine is according to ABO formula blood type and structure such as different report (for example non-patent literature 4) etc.s.But whether this screening method is suitable for the purposes of milk-acid bacteria beyond research that is fit to each blood type, and Shang Weijian has research.
Patent documentation 1: TOHKEMY 2004-101249 communique
Patent Document 1: Saito Tadao Ito open Min, Tateno Yoshio Yamazaki Yukiko, Hiroshi Suites intestinal origin Full? Lactobacillus? Gasseri) (ga Center re-bacteria) concise analyzes ru ra ku Suites a su-owned Chemistry Department Full New Ordinary road Issuance see (human intestinal origin plus Lactobacillus lactose utilization system in the discovery of new ways), Biotechnology, Society, (2001), 79 (6), 172 - 173
Non-patent literature 2:Marteau P; Minekus M, Havenaar R, Huis in ' t Veld JH.; Survival of lactic acid bacteria in a dynamic model of the stomach andsmall intestine:validation and the effects of bile.; J Dairy Sci, (1997) 80 (6), 1031～1037 pages
Non-patent literature 3:Takahashi N, Saito T, Ohwada S; Ota H, Hashiba H, ItohT.; " A new screening method for the selection of Lactobacillusacidophilus group lactic acid bacteria with high adhesion to humancolonic mucosa. ", Biosci Biotechnol Biochem., (1996).; 60 (9)., 1434～1438 pages
Non-patent literature 4: day wild pure son; On the digest tube system チ Application blood type sugar lock sow with a drill make-bacterial infection メ カ ニ ズ system separates bright To け て (the blood type sugar chain structure on the alimentary canal mucoprotein-bacterial infection mechanism progress); Biochemical; The biochemical meeting of the team legal person of society Japan; (1999) 71(4), 274～277 pages
Summary of the invention
Invent problem to be solved
The object of the present invention is to provide milk-acid bacteria with ability of improving lactose intolerance.
Solve the means of problem
The inventor etc. have carried out deep research for solving above-mentioned problem, and the result finds: in the lactobacillus milk-acid bacteria of a part, its enteron aisle adhesivity and lactose decomposing enzyme activity all strengthen, and have accomplished the present invention based on this understanding.
That is, the present invention comprises following content.
Screen the method for the milk-acid bacteria of improvement ability, it is characterized in that: the active all enhanced bacterium of screening enteron aisle adhesivity and lactose decomposing enzyme from the lactobacillus milk-acid bacteria with lactose intolerance.
In this method; More preferably in the screening of this bacterium; Use the surface plasma body resonant vibration analysis to measure at least a binding ability in milk-acid bacteria and people A type enteron aisle Saliva Orthana, human B-type enteron aisle Saliva Orthana and the people O type enteron aisle Saliva Orthana, the RU value of this binding ability of expression is screened as enteron aisle adhesivity enhanced bacterium for the bacterium more than the 100RU.
In this method, enhanced lactose decomposing enzyme activity is preferably at least a lactase activity in beta-galactosidase enzymes, phosphoric acid-beta-galactosidase enzymes and the phosphoric acid-beta-glucosidase enzyme through the bacterium of screening.
In this method, the lactobacillus milk-acid bacteria of preferably screening is Lactobacillus gasseri (Lactobacillus gasseri) or mucous membrane probiotic lactobacillus (Lactobacillus mucosae).
Milk-acid bacteria, it is by above-mentioned [1] described method acquisition, enteron aisle adhesivity and the active all enhanced milk-acid bacterias of lactose decomposing enzyme.
Milk-acid bacteria, it is any one milk-acid bacteria in Lactobacillus gasseri OLL2836 strain (preserving number NITE BP-241), Lactobacillus gasseri OLL2948 strain (preserving number NITE BP-242) and the mucous membrane probiotic lactobacillus OLL2848 strain (preserving number MTE BP-243).
Lactose intolerance activator, this activator contain a kind of above-mentioned [3] described milk-acid bacteria at least.
This lactose intolerance activator more preferably contains three kinds of milk-acid bacterias of Lactobacillus gasseri OLL2836 strain (preserving number MTE BP-241), Lactobacillus gasseri OLL2948 strain (preserving number NITE BP-242) and mucous membrane probiotic lactobacillus OLL2848 strain (preserving number NITE BP-243) at least.
Diet article, these diet article contain a kind of above-mentioned [3] described milk-acid bacteria at least.
These diet article more preferably contain three kinds of milk-acid bacterias of Lactobacillus gasseri OLL2836 strain (preserving number NITEBP-241), Lactobacillus gasseri OLL2948 strain (preserving number NITE BP-242) and mucous membrane probiotic lactobacillus OLL2848 strain (preserving number NITE BP-243) at least.
These diet article are preferably milk formula, junior food, nursing women's food, the elderly's food, patient's food, health functional food, dietary supplements, fermented-milk or lactobacillus drink.
These diet article especially are suitable as and improve the diet article that lactose intolerance is used.
The invention effect
According to screening method of the present invention, can screen efficiently the effective lactobacillus strain of the improvement of lactose intolerance.Use lactobacillus strain of the present invention, can also prepare effective medicine of the improvement of lactose intolerance or various diet article.
This specification sheets comprises the disclosure that Japan as the basis of claim of priority of the present invention speciallys permit out hope 2006-175897 number.
The accompanying drawing summary
Fig. 1 representes P-β-gal activity test of result carry out to(for) the high representative bacterial strain of enteron aisle adhesivity.
Fig. 2 representes P-β-gLc activity test of result carry out to(for) the high representative bacterial strain of enteron aisle adhesivity.
Fig. 3 representes β-gal activity test of result carry out to(for) the high representative bacterial strain of enteron aisle adhesivity.
The best mode that carries out an invention
Below, specify the present invention.
1. screening has the method that lactose intolerance improves the milk-acid bacteria of ability
The present invention relates to through the active all enhanced bacterium of screening enteron aisle adhesivity and lactose decomposing enzyme from the lactobacillus milk-acid bacteria, the method for screening milk-acid bacteria with lactose intolerance improvement ability.
In the method for the present invention, " the lactobacillus milk-acid bacteria " of supplying with screening is meant that the taxonomy method according to routine is classified as lactobacillus (Lactobacillus) or belongs to the bacterium of any bacterial species of lactobacillus.Do not limit this taxonomy method is special; Comprise known employed based on the sorting technique of morphological classification method or physiology, biochemical proterties in the past in the classification of lactobacillus bacterium; For example for the bacterium that does not carry out strain identification; The sorting technique of the molecular systematics of the homology analysis that employing is carried out based on the nucleotide sequence with 16SrDNA sequence, particularly V3 district, this can clearly classify, and is more preferred.The object lesson that belongs to the bacterial species of lactobacillus milk-acid bacteria has: lactobicillus bulgaricus (lactobicillus bulgaricus (Lactobacillus bulgaricus) or lactobacillus delbruockii subspecies bulgaricus (Lactobacillusdelbrueckii subsp.Bulgaricus)), lactobacillus delbruckii (lactobacillus delbruckii (Lactobacillusdelbrueckii) or Lactobacillus delbrueckii subsp. (Lactobacillus delbrueckii subsp.Delbrueckii)), Lactobacterium acidophilum (Lactobacillus acidophilus), lactobacterium casei (Lactobacillus casei), plant lactobacillus (Lactobacillus plantarum), short lactobacillus (Lactobacillus brevis), Lactobacillus buchneri (Lactobacillus buchneri), lactobacillus fermentum (Lactobacillus fermentum), lactobacterium helveticus (Lactobacillus helveticus), newborn probiotic lactobacillus (Lactococcus lactis), lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus gasseri (Lactobacillus gasseri), mucous membrane probiotic lactobacillus (Lactobacillus mucosae) etc., but be not limited to this.In the method for the present invention, as the preferred intestinal tract milk-acid bacteria of lactobacillus milk-acid bacteria, especially preferred Lactobacillus gasseri and mucous membrane probiotic lactobacillus.In present method, " the lactobacillus milk-acid bacteria " of supplying with screening can be cell group or the cell mixture that contains multiple lactobacillus lactic-acid bacteria cells, also can be the set of indivedual bacterial strains of lactobacillus milk-acid bacteria.
Among the present invention,, measure enteron aisle adhesivity and lactose decomposing enzyme activity, screen their equal enhanced bacterium for above-mentioned lactobacillus milk-acid bacteria.
Here, " enteron aisle adhesivity " is meant the binding ability that milk-acid bacteria is surperficial with the intestinal epithelial cells that is tried body.In the enteron aisle adhesivity of the milk-acid bacteria that the present invention relates to is measured; Representational is the mucinous binding ability of measuring in milk-acid bacteria and people A type enteron aisle Saliva Orthana, human B-type enteron aisle Saliva Orthana and the people O type enteron aisle Saliva Orthana of at least a enteron aisle, and the measured value that can use gained is as the adhering value of expression enteron aisle.The mensuration of milk-acid bacteria and the mucinous binding ability of enteron aisle for example can be carried out through the method for utilizing the surface plasma body resonant vibration analysis of record in patent documentation 1 and the International Application No. WO 2006/067940.When adopting surface plasma body resonant vibration to analyze, will represent that preferably the RU value of milk-acid bacteria and enteron aisle Saliva Orthana binding ability is the candidate strain of the bacterium more than the 100RU as enteron aisle adhesivity enhanced bacterium, conduct screening lactobacillus strain.
Below, further specify mensuration system with the mucinous binding ability of this enteron aisle.At first; In order to prepare people's enteron aisle Saliva Orthana of different blood types; Acquisition tables layer segment from people's enteron aisle of each blood type (A type, Type B and O type); Solubilizing agent such as use Guanidinium hydrochloride carry out gel-filtration, are index with protein adsorption (absorbancy of 280nm) and neutral sugar content height, gather target components and purifying.The detailed content of this gel-filtration purifying for example can be with reference to people such as PurushothamanS.S.; " Adherence of Shigella dysenteriae 1 to Human ColonicMucin. " Curr.Miorobiol.; 42 (6), the mucinous purification process of people's large intestine of record in 381～387 pages (2001).Behind the purifying, more preferably use anti-blood type antigen-antibody, confirm on people's enteron aisle Saliva Orthana of preparation, to express blood type antigen.The method that object lesson can be given an example and put down in writing among the embodiment.Blood type antigen can use commercially available sugar chain probe (for example biochemical industrial preparation) or Neoglycoproteins Blood Group A Trisaccaride-BSA (for example Calbiochem company) or Neoglycoproteins Blood Group B Trisaccaride-BSA (for example Calbiochem company) etc.As antigen, can prepare anti-blood type antigen-antibody with it according to various preparation method for antibody well known in the art.
In the screening method of the present invention; With people A type enteron aisle Saliva Orthana, human B-type enteron aisle Saliva Orthana, the mucinous people separately of people O type enteron aisle enteron aisle Saliva Orthana as probe; Carry out the surface plasma body resonant vibration spectrum analysis, can measure milk-acid bacteria and the mucinous binding ability of these people's enteron aisles thus.BIACORE system (BIACORE company) can be used in the surface plasma body resonant vibration spectrum analysis, for example BIACORE1000 can be used as biosensor (the intermolecular mutual analytical equipment of organism).
The BIACORE system is according to the principle of surface plasma body resonant vibration spectrum (SPR), need not applying marking, promptly can monitor the analytical equipment of interaction between the organism molecule (combine and dissociate) in real time.Be the molecular interaction of measuring between part (below be also referred to as probe) and the assay specifically.Among the present invention, use milk-acid bacteria as assay respectively, end user's large intestine Saliva Orthana (A type, Type B, O type) is as part.About the principle of surface plasma body resonant vibration, the report of the existing a lot of public publications of its circumstantial letter.The principle of simply this device being described and being adopted then is: make material on golden film, combine/dissociate, measure because the reflected light change of refractive that the quality change on this golden film that combines/dissociate to cause is followed is calculated and golden membrane-bound amount of substance thus.More particularly, in this device, the stream that the glass substrate that is pasted with the golden film that is called as sensing chip is arranged on flow through sample or reagent midway; And contact with this fluxion; Sample etc. is flow through, simultaneously through prism etc. with the polarisation of 760nm with the wedge shape light harvesting on sensing chip, on golden film, reflect; Monitor this catoptrical specific refractory power, then the ratio that is varied to of material binding capacity shows on the angle variation of surface plasma body resonant vibration spectrum and the golden film.Here; In the BIACORE system, at first make probe flow through stream, in advance with probe stationary on the golden film of sensing chip; Then seal the unconjugated position of probe; Will study with probe then combine/material (assay) of dissociation reaction flows into stream, monitors catoptrical specific refractory power at different time, can be calculated the binding capacity of probe and assay thus by this variable quantity.In this system, the variation of 0.1 ° of the angle of surface plasma body resonant vibration spectrum is defined as 1000 resonance units (RU), according to representing catoptrical variations in refractive index as the value (RU value) of unit with this resonance units.Here, 1 resonance units (RU) is equivalent to the lip-deep 1pg/1mm of sensing chip 2Quality change.That is the poor calculating of the RU value when combining to accomplish when (before adding) before, the binding capacity on material and the sensing chip gold film can be combined by material.Use the BIACORE system, the binding capacity that then can obtain probe (the present invention is each one enteron aisle Saliva Orthana) and assay (being milk-acid bacteria among the present invention) is as 1mm 2The quality change of sensing chip (pg), i.e. RU value, this binding capacity (RU value) is equivalent to the binding ability of assay and probe.
Need to prove; The sensing chip basis of in the BIACORE system, using has several kinds with this sensing chip fixed material (probe), fixing means, application target etc.; The CM5 that for example removes standard (pastes golden film (being typically 50nm), possesses carboxyl methyl VISOSE layer (being typically 100nm) on its surface on the glass surface.The amino that carboxyl and part had, thiol group, aldehyde radical or carboxyl via this Sensor Chip CM 5 layer is had are fixed on part (probe) on the sensing chip) outside, also have CM4, CM3, C1, SA, NTA, L1, HPA etc.
Among the present invention, the enteron aisle Saliva Orthana as probe stationary on sensing chip the time, for example can be implemented according to the working instructions of the manufacturers of BIACORE system.Fixing means for example can be the method for physical adsorption or the method for adsorbing through covalent linkage.One of them example is: if use CM5 (BIACORE company) as sensing chip; Preferably at first N-ethyl-N '-3-dimethylaminopropyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) were mixed by 1: 1; This mix reagent (EDC/NHF) is flowed into stream; Make the activated carboxylic in the Sensor Chip CM 5 on the sensing chip, prepare the mixing solutions that in fixing, adds people's enteron aisle Saliva Orthana solution (using A-HCM, B-HCM, O-HCM respectively) gained then, further flow in the stream with acetate buffer (pH 4.0); Through the amine linked reaction, the carboxyl position covalent attachment on people's enteron aisle Saliva Orthana and the sensing chip.And, using ethanolamine hydrochloric salt-NaOH (pH8.5), the fixedly sensing chip of enteron aisle Saliva Orthana (HCM) is processed at the carboxyl position on the unconjugated sensing chip of sealing probe.Among the present invention, do not limit the fixed amount of HCM on sensing chip is special, but be preferably 1000～2000RU.
Then, the sample (assay solution) that will contain the milk-acid bacteria thalline flows in the stream, the HCM (probe) on the sensing chip of fixing each HCM of calculating and the binding capacity of the milk-acid bacteria thalline (assay) in the assay solution.An example of the condition determination of the BIACORE1000 that can in this is analyzed, use is as follows, but is not limited to this.
Electrophoretic buffer: HBS-EP damping fluid (pH 7.4)
The addition of assay solution (sample): 20 μ L
Flow velocity: 3 μ L/ minutes
Temperature of reaction: 25 ℃
Regeneration soln: 1M guanidinesalt acid salt solution 5 μ L
According to combination/dissociation curve that said determination obtains, can use by the RU value after the assay combination and deduct the preceding RU value gained RU value of assay combination as expression assay milk-acid bacteria and as the mucinous every 1mm of each enteron aisle of probe 2Binding capacity (pg), that is, and the value of this milk-acid bacteria and the mucinous binding ability of enteron aisle.Among the present invention; The mucinous binding ability of milk-acid bacteria and enteron aisle with respect to people A type enteron aisle Saliva Orthana, Type B enteron aisle Saliva Orthana, O type enteron aisle mucinous at least one when showing the value more than the 100RU, then can judge " enhancings of enteron aisle adhesivity " or " enteron aisle adhesivity height ".
Need to prove that the RU value can change according to the condition determination of BIACORE system.For example 20～40 ℃ of temperature condition of the present invention, preferred 20～30 ℃, more preferably 23～28 ℃.The concentration of preferred assay solution (sample) is 0.1～0.5mg/mL in present method, and preferred flow velocity is 3～10 μ L/ minutes in present method.If in above-mentioned scope, even then change above-mentioned each condition, the RU value can not change yet, and can be that benchmark judges whether the enteron aisle adhesivity is high with " 100RU " therefore.And, even when each condition of the concentration of assay solution changes outside above-mentioned scope, if show and the equal binding ability of 100RU that then the enteron aisle adhesivity of this milk-acid bacteria strengthens when being scaled the RU value under the above-mentioned condition.And in the screening method of the present invention, then the enteron aisle adhesivity is also high for the high bacterium of RU value,, we can say that the enteron aisle adhesivity strengthens than common milk-acid bacteria that is.Among the present invention, preferred screening to the mucinous binding ability of at least a people's enteron aisle be shown as more than the 100RU, more preferably the milk-acid bacteria of the RU value more than the 300RU, more than the preferred especially 1000RU is as enteron aisle adhesivity enhanced bacterium.
And; In the screening method of the present invention; (((mensuration of the lactase activity (lactose decomposing enzyme is active) of at least a lactose decomposing enzyme among the P-β-glc) is screened the bacterial strain of this increased activity for P-β-gal) and phosphoric acid-beta-glucosidase enzyme for β-gal), phosphoric acid-beta-galactosidase enzymes to carry out beta-galactosidase enzymes for above-mentioned lactobacillus milk-acid bacteria.
Specifically; According to the Fisher method, the freeze-drying thalline is suspended in the 0.05M phosphoric acid buffer, it is added toluene-acetone mixed solution (1: 9 (v/v)); Vigorous stirring; If be used to measure the lactase activity of β-gal, then in this suspension-s, add the phosphoric acid buffer that is added with ortho-nitrophenyl base-β-D-galactopyranoside (ONPGal), if be used to measure the lactase activity of P-β-gal; Then in this suspension-s, add the phosphoric acid buffer that is added with ortho-nitrophenyl base-β-D-galactopyranoside-6-SULPHOSUCCINIC ACID ESTER (ONPGal-6P); Perhaps, then in this suspension-s, add the phosphoric acid buffer that is added with ortho-nitrophenyl base-β-D-glycopyranoside-6-SULPHOSUCCINIC ACID ESTER (ONPGlc-6P), reacted 15 minutes down at 37 ℃ if be used to measure the lactase activity of P-β-glc.The reaction back adds 0.5 M sodium carbonate solution, and stopped reaction is through centrifugal (6; 000 * rpm, 10 minutes, 4 ℃) remove the thalline composition; Measure the absorbancy of the 405nm of supernatant through extinction photometer (for example Multiskan MS-UV (Labsystems, Helsinki, Finland)).Free 1 minute o-NP (ONP) of 1 μ mol is defined as 1 unit, is scaled activity value.Calculate contained every 1mg activity of proteins value in activity value and the culture supernatant of every 1mg thalline respectively by this activity value.
Need to prove that proteinic quantitatively can be MicroBCA protein determination kit (Pierce, the Rockford that utilizes according to the BCA method; The Illinois; The U.S.), after reaction 2 hours, use extinction photometer (for example Multiskan MS-UV) to measure the absorbancy under the 540nm.Calibration curve can use bovine serum albumin (BSA) to make.Can calculate contained proteinic amount in the supernatant according to this calibration curve.
Can be by the contained extra high milk-acid bacteria of every 1mg activity of proteins value this lactase activity of screening (common milk-acid bacteria strengthens the lactose decomposing enzyme specific activity) in the activity value of β-gal, P-β-gal and the P-β-glc of every 1mg thalline of aforementioned calculation and the culture supernatant.
In every 1mg protein in the preferred especially screening and culturing supernatant for example the lactase activity of β-gal (it is active to be also referred to as β-gal among the present invention) be the bacterial strain that 15 units are above, preferred 100 units are above, but be not limited to this.The lactase activity (it is active to be also referred to as P-β-gal among the present invention) of P-β-gal is shown as the bacterial strain that 15 units are above, preferred 45 units are above in every 1mg protein in the also preferred especially screening and culturing supernatant.And the lactase activity (it is active to be also referred to as P-β-glc among the present invention) of P-β-glc is shown as the bacterial strain that 25 units are above, preferred 50 units are above in the every 1mg protein in the preferred especially screening and culturing supernatant.
In the method for the present invention; As stated; Measure enteron aisle adhesivity and lactose decomposing enzyme activity respectively for above-mentioned lactobacillus milk-acid bacteria (more preferably Lactobacillus gasseri or mucous membrane probiotic lactobacillus), the result can screen enteron aisle adhesivity and the active all enhanced lactobacillus strains of lactose decomposing enzyme efficiently.The present invention also relates to the lactobacillus strain of above-mentioned screening.The above-mentioned lactobacillus milk-acid bacteria that obtains well breeds in enteron aisle and settles down, and possesses the ability that can reduce lactose very efficiently, and is therefore very effective for the improvement of lactose intolerance.Among the present invention; " improvement of lactose intolerance " is meant when picked-up contains the diet article (milk-product etc.) of lactose; The frequency that the symptom of digestive tract of the discomfort of lactose intolerance (stomachache, diarrhoea, the rumble sense of belly rumble etc.) occurs reduces or suppresses morbidity fully, and the severity of symptom is alleviated.In addition, " the improvement ability of lactose intolerance " is meant that milk-acid bacteria has the ability that can improve lactose intolerance as stated.
Among the present invention,, can actually obtain all lactobacillus milk-acid bacterias of remarkable three kinds of bacterial strains of enhanced of enteron aisle adhesivity and lactose decomposing enzyme activity through adopting above-mentioned screening method.These three kinds of milk-acid bacterias are Lactobacillus gasseri OLL2836 strain (below be also referred to as the OLL2836 strain), Lactobacillus gasseri OLL2948 strain (below be also referred to as the OLL2948 strain) and mucous membrane probiotic lactobacillus OLL2848 strain (below be also referred to as the OLL2848 strain).These lactobacillus strains are deposited in the independent administrative legal person's products assessment technique basal disc organization and specially permit mikrobe sustenance center.Relevant their preservation information is following.
(1) preservation mechanism name: the independent administrative legal person's products assessment technique basal disc organization speciallys permit mikrobe sustenance center
(2) the communication address of preservation mechanism: Chiba,Japan county wood is the か of Jinshi City ず さ Sickle foot 2-5-8 more
(3) preservation day (former preservation day): on June 9th, 2006
(4) preserving number with receive number:
Lactobacillus gasseri (Lactobacillus gasseri) OLL2836 strain:
Preserving number NITE BP-241 (the reception NITE AP-241 of former preservation)
Lactobacillus gasseri (Lactobacillus gasseri) OLL2948 strain:
Preserving number NITE BP-242 (the reception NITE AP-242 of former preservation)
Mucous membrane probiotic lactobacillus (Lactobacillus mucosae) OLL2848 strain:
Preserving number NITE BP-243 (the reception NITE AP-243 of former preservation)
These OLL2836 strains, OLL2948 strain and the OLL2848 strain of preservation have the character shown in the following table 1.
[table 1]
Figure G2007800237640D00121
(Lactobacilli MRS Broth:MRS substratum, for example Difco is to be suitable for the substratum that these lactobacillus strains are cultivated Ref.NO.288160) to milk-acid bacteria MRS meat soup in the table 1.The typical case of MRS substratum forms as shown in table 2.
[table 2]
Figure G2007800237640D00122
The improvement ability of three kinds of its lactose intolerances of bacterial strain shown in the table 1 is high especially, therefore is particularly conducive in the present invention and utilizes.
2. the lactose intolerance activator that contains milk-acid bacteria of the present invention
Milk-acid bacteria administered through oral of the present invention gives, and can under existing state, arrive intestines, settles down on intestines, and performance intensive lactose decomposing enzyme is active, and the result can improve lactose intolerance.In addition, in the fermented product that uses milk-acid bacteria preparation of the present invention,, improves the high lactose degrading activity owing to making the decomposition efficiency of lactose.
Therefore, the present invention relates to the lactose intolerance activator, that this lactose intolerance activator contains is that above-mentioned screening method obtains by using, the equal remarkable enhanced lactobacillus milk-acid bacteria (milk-acid bacteria of the present invention) of enteron aisle adhesivity and lactose decomposing enzyme activity.Lactose intolerance activator of the present invention is through perhaps having the body that tried of its dubiety to give to the demonstration lactose intolerance; The effect of lactose intolerance can improve; Promptly; The frequency of symptom of digestive tract (stomachache, diarrhoea, the rumble sense of the belly rumble etc.) appearance of the discomfort of lactose intolerance is reduced, perhaps suppress morbidity fully, perhaps can alleviate the severity of the symptom of lactose intolerance.Lactose intolerance activator of the present invention also has the effect of the morbidity of prevention lactose intolerance.
Lactose intolerance activator of the present invention contains a kind of (one more than the bacterial strain) milk-acid bacteria of the present invention at least.Lactose intolerance activator of the present invention preferably contains and is selected from least a as milk-acid bacteria of the present invention of Lactobacillus gasseri OLL2836 strain (preserving number NITE BP-241), Lactobacillus gasseri OLL2948 strain (preserving number NITE BP-242) and mucous membrane probiotic lactobacillus OLL2848 strain (preserving number NITE BP-243).In the particularly preferred embodiment, lactose intolerance activator of the present invention contains three kinds of milk-acid bacterias of Lactobacillus gasseri OLL2836 strain (preserving number NITE BP-241) and Lactobacillus gasseri OLL2948 strain (preserving number NITE BP-242), mucous membrane probiotic lactobacillus OLL2848 strain (preserving number NITE BP-243) at least.The lactose intolerance activator that these three kinds of milk-acid bacteria combinations are contained can also combine with the antigenic enteron aisle Saliva Orthana of any blood type with A type, Type B, O type, and is therefore effective for being tried body widely.
Lactose intolerance activator of the present invention can be the compsn that contains the purifying thalline of milk-acid bacteria of the present invention, also can be the compsn that contains the fermented product, culture or their enriched material that use this milk-acid bacteria preparation.The contained milk-acid bacteria of the present invention of lactose intolerance activator of the present invention can be that the viable bacteria body also can be dead thalline, can be that moistening thalline also can be dry thalline.But in the preferred embodiment, lactose intolerance activator of the present invention is the compsn that contains milk-acid bacteria of the present invention with existing state.Lactose intolerance activator of the present invention can be any forms such as liquid, gel, Powdered, particulate state, solid-like, capsule shape or sheet, but is not limited to this.
Lactose intolerance activator of the present invention adds through an amount of, can make diet article or pharmaceutical composition have lactose intolerance improvement effect.Lactose intolerance activator of the present invention also can contain acceptable carrier or additive on the pharmaceutical prepn except that milk-acid bacteria of the present invention or its culture, fermented product or their enriched material etc. as effective constituent.The example of above-mentioned carrier and additive has: in addition the tensio-active agent of water, the acceptable organic solvent of medicine, collagen, Z 150PH, Vinylpyrrolidone polymer, Carbopol ETD2050, sodium alginate, SG, sodium starch glycolate, pectin, xanthenes glue, Sudan Gum-arabic, casein, gelatin, agar, glycerine, Ucar 35, polyoxyethylene glycol, Vaseline, paraffin, stearyl alcohol, Triple Pressed Stearic Acid, human serum albumin, mannitol, sorbyl alcohol, CMC 99.5, useful as drug additive etc. also have artificial cell works such as liposome etc.The additive that uses can be according to the formulation of preparation suitably or combination selection.
Lactose intolerance activator of the present invention can be oral or non-oral (for example give in the stomach or intestines in give etc.) gives, but special preferred oral gives.The lactose intolerance activator of the present invention of orally give can be formulations such as liquid preparation such as solid preparations such as tablet, granule, powder, pill, capsule, gelifying agent or solution, suspensoid, syrup.When using, can supply with the form of the dry thing of dissolved again when using lactose intolerance activator of the present invention with liquid preparation.
In the above-mentioned formulation, solid preparation for oral use can contain additives such as normally used sticker, vehicle, lubricant, disintegrating agent, wetting agent on the pharmacopedics.Liquid preparation for oral use can contain additives such as normally used stablizer, buffer reagent, correctives, preservatives, perfume compound, tinting material on the pharmacopedics.
The administered dose of lactose intolerance activator of the present invention according to the age that gives object with body weight, give approach, give number of times and different, can on a large scale, change according to those skilled in the art's understanding.For example during orally give, in the lactose intolerance activator of the present invention the administered dose of contained milk-acid bacteria of the present invention be 1～1000mg/kg/ days comparatively suitable.Lactose intolerance activator of the present invention can give by single, but also can give repeatedly with 6～8 hours interval.
Object to giving lactose intolerance activator of the present invention is unqualified, but is preferably the Mammals that comprises people, poultry, pet animals, experiment (test) animal etc.And, for lactose intolerance or infantile period, Adulthood, senile Mammals that its dubiety arranged also preferably as the body that tried of the present invention.More preferably owing to the low Mammals of the lactose resolving power that causes such as old or ill, or the Mammals of inherited genetic factors or environmental factors with lactose intolerance also as the body that tried that gives lactose intolerance activator of the present invention.Therefore lactose intolerance activator few side effects of the present invention can use aspect utilizing continuously very effectively.
3. the diet article that contain milk-acid bacteria of the present invention
The present invention also relates to the diet article, these diet article contain enteron aisle adhesivity and all remarkable enhanced lactobacillus milk-acid bacteria of lactose decomposing enzyme activity that obtains with above-mentioned screening method.Diet article of the present invention are particularly suitable for improving the diet article of lactose intolerance, but are not limited to this." be used to improve lactose intolerance " and be meant tried body as giving object with what show lactose intolerance or its dubiety arranged, the frequency that purpose is to make the symptom of digestive tract (stomachache, diarrhoea, the rumble sense of belly rumble etc.) of the discomfort of lactose intolerance to occur reduces, or stop morbidity fully, perhaps the severity of symptom of lactose intolerance alleviated.
" diet article " among the present invention are unqualified, do not limit for the kind of the diet article that contain milk-acid bacteria of the present invention that comprise beverage, food and functional foodstuff is special.For example, be beverages such as beverage (comprising the fruit juice of orange, apple, grape etc. or the beverage of vegetables juice such as tomato, Radix Dauci Sativae), alcoholic beverage (beer, sparkling wine, wine etc.), soda pop and refreshment drink as can give an example fermented-milk (yogourt etc.), lactobacillus drink, milky-drinks (coffee cow's milk, fruit cow's milk etc.), teas beverage (green tea, black tea and oolong tea etc.), fruit/vegetable of the beverage that contains milk-acid bacteria of the present invention.The preparing methods of various beverages etc. can be with reference to existing book of reference, " up-to-date soft drink " (2003) (Co., Ltd.'s light beautiful jade) etc. for example.
Not limiting the food that contains milk-acid bacteria of the present invention is special, can be that fresh product also can be a processed food, but preferred especially food for example has: can milk-acid bacteria of the present invention be sent to fermented-milk or lactobacillus drink to intestines with the state of survival.The food that contains milk-acid bacteria of the present invention can be the introduction (starter) of milk-product such as preparation yogourt or cheese, fermented-milk.
In addition, the diet article that contain milk-acid bacteria of the present invention are especially preferably as functional foodstuff." functional foodstuff " of the present invention is meant that organism is had certain functional food, for example comprises specific food for health care (comprising special protect [the specific food for health care] of conditional Japan) and comprises the health functional food of trophic function food, special purposes food, dietary supplement, healthy complementary food, dietary supplements (for example various formulations such as tablet, coating, coated tablet, capsule and solution) and beauty food so-called all heath food such as (for example diet food).Functional foodstuff of the present invention also comprises the heath food that the health that is applicable to the codex alimentarius that meets CODEX (the FAO/WHO combination food code council) is stressed sign (health is claimed, Health claim).
As functional foodstuff of the present invention, preferably more specifically example has: special purposes food such as patient's food, pregnant and lying-in women/lactating women's powder is newborn, infant formula is newborn, the elderly's food.Milk-acid bacteria of the present invention can be improved environment in the intestines gradually with the effect of gentleness; Lactose resolving power in the intestines is improved; Improve lactose intolerance; Also can alleviate its symptom; Therefore be particularly suitable for the application (for example can add in the raw material of common baby formula breast) in the weak infant's lactose intolerance of treatment muscle power of Infant Formula Enterprises breast or liquid formulations breast, or the pregnant woman of lactating women's powder breast etc. with or the food used of lactating women, and the elderly's food or the patient's food application in low the elderly of muscle power or patient's lactose intolerance treatment.
As preferred functional foodstuff of the present invention, can also further give an example: be used for the infant's dietary supplements that is tried body, pregnant and lying-in women/lactating women's dietary supplements, the elderly's dietary supplements and patient's dietary supplements congenital or acquired character factor demonstration lactose intolerance.
The preferred example that contains the functional foodstuff of milk-acid bacteria of the present invention has health functional food.The health functional food system be based on domestic and international trend, with the conformability of in the past food system specific for health care, not only consider common food, be the object setting also with the food of processing shapes such as tablet, capsule.Under this system, health functional food comprises two types of specific food for health care (permission type individually) and trophic function food (specification reference model).And, also comprise the special new types such as [specific food for health care] of protecting of sub conditione.
Functional foodstuff of the present invention preferably can import milk-acid bacteria of the present invention in the intestines and settles down, and lactose resolving power in the intestines (preferred persistence) is improved, and the result has the effect of improving lactose intolerance.Purposes beyond functional foodstuff of the present invention can also improve with lactose intolerance is as first purpose.Functional foodstuff of the present invention (preferred specific food for health care or conditional special protect [specific food for health care]) can be put down in writing or mark the effect that improves lactose resolving power in the intestines, improves lactose intolerance or its symptom thus.Above-mentioned record or mark can obtain the mark approval according to the regulation in the health functional food system.For example, in the functional foodstuff of the present invention, should put down in writing " be fit to needs and regulate belly state person " " well keeping the belly state " records such as " suitable should not drink cow's milk person " of " regulating environment in the intestines ", but be not limited to this.
Functional foodstuff of the present invention can be solid preparations such as tablet, granule, powder, pill, capsule; Liquid preparations such as solution, suspensoid, syrup; The perhaps shape of preparation such as gelifying agent can be the shape (for example fermented-milk, beverage, Powdered tealeaves, dessert etc.) of common diet article.
Do not limit the use level of milk-acid bacteria of the present invention in the diet article is special, can be according to each situation and difference.Concrete use level can be considered kind or the required taste or the mouthfeel of diet article, is suitably confirmed by those skilled in the art.But the total amount of common milk-acid bacteria of the present invention is comparatively suitable with the proportional quantity of 0.001～100% (weight), particularly 0.1～100% (weight).
Milk-acid bacteria of the present invention can contain in the diet article through the available proper method arbitrarily in this area.For example, can milk-acid bacteria of the present invention be blended directly in the raw material of diet article.Milk-acid bacteria of the present invention can be coated with, coats, permeates or be sprayed on the diet article.Milk-acid bacteria of the present invention can be evenly dispersed in the diet article, also can locally exist.Also can process the preparation of capsule of having added milk-acid bacteria of the present invention etc.Can be with parcel milk-acid bacterias of the present invention such as edible film or edible seed dressing agents.After adding appropriate excipients etc. in the milk-acid bacteria of the present invention, can be shaped to shapes such as tablet.The diet article that contain milk-acid bacteria of the present invention can further be processed, and above-mentioned processed goods is also contained in the scope of the present invention.And the diet article that especially preferably contain the milk-fermented thing that uses milk-acid bacteria preparation of the present invention.In a word, diet article of the present invention especially preferably contain milk-acid bacteria of the present invention with existing state, but are not limited to this.
In the preparation of diet article of the present invention, can use various additives commonly used in the diet article.Unqualified to additive, can further add developer (Sodium Nitrite etc.), tinting material (cape jasmine dyestuff, red 102 etc.), spices (orange spices etc.), sweeting agent (sweet Stevia, ASPARTAME POWDER BP/USP etc.), sanitas (sodium acetate, Sorbic Acid etc.), emulsifying agent (sodium chondroitin sulfate, propylene glycol fatty acid ester etc.), inhibitor (EDTA disodium, vitamins C etc.), pH regulator agent (Hydrocerol A etc.), chemical seasonings (Sodium Inosinate etc.), thickening material (xanthenes glue etc.), swelling agent (lime carbonate etc.), skimmer (calcium phosphate), sticker (sodium polyphosphate etc.), nutrition-fortifying agent (Creta Preparata, vitamin A etc.) etc.And can further add functional materials such as Radix Ginseng extract, Radix Et Caulis Acanthopanacis Senticosi extract, eucalyptus extract, bark of eucommia tea extract.
Diet article of the present invention contain a kind of (one more than the bacterial strain) milk-acid bacteria of the present invention at least.In the diet article of the present invention; As milk-acid bacteria of the present invention, preferably contain and be selected from least a of Lactobacillus gasseri OLL283 6 strains (preserving number NITE BP-241), Lactobacillus gasseri OLL2948 strain (preserving number NITE BP-242) and mucous membrane probiotic lactobacillus OLL2848 strain (preserving number NITE BP-243).Especially preferred embodiment, diet article of the present invention at least all contain three kinds of milk-acid bacterias of Lactobacillus gasseri OLL2836 strain (preserving number NITE BP-241), Lactobacillus gasseri OLL2948 strain (preserving number NITE BP-242) and mucous membrane probiotic lactobacillus OLL2848 strain (preserving number NITE BP-243).The diet article that this three kinds of milk-acid bacterias combination is contained can also with have A type, Type B, the antigenic enteron aisle Saliva Orthana of any blood type of O type and combine, so effective to being tried body widely.
To picked-up or give diet article of the present invention tried that body is not special to be limited, be preferably and comprise people, domestic animal (pig, horse etc.), pet animals (dog, cat etc.), test (test) animal the Mammals of (rodent such as mouse, rat or rabbit etc.).And; The infantile period, Adulthood, senile Mammals that also are preferably lactose intolerance or its dubiety arranged are as the body that tried of the present invention; In addition, owing to old and ill etc. cause the low Mammals of lactose resolving power, or the Mammals of inherited genetic factors or environmental factors with lactose intolerance also preferably as the body that tried that absorbs or give diet article of the present invention.
Embodiment
Below, adopt embodiment further to specify the present invention.But technical scope of the present invention is not limited to these embodiment.
[embodiment 1] supplies cultivation and the preparation of examination milk-acid bacteria
Below employed confession examination milk-acid bacteria used the lactobacillus strain accepting Mingzhi Dairy Co., Ltd and provide, preserved the lactobacillus strain that facility (JCM), Agriculture, Forestry and Fisheries Ministry livestock products experiment (NIAI) and Something English bacterium DSMZ (NCFB) obtain by American type culture collection (ATCC), RIKEN's microflora from the people; And the inventor etc. uses modification LBS agar, by the isolating totally 104 strain bacterial strains of child's ight soil.These supply the examination milk-acid bacteria except that the various bacterial strains that belong to lactobicillus bulgaricus (Lactobacillus bulgaricus), Lactobacterium acidophilum (Lactobacillusacidophilus), newborn probiotic lactobacillus (Lactococcus lactis), Lactobacillus gasseri (Lactobacillusgasseri), lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus johnsonii (Lactobacillus johnsonii) and mucous membrane probiotic lactobacillus (Lactobacillus mucosae), also comprise the unidentified bacterial strain of bacterial classification.Need to prove that the bacterial strain that the bacterial strain name is recited as OLL or MEP is expressed as the bacterial strain that Mingzhi Dairy Co., Ltd possesses.
Each supplies thalline of examination milk-acid bacteria to be scattered in 10% (w/v) degreasing milk medium (Snow Brand Milk Products Co., Ltd, Sapporo) of sterilization, before using in deep refrigeration, preserve down at-80 ℃.
In the cultivation, each bacterial strain is in MRS substratum (Difco, Detroit; The state of Michigan, the U.S.) middle succeeding transfer culture three times (37 ℃, 24 hours); Twice of succeeding transfer culture (37 ℃, 24 hours) in MRSL substratum (2% glucose sugar being replaced into the MRS substratum of lactose gained) then.Then, the gained bacterial culture fluid is inoculated in the freshly prepd MRSL substratum with 1%, cultivated 18 hours down at 37 ℃ then.This nutrient solution of preparation carries out centrifugal (6,000 * g, 20 minutes, 4 ℃) like this, and the thalline of collecting is cleaned with 0.05M phosphoric acid buffer (pH 6.8), is suspended in then in the zero(ppm) water, carries out lyophilize then.
The screening of [embodiment 2] enteron aisle adhesivity bacterial strain
Employing waits being used to of being developed to study the mensuration system with the mucinous binding ability of enteron aisle by inventor; By the bacterial strain of screening enteron aisle adhesivity high (the RU value is more than 100) in the above-mentioned confession examination lactobacillus strain, employed mensuration system write up is in International Application No. WO 2006/067940.
Specifically, through using biosensor BIACORE1000 (BIACORE company), milk-acid bacteria and people A type enteron aisle Saliva Orthana, human B-type enteron aisle Saliva Orthana, the mucinous binding ability of people O type enteron aisle are carried out the surface plasma body resonant vibration spectrum analysis and measure.
1. the preparation of assay solution
After playing bacterium, at first, cultivated 12 hours down, divide with 500 μ L then and annotate in the centrifuge tube of 1.5mL capacity at 37 ℃ with each bacterial strain subculture three times in the MRS substratum.This nutrient solution is carried out centrifugal (6,000rpm, 4 ℃, 10 minutes), remove supernatant, the gained thalline is cleaned twice with PBS damping fluid (pH 7.2), be suspended in the zero(ppm) water lyophilize then.This thalline is prepared into 0.1mg/mL concentration with HBS-EP damping fluid (0.01M HEPES (pH7.4), 0.15M NaCl, 3mM EDTA, 0.005% tensio-active agent P20), as assay solution.
2. the preparation of people's enteron aisle Saliva Orthana (HCM) and be fixed with the making of the sensing chip of HCM
Carry out the mucinous preparation of people's enteron aisle and people's enteron aisle Saliva Orthana fixing on sensing chip.People A type enteron aisle (is the people's of A type large intestine from blood type), human B-type enteron aisle (is the people's of Type B large intestine from blood type) and people O type enteron aisle (is the people's of O type large intestine from blood type) are as the collection of specimens sample, accept the dispensing of research section of department of medial science of big institute of northeastern Japan university.Need to prove that the collection of this sample is implemented, and has obtained patient's agreement after Ethics Committee's approval of research section of department of medial science of big institute of northeastern Japan university.Scrape through the top layer and to follow the example of, gather mucus Saliva Orthana layer by their normal position of large intestine of enteron aisle.Mucus Saliva Orthana layer is through Folch solvent (chloroform-methanol mixed solution (2: 1 (v/v)); People such as J.Folch.: J.Biol.Chem. (1957) 226,497～500 pages) and diethyl ether carry out the degreasing after drying, extracted 2 hours down at 37 ℃ with 4 M guanidine hydrochloride solutions.Then carry out purifying through gel-filtration for this extract.The gel-filtration method of purification is according to people such as Purushothaman S.S.; " Adherence ofShigella dysenteriae 1 to Human Colonic Mucin. " Curr.Miorobiol.; 42 (6), the mucinous purification process of people's large intestine that 381～387 pages (2001) are put down in writing is implemented.Fluidised bed uses 4 M guanidine hydrochloride solutions, and the post use Toyopearl HW-65F that gel filtration chromatography is used (90cm * 2.6cm, Tosoh.Tokyo.Japan).For gained post extracting solution; Measure neutral sugar through phenol sulfuric acid process (absorbancy of 490nm is measured in the reaction back); Through the absorbance measurement protein of 280nm, the result selects to have protein adsorption (absorbancy of 280nm) and the highest peak of neutral sugar content; Further about with molecular weight in the gel filtration chromatography is index more than 2,000,000, obtains large intestine Saliva Orthana component.The purifying thing that obtains like this is corresponding with the people's in source blood type, processes people A type enteron aisle Saliva Orthana, human B-type enteron aisle Saliva Orthana, people O type enteron aisle Saliva Orthana respectively.Below, in these people's enteron aisle Saliva Orthanas (human colon muci:HCM), A type enteron aisle Saliva Orthana is called A-HCM, Type B enteron aisle Saliva Orthana is called B-HCM, O type enteron aisle Saliva Orthana is called O-HCM.For each HCM of gained, confirm the human blood type matrix antigen property of each blood type through antigen antibody reaction.
Above-mentioned each HCM that obtains, carries out through the amine coupling method with on the sensing chip fixedly being in biosensor BIACORE1000 (BIACORE company) at BIACORE.At first; In the sensing chip CM5 that has imported the Sensor Chip CM 5 base in advance (BIACORE company), feed the mix reagent (EDC/NHS) that 50 μ L (75.0mg/mL) N-ethyl-N '-3-dimethylaminopropyl-carbodiimide hydrochlorides (EDC) and 50 μ L (11.5mg/mL) N-hydroxy-succinamides (NHS) are mixed, make the activated carboxylic in the Sensor Chip CM 5.Then; To the fixing solution of HCM arbitrarily (A-HCM, B-HCM, O-HCM) of 120 μ L, preparation mixing solutions with the above-mentioned purifying of adding 30 μ L in the acetate buffer (10mM, pH 4.0); Flow into sensing chip, make the carboxyl covalent attachment on HCM and the sensing chip through the amine linked reaction.And, use 1M ethanolamine hydrochloric salt-NaOH (pH 8.5), carry out the sealing of the residual activity group at the position on the sensing chip of bonding probes not.Use the HBS-EP damping fluid as electrophoretic buffer.HCM fixed amount to represent with the difference of adding ethanolamine solutions display data (reportpoint) afterwards before the importing EDC/NHS is 1000～2000RU.Then, the sensing chip that is fixed with HCM that obtains like this is used for the test of above-mentioned assay solution.
3. surface plasma body resonant vibration spectrum analysis
Further use biosensor BIACORE1000 (BIACORE company), the binding capacity of the milk-acid bacteria thalline in HCM on the sensing chip that respectively is fixed with HCM and the above-mentioned assay solution is analyzed.The condition determination of the BIACORE1000 that uses during this is analyzed is as follows.
Electrophoretic buffer: HBS-EP damping fluid (pH 7.4)
The addition of assay solution (sample): 20 μ L
Flow velocity: 3 μ L/ minutes
Temperature of reaction: 25 ℃
Regeneration soln: 1M guanidinesalt acid salt solution 5 μ L
In the analysis of carrying out through the BIACORE system, the binding capacity of assay and probe (interaction) is through being the value representation of unit with resonance units (RU).1 resonance units (RU) is meant every 1mm 2Be combined with the 1pg material on the sensing chip surface.That is, according to the combination/dissociation curve that is obtained by this analysis, the value that deducts the RU value gained before combining by the RU value after combining is equivalent to as each milk-acid bacteria of assay and as every 1mm of probe 2The mucinous binding capacity of each enteron aisle.This binding capacity is equivalent to this milk-acid bacteria and the mucinous binding ability of each enteron aisle.
In this analytical results, supply to screen the examination milk-acid bacteria bacterial strain that shows the value more than the 100RU with the mucinous binding ability of at least a enteron aisle from 104 strains.We can say in the bacterial strain of screening that the enteron aisle adhesivity strengthens (the enteron aisle adhesivity is high) than other average lactobacillus strain.The representative example of the bacterial strain of screening and analytical results thereof are shown in Fig. 1～3.
Synthetic and the purifying of the ONPGlc-6P that uses in [embodiment 3] P-β-glc determination of activity
Below, attempt further screening and have the high and active bacterial strain of high lactose lytic enzyme of enteron aisle adhesivity.Here, in the present embodiment, the preparation of the ONPGlc-6P that at first carries out using in this determination of activity.
1.ONPGlc-6P chemosynthesis
Compound method (Hengstenberg according to Hengstenberg and Morse; W. and M.L.Morse.; Synthesis of o-Nitrophenyl-beta-D-galactopyranoside 6-phosphate; Carbohydr.Res., 10,463～465 pages (1969)) the modification method chemosynthesis at phosphoric acid-beta-glucosidase enzyme (ortho-nitrophenyl base-β-D-glycopyranoside-6-SULPHOSUCCINIC ACID ESTER (ONPGlc-6P) that uses in the determination of activity of P-β-glc).Specifically, at first with 0.9g ortho-nitrophenyl base-β-D-glycopyranoside (ONPGlc, Sigma, St. Louis, the U.S.) be dissolved in advance in cooled on ice, blended contains in the 7.5mL trimethyl phosphite 99 of 54 μ L zero(ppm) water and 840 μ L Phosphorus Oxychlorides then.Then this mixed solution was stirred on ice 5 hours, react.The reaction back adds borneol (zero(ppm) water), uses pH meter neutralization solution (pH 7.0), termination reaction while drip strong aqua.
2.ONPGlc-6P purifying
For the solution that makes the stopping of reaction in above-mentioned, the concentrate drying that uses rotatory evaporator (Tokyo natural sciences machinery, Tokyo) to carry out repeatedly under 40 ℃ solidifies, and removes the ortho-nitrophenyl base (ONP) that generates in the dereaction.Then, spissated reaction solution is mixed with the 50g activated carbon, under 4 ℃, left standstill 2 hours, the ONPGlc-6P of chemosynthesis and unreacted ONPGlc are adsorbed on the activated carbon, with its zero(ppm) water (2L) washing, carry out desalination then with 20 times of amounts.The wash-out that is adsorbed on the ONPGlc-6P on the activated carbon is to use pyridine: water: (1: 1: 1 (v/v/v) 600mL) carries out alcohol mixeding liquid.Elutriant concentrates through rotatory evaporator down at 40 ℃ repeatedly, removes pyridine.Then, supply with the preparation ply of paper and analyse (PPC).That is, use 100 μ L micropipets, the ONPGlc-6P of the above-mentioned thick purifying shape that is in line is coated on PPC with (Whatman on the filter paper; 3MM; 46 * 57cm, Maidstone, England); Use butanols: pyridine: water (6: 4: 3 (v/v/v)) carries out one-dimensional development as launching solvent through rise method.Expansion needs 2 day time, after launching to finish, in the ventilating chamber, makes this filter paper dry, uses UV transilluminator (302nm, FUNAKOSHI, Tokyo) to confirm the band of ONPGlc-6P and ONPGlc then, only cuts the band of ONPGlc-6P.The filter paper that cuts is dipped in the zero(ppm) water,, carries out the extraction of ONPGlc-6P thus 4 ℃ of following stirred overnight.Remove filter paper after the extraction, concentrate water layer, use then, remove by gel filtration and mix the byproduct of reaction that exists with the TOYOPEARL HW40S post (2.6 φ * 90cm, eastern ソ one, Tokyo) of distilled water as mobile phase by rotary evaporator.After the gel-filtration each elution fraction is used thin-layer chromatography (TLC, silica gel 60,10 * 10cm, Merck, Darmstadt, Germany), reclaim the ONPGlc-6P component.Use butanols in the expansion solvent of TLC: the 2-propyl alcohol: water (3: 12: 4 (v/v/v)) carries out one-dimensional development.After air-dry, 5% (v/v) sulfuric acid-methanol solution of on thin layer plate, spraying equably detected at moisture eliminator (Yamato, the Tokyo) internal heating of heating to 150 ℃ in 3 minutes.Purifying ONPGlc-6P is processed in the lyophilize of the ONPGlc-6P component that reclaims.Confirm purity for these purifying article through TLC, through 1H-NMR carries out the affirmation of structure.
[embodiment 4] β-gal, P-β-gal and the active mensuration of P-β-glc
1 04 strains for preparation among the embodiment 1 supply the examination milk-acid bacteria, use the Fisher method to carry out the mensuration of the lactase activity of β-gal, P-β-gal and P-β-glc.That is,, 0.5mg freeze-drying thalline is suspended in the 1.0mL 0.05M phosphoric acid buffer (pH 6.8), adds 50 μ L toluene-acetone mixed solutions (1: 9 (v/v)), vigorous stirring 3 minutes each bacterial strain.In 25 these suspension-s of μ L, add the 0.05M phosphoric acid buffer (pH 6.8) that 100 μ L contain the ortho-nitrophenyl base-β-D-glycopyranoside-6-SULPHOSUCCINIC ACID ESTER (ONPGlc-6P) of ortho-nitrophenyl base-β-D-galactopyranoside (ONPGal), ortho-nitrophenyl base-β-D-galactopyranoside-6-SULPHOSUCCINIC ACID ESTER (ONPGal-6P) or above-mentioned preparation, reacted 15 minutes down at 37 ℃.The reaction back adds 125 μ L 0.5M sodium carbonate solution stopped reactions, removes the thalline composition through centrifugal (6,000 * rpm, 10 minutes, 4 ℃), uses MultiskanMS-UV (Labsystems, Helsinki, Finland) to measure the absorbancy of the 405nm of supernatant.Being defined as 1 unit with free 1 μ mol o-NP (ONP) in 1 minute, is unit with this unit, calculates contained every 1mg activity of proteins value in activity value and the culture supernatant of every 1mg thalline respectively.
Need to prove that the proteinic Micro BCA protein determination kit (Pierce, Rockford, Illinois, the U.S.) that quantitatively is to use based on the BCA method after reaction 2 hours, uses Multiskan MS-UV to measure the absorbancy of 540nm.Calibration curve is to use bovine serum albumin (BSA) to make, and calculates proteinic amount thus.Provided the result of the lactase activity mensuration of the typical example of the bacterial strain of screening among the embodiment 2 in Fig. 1～3.
The screening of [embodiment 5] lactobacillus strain
Adhere to living and β-gal, P-β-gal and the active mensuration result of P-β-glc according to the enteron aisle shown in the foregoing description, carry out the screening of the also high lactobacillus strain of enteron aisle adhesivity height and lactase activity.As a result, all show remarkable highly active bacterial strain, screened three kinds of lactobacillus strains (OLL2836 strain, OLL2948 strain and OLL2848 strain) (Fig. 1～3) as any lactase activity among enteron aisle adhesivity height and β-gal, P-β-gal and the P-β-glc.And most of lactase activity is than higher lactobacillus strain, and its enteron aisle adhesivity low (for any type people's enteron aisle Saliva Orthana of A type, Type B, O type, the RU value all is lower than 100RU) therefore is excluded.
Shown in Fig. 1～3; The OLL2836 strain shows that the proteinic P-β of 46.576 units/mg-gal is active; The OLL2948 strain shows that the proteinic P-β of 50.194 units/mg-glc is active; The OLL2848 strain shows that the proteinic β of 107.090 units/mg-gal is active, but other high lactobacillus strain comparison of these activity values and enteron aisle adhesivity shows very high value respectively.And the adhering RU value of the demonstration enteron aisle of these bacterial strains also is to show very high value.Specifically, OLL2836 strain and human B-type enteron aisle Saliva Orthana and people O type enteron aisle Saliva Orthana show especially strong binding ability, and OLL2948 strain and OLL2848 strain and people A type enteron aisle Saliva Orthana show strong especially binding ability.
Show that by above result for these lactobacillus strains, they are settled in the enteron aisle separately respectively; Be expected to obtain the effect that continues to reduce lactose; In addition, through these three kinds of strain combinations are used, tried all to have shown in the body that at any blood type can obtain high lactose decomposes effect.
The strain identification test is further carried out in OLL2836 strain, OLL2948 strain and OLL2848 strain; The result who identifies shows: OLL2836 strain and OLL2948 strain belong to Lactobacillus gasseri (Lactobacillus gasseri), and the OLL2848 strain belongs to mucous membrane probiotic lactobacillus (Lactobacillusmucosae) (stating embodiment after the reference).These three kinds of lactobacillus strains are preserved in independent administrative corporation's goods evaluation technique basal disc assessing mechanism special permission mikrobe sustenance center (Chiba,Japan county wood is the か of Jinshi City ず さ Sickle foot 2-5-8 more) respectively at application on June 9th, 2006 (former preservation day).The reception of this preservation application is number as follows: the OLL2836 strain is NITE AP-241, and the OLL2948 strain is NITEAP-242, and the OLL2848 strain is NITE AP-243; Preserving number is respectively: MTE P-241, NITE P-242, NITE P-243.These preservation strains shift on March 27th, 2007 and are the preservation (international preservation) according to budapest treaty, with on June 9th, 2006 as the preservation of former preservation day.This world preserving number is following: the OLL2836 strain is NITE BP-241, and the OLL2948 strain is NITE BP-242, and the OLL2848 strain is NITE BP-243.The microbiological property of these bacterial strains has been confirmed as like above-mentioned table 1 said.
As stated, the screening method of the application of the invention can obtain the also high lactobacillus strain of enteron aisle adhesivity height and lactase activity.And β-gal, P-β-gal and P-β-glc are measured lactase activity; Screen three kinds of lactose decomposing enzymes and show remarkable highly active bacterial strain respectively; Through strain combinations, can also obtain to make up applicable to the very effective milk-acid bacteria of being tried body widely with gained.Think that the lactobacillus strain that obtains in the method for the present invention is settled in the enteron aisle, the effective probiotic lactic acid bacterial strain as continuing to reduce lactose can be effective to improve lactose intolerance.
[embodiment 6] strain identification
Strain identification is carried out in three kinds of lactobacillus strain OLL2836 strain, OLL2948 strain, OLL2848 strain for screening among the embodiment 5.Evaluation is based on amplification, the sequence in V3 district to be confirmed, carries out through 16S rDNA nucleotide sequence analysis.Below, to set forth as example with the test of using OLL2848 strain and OLL2948 strain to carry out, the OLL2836 strain is also basically likewise identified.
1. detect the 16S rDNA fragment of screening strain through direct PCR method
Succeeding transfer culture is carried out in OLL2848 strain and OLL2948 strain as stated on the MRS substratum, carry out direct PCR then.With injecting 4 μ LmilliQ water in the micro tube of 0.6mL capacity respectively, be suspended in wherein respectively using the sterilization toothpick to scrape the thalline of getting.In this suspension-s, add Taq archaeal dna polymerase (TaKaRa, capital of a country), 10 * PCR damping fluid, dNTP Mix (TaKaRa, capital of a country), one group of primer are processed the PCR reaction solution.One group of primer is to use universal primer-27F (5 '-GAGTTTGATCCTGGCTCAG-3 ', SEQ ID NO.1) and 518R (5 '-ATTACCGCGGCTGCTGG-3 ', SEQ ID NO.2).The PCR condition is to carry out 1 [95 ℃ 10 minutes, 55 ℃ 3 minutes, 72 ℃ 1 minute] circulation, and 39 [95 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds] circulations circulate with 1 [72 ℃ 1 minute].
2. agarose gel electrophoresis
Then, will carry out electrophoresis through the dna fragmentation of above PCR reaction amplification.Electrophoresis be with 2% sepharose as running gel, electrophoretic buffer uses 1 * tbe buffer liquid (90mM Tris-boric acid, 2mM EDTA, pH 8.0), under the constant voltage of 100V, carries out.Electrophoresis apparatus uses Mupid-2 (Cosmo Bio, Tokyo).Molecular weight marker uses 100b DNA (TaKaRa, capital of a country).The dyeing of the gel behind the electrophoresis is in ethidium bromide (EB) solution (0.5 μ g/mL), to soak to carry out in 10 minutes.
3. by extracting DNA in the sepharose
Carry out UV irradiation through the gel of UV transilluminator TM-20 (FUNAKOSHI medicine Co., Ltd., Tokyo) after, confirm that the target dna fragment also cuts agarose gel electrophoresis and dyeing.Extract DNA through dna fragmentation purification kit MagExtractor (TOYOBO, Osaka).That is, the sepharose that cuts is transferred in the pipe of 1.5mL capacity, adds 400 μ L adsorption liquids then, heat, make it to dissolve fully to 55 ℃.The magnetic beads that then in this reaction solution, adds 15 μ L stirs, and at room temperature places then 1 minute.Centrifugal (6,000rpm, 5 seconds, 4 ℃) are removed supernatant then, and the washings that adds 500 μ L stirs.Recentrifuge (6,000rpm, 5 seconds, 4 ℃) adds 75% ethanol of 1mL then, stirs, and centrifugal (15,000rpm, 1 minute, 4 ℃) are removed supernatant then.Add the zero(ppm) water of 15 μ L and stir, supernatant that then will be through centrifugal (15,000rpm, 1 minute, 4 ℃) recovery is as purify DNA solution.
4.DNA confirming and homology analysis of sequence
For the DNA of purifying, use primer 2 7F (5 '-GAGTTTGATCCTGGCTCAG-3 ', SEQ ID NO.3) to carry out nucleotide sequence and confirm.The definite of nucleotide sequence undertaken by OPERONBAIOTEKUNOROJI Co., Ltd..Like this, the nucleotides sequence in the V3 district of the 16S rDNA that the OLL2848 strain is confirmed is classified SEQ ID NO.4 as, and the nucleotides sequence in the V3 district of the definite 16S rDNA of OLL2948 strain is classified as SEQ ID NO.5.For these nucleotide sequences, on the website of Japanese DNA DB (DDBJ), carry out homology analysis through program BLASTN.
As a result, the sequence in the V3 district of the nucleotide sequence in the V3 district of OLL2848 strain and mucous membrane probiotic lactobacillus shows 99.6% homology.And the sequence in the V3 district of the nucleotide sequence in the V3 district of OLL2948 strain and Lactobacillus gasseri shows 99.2% homology.Can identify thus: the OLL2848 strain belongs to the mucous membrane probiotic lactobacillus, and the OLL2948 strain belongs to Lactobacillus gasseri.
Industrial applicability
Method of the present invention can be used for efficiently screening improving the effective lactobacillus strain of lactose intolerance.The lactobacillus strain that is obtained by method of the present invention can give the patient easily, and can be used as the medicine that improves lactose intolerance sustainably or the effective constituent of functional foodstuff is advantageously used.
The free text of sequence table
The sequence of SEQ ID NO.1～3 is a primer.
All all being incorporated in this explanation of all publications, patent and the patented claim of quoting in this specification sheets through reference.
Sequence table
< 110>Food Science Inst Foundation
Corporate National University Northeastern University
 
< 120>be used to improve the milk-acid bacteria of lactose intolerance
 
<130>PH-3099-PCT
 
<150>JP?2006-175897
<151>2006-06-26
 
<160>5
 
<170>PatentIn?Ver.2.1
 
<210>1
<211>19
<212>DNA
< 213>artificial sequence
 
<220>
< 223>kind of artificial sequence: primer
 
<220>
< 223>contriver: Saito, Tadao; Kitazawa, Haruki
Contriver: Kawai, Yasushi; Itoh, Hiroyuki
 
<400>1
gagtttgatc?ctggctcag 19
 
<210>2
<211>17
<212>DNA
< 213>artificial sequence
 
<220>
< 223>kind of artificial sequence: primer
 
<400>2
attaccgcgg?ctgctgg 17
 
<210>3
<211>19
<212>DNA
< 213>artificial sequence
 
<220>
< 223>kind of artificial sequence: primer
 
<400>3
gagtttgatc?ctggctcag 19
 
<210>4
<211>559
<212>DNA
< 213>mucous membrane probiotic lactobacillus
 
<400>4
tgggggaggg?gggggggggg?ggggccggcg?gtgtgtctct?atatacatgc?aagtccgaac?60
gcgttggccc?aactgattga?acgtgcttgc?acggaacttg?acgttggttt?accagcgagt?120
ggcggacggg?tgagtaacac?gtaggtaacc?tgccccaaag?cgggggataa?catttggaaa?180
cagatgctaa?taccgcataa?caatttgaat?cgcatgattc?aaatttaaaa?gatggcttcg?240
gctatcactt?tgggatggac?ctgcggcgca?ttagcttgtt?ggtagggtaa?cggcctacca?300
aggctgtgat?gcgtagccga?gttgagagac?tgatcggcca?caatggaact?gagacacggt?360
ccatactcct?acgggaggca?gcagtaggga?atcttccaca?atgggcgcaa?gcctgatgga?420
gcaacaccgc?gtgagtgaag?aagggtttcg?gctcgtaaag?ctctgttgtt?agagaagaac?480
gtgcgtgaga?gcaactgttc?acgcagtgac?ggtatctaac?cagaaagtca?cggctaacta?540
cgtgccagca?gcccgcggt 559
 
<210>5
<211>524
<212>DNA
< 213>Lactobacillus gasseri
 
<400>5
ggcgtgcctt?atacatgcaa?gccgagcgag?cttgcctaga?tgaattttgg?tgcttgcacc?60
acgatgaaac?tagatacaag?cgagcggcgg?acgggtgagt?aacacgtggg?taacctgccc?120
aagagactgg?gataacacct?ggaaacagat?gctaataccg?gataacaaca?ctagacgcat?180
gtctagagtt?taaaagatgg?ttctgctatc?actcttggat?ggacctgcgg?tgcattagct?240
agttggtaag?gtaacggctt?accaaggcaa?tgatgcatag?ccgagttgag?agactgatcg?300
gccacattgg?gactgagaca?cggcccaaac?tcctacggga?ggcagcagta?gggaatcttc?360
cacaatggac?gcaagtctga?tggagcaacg?ccgcgtgagt?gaagaagggt?ttcggctcgt?420
aaagctctgt?tggtagtgaa?gaaagataga?ggtagtaact?ggcctttatt?tgacggtaat?480
tacttagaaa?gtcacggcta?actacgtgcc?agcagcccgc?ggta 524

Claims (6)

1. lactobacillus milk-acid bacteria; It is enteron aisle adhesivity and lactose decomposing enzyme active all enhanced Lactobacillus gasseri (Lactobacillus gasseri) or mucous membrane probiotic lactobacillus (Lactobacillus mucosae); Described lactobacillus milk-acid bacteria uses RU value that the surface plasma body resonant vibration analysis measures at least a binding ability in milk-acid bacteria and people A type enteron aisle Saliva Orthana, human B-type enteron aisle Saliva Orthana and the people O type enteron aisle Saliva Orthana as more than the 300RU; And have and be selected from following at least a lactase activity; Wherein said lactase activity is: the lactase activity that the lactase activity of β-gal is shown as P-β-gal in the above lactase activity of 100 units, the every 1mg protein in the culture supernatant in the every 1mg protein in the culture supernatant is shown as the above lactase activity of 45 units; And the lactase activity of P-β-glc is shown as the above lactase activity of 50 units in the every 1mg protein in the culture supernatant, wherein said milk-acid bacteria be preserving number be the Lactobacillus gasseri OLL2836 strain of NITE BP-241, Lactobacillus gasseri OLL2948 strain that preserving number is NITE BP-242 and preserving number be NITE BP-243 mucous membrane probiotic lactobacillus OLL2848 strain in any one milk-acid bacteria.
2. lactose intolerance activator, this activator contain any one the milk-acid bacteria in the mucous membrane probiotic lactobacillus OLL2848 strain that Lactobacillus gasseri OLL2836 strain that preserving number is NITE BP-241, Lactobacillus gasseri OLL2948 strain that preserving number is NITE BP-242 and preserving number be NITE BP-243 at least.
3. the described lactose intolerance activator of claim 2, this activator contain three kinds of milk-acid bacterias of the mucous membrane probiotic lactobacillus OLL2848 strain that Lactobacillus gasseri OLL2836 strain that preserving number is NITE BP-241, Lactobacillus gasseri OLL2948 strain that preserving number is NITEBP-242 and preserving number be NITE BP-243 at least.
4. be used to improve the diet article of lactose intolerance, these diet article contain any one the milk-acid bacteria in the mucous membrane probiotic lactobacillus OLL2848 strain that Lactobacillus gasseri OLL2836 strain that preserving number is NITE BP-241, Lactobacillus gasseri OLL2948 strain that preserving number is NITE BP-242 and preserving number be NITE BP-243 at least.
5. described diet article of claim 4, these diet article contain three kinds of milk-acid bacterias of the mucous membrane probiotic lactobacillus OLL2848 strain that Lactobacillus gasseri OLL2836 strain that preserving number is NITEBP-241, Lactobacillus gasseri OLL2948 strain that preserving number is NITE BP-242 and preserving number be NITE BP-243 at least.
6. claim 4 or 5 described diet article, these diet article are selected from milk formula, junior food, nursing women's food, the elderly's food, patient's food, health functional food, dietary supplements, fermented-milk and lactobacillus drink.
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JP6230050B2 (en) * 2013-10-09 2017-11-15 国立研究開発法人農業・食品産業技術総合研究機構 Lactic acid bacteria with gastrointestinal mucosa adhesion
SI24570A (en) 2013-12-23 2015-06-30 Medis D.O.O. New strains of the genus Lactobacillus and use thereof
AU2015226184A1 (en) * 2014-03-05 2016-09-01 Dsm Ip Assets B.V. Liquid lactase compositions
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US20230149482A1 (en) 2020-04-08 2023-05-18 Megmilk Snow Brand Co., Ltd. Composition for improving gut microbiota
CN111979145B (en) * 2020-08-07 2022-06-28 上海交通大学 Human-derived lactobacillus mucosae and application thereof
WO2024013211A1 (en) 2022-07-12 2024-01-18 Novozymes A/S Method for providing relief from lactose induced abdominal discomfort

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006059408A1 (en) * 2004-12-01 2006-06-08 Meiji Dairies Corporation Lactic acid bacterium binding to human abo blood types

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2711095B2 (en) * 1986-09-27 1998-02-10 ユニチカ株式会社 Production method of growth promoter of bifidobacterium
JP3436581B2 (en) * 1994-03-18 2003-08-11 雪印乳業株式会社 Method for producing cells with high lactose-degrading enzyme activity
US8871266B2 (en) * 2003-10-01 2014-10-28 Commonwealth Scientific & Industrial Research Organisation Probiotic storage and delivery

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006059408A1 (en) * 2004-12-01 2006-06-08 Meiji Dairies Corporation Lactic acid bacterium binding to human abo blood types

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Colum Dunne et al..In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings.《Am J Clin Nutr》.2001,第73卷386S-392S. *
D R Mack et al.Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro.《Gut》.2003,第52卷827-833. *
Hideaki Uchida et al..Lactobacilli binding human A-antigen expressed in intestinal mucosa.《Research in Microbiology》.2006,第157卷659-665. *
Stefan Roos et al.Lactobacillus mucosae sp. Nov., a new species with in vitro mucus-binding activity isolated from pig intestine.《International Journal of Systematic and Evolutionary Microbiology》.2000,第50卷251-258. *
StefanRoosetal.Lactobacillusmucosaesp.Nov. a new species with in vitro mucus-binding activity isolated from pig intestine.《International Journal of Systematic and Evolutionary Microbiology》.2000

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