CN101479382A - Lactic acid bacterium for amelioration of lactose intolerance - Google Patents
Lactic acid bacterium for amelioration of lactose intolerance Download PDFInfo
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- CN101479382A CN101479382A CNA2007800237640A CN200780023764A CN101479382A CN 101479382 A CN101479382 A CN 101479382A CN A2007800237640 A CNA2007800237640 A CN A2007800237640A CN 200780023764 A CN200780023764 A CN 200780023764A CN 101479382 A CN101479382 A CN 101479382A
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- milk
- strain
- acid bacteria
- lactobacillus
- enteron aisle
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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
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 at infantile period also as seen.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, becomes very big problem aspect nutrition.Osteoporosis is to threaten one of maximum disease along with the booming Japan of aging, from reducing the angle of senile osteoporosis disease risk, promotes the positive picked-up of milk-product to be even more important.
Lactose decomposes, absorbs by the lactose decomposing enzyme that produces in the cell of small intestine internal layer 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 so that the symptom of lactose intolerance to take place, and, by orally ingestible constantly, interior these bacterium of intestines are slowly bred, and the lactose capacity of decomposition 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 participate 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 survive in people's enteron aisle 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, in order to improve lactose intolerance by the orally give milk-acid bacteria, lactase activity height just not also must have to continue the milk-acid bacteria that reduces lactose in intestines, 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: by 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 for people's initial stage being infected and being 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
The loyal husband of non-patent literature 1: Lent rattan, the spacious Min, Hall Ye Righteousness man of her rattan, mountain Qi You Ji ヒ ト Intestinal pipe origin Lactobacillus gasseri) (ガ セ リ bacterium) To お け ゐ ラ Network ト — ス Capitalization Xi Xin Longitude Lu development See (discovery of the new way of the lactose utilization system in the Lactobacillus gasseri of people's enteron aisle origin), biological engineering Hui Chi, (2001), 79 (6), 172～173 pages
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, on the alimentary canal system チ Application blood type Tang Lock Agencies make-the Fine bacterium infects メ カ ニ ズ system and separates bright To け て (the blood type sugar chain structure on the digestive tube Saliva Orthana-infectation of bacteria 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 found that: in the lactobacillus milk-acid bacteria of a part, its enteron aisle adhesivity and lactose decomposing enzyme activity all strengthen, and have finished the present invention based on this understanding.
That is, the present invention comprises following content.
[1] screening has the method for milk-acid bacteria of the improvement ability of lactose intolerance, 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.
In this method, more preferably in the screening of this bacterium, 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 is measured in the analysis of use surface plasma body resonant vibration, and 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 by the bacterium of screening.
In this method, the lactobacillus milk-acid bacteria of preferably screening is Lactobacillus gasseri (Lactobacillus gasseri) or mucous membrane Bacterium lacticum (Lactobacillus mucosae).
[2] 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.
[3] 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 Bacterium lacticum OLL2848 strain (preserving number NITE BP-243).
[4] 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 NITE BP-241), Lactobacillus gasseri OLL2948 strain (preserving number NITE BP-242) and mucous membrane Bacterium lacticum OLL2848 strain (preserving number NITE BP-243) at least.
[5] diet product, these diet product contain a kind of above-mentioned [3] described milk-acid bacteria at least.
These diet product 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 Bacterium lacticum OLL2848 strain (preserving number NITE BP-243) at least.
These diet product are preferably infant food, 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 product especially are suitable as and improve the diet product 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 product.
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 represents P-β-gal activity test of result carry out to(for) the high representative bacterial strain of enteron aisle adhesivity.
Fig. 2 represents P-β-gLc activity test of result carry out to(for) the high representative bacterial strain of enteron aisle adhesivity.
Fig. 3 represents 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, describe the present invention in detail.
1. screening has the method that lactose intolerance improves the milk-acid bacteria of ability
The present invention relates to by 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.This taxonomy method is not particularly limited, 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: lactobacillus bulgaricus (lactobacillus 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), breast Bacterium lacticum (Lactococcus lactis), lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus gasseri (Lactobacillus gasseri), mucous membrane Bacterium lacticum (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 Bacterium lacticum.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 milk-acid bacteria and tried the binding ability on the intestinal epithelial cells surface of 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 undertaken by 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, with protein adsorption (absorbancy of 280nm) and neutral sugar content height is index, gathers 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.Object lesson can exemplify the method for putting 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 (combination 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 that simply this device is described and is adopted then is: make material on golden film in conjunction with/dissociate, measure because the reflected light change of refractive that the quality change on this combination/golden film that causes that dissociates 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 by 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 when, the binding capacity on material and the sensing chip gold film can combine preceding (before adding) by material and the difference calculating of the RU value when finishing.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 that uses in the BIACORE system is according to having several 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 dextran 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 by 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 then to fixing with acetate buffer (pH4.0) in adding people enteron aisle Saliva Orthana solution (use A-HCM respectively, B-HCM, O-HCM) mixing solutions of gained, further flow in the stream, by 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 made at the carboxyl position on the unconjugated sensing chip of sealing probe.Among the present invention, the fixed amount of HCM on sensing chip is not particularly limited, but is preferably 1000～2000RU.
Then, the sample (assay solution) that will contain the milk-acid bacteria thalline flows in the stream, calculates the binding capacity of the milk-acid bacteria thalline (assay) in HCM (probe) and the assay solution on the sensing chip of fixing each HCM.An example of the condition determination of the BIACORE1000 that can use in this is analyzed is as follows, but is not limited to this.
Electrophoretic buffer: HBS-EP damping fluid (pH7.4)
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 assay in conjunction with preceding RU value gained RU value conduct 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 value more than the 100RU, then can judge " enhancing 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 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 activity) 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, 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, add the phosphoric acid buffer that is added with ortho-nitrophenyl base-β-D-galactopyranoside-6-phosphoric acid ester (ONPGal-6P), perhaps if be used to measure the lactase activity of P-β-glc, then in this suspension, add the phosphoric acid buffer that is added with ortho-nitrophenyl base-β-D-glycopyranoside-6-phosphoric acid ester (ONPGlc-6P), reacted 15 minutes down at 37 ℃.The reaction back adds the 0.5M sodium carbonate solution, and stopped reaction is by centrifugal (6,000 * rpm, 10 minutes, 4 ℃) remove the thalline composition, measure the absorbancy of the 405nm of supernatant by 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 the activity value of every 1mg thalline and the culture supernatant 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 the absorbancy under extinction photometer (for example Multiskan MS-UV) the mensuration 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 screen the extra high milk-acid bacteria of this lactase activity (common milk-acid bacteria strengthens the lactose decomposing enzyme specific activity) by contained every 1mg activity of proteins value in the activity value of β-gal, the P-β-gal of every 1mg thalline of aforementioned calculation and P-β-glc and the culture supernatant.
In every 1mg protein in the preferred especially screening and culturing supernatant for example the lactase activity of β-gal (being also referred to as β-gal activity 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 (being also referred to as P-β-gal activity 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 (being also referred to as P-β-glc activity 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 mentioned above, measure enteron aisle adhesivity and lactose decomposing enzyme activity respectively for above-mentioned lactobacillus milk-acid bacteria (more preferably Lactobacillus gasseri or mucous membrane Bacterium lacticum), 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 product (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 mentioned above.
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 by 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 Bacterium lacticum 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 microorganism sustenance center.Relevant their preservation information is as follows.
(1) preservation mechanism name: the independent administrative legal person's products assessment technique basal disc organization speciallys permit microorganism 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 and 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 Bacterium lacticum (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]
(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]
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 of the present invention can arrive intestines by orally give under existing state, settles down on intestines, and performance intensive lactose decomposing enzyme activity, 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 by to showing lactose intolerance or having the body that tried of its dubiety to give, 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 Bacterium lacticum 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 Bacterium lacticum 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 composition that contains the purifying thalline of milk-acid bacteria of the present invention, also can be the composition 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 composition 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 state, capsule shape or sheet, but is not limited to this.
Lactose intolerance activator of the present invention adds by an amount of, can make diet product 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 preparation 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: water, the acceptable organic solvent of medicine, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble glucan, sodium starch glycolate, pectin, xanthenes glue, Sudan Gum-arabic, casein, gelatin, agar, glycerine, propylene glycol, polyoxyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbyl alcohol, carboxymethyl cellulose, in addition the tensio-active agents of 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 binding agent, 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.
Unqualified to the object that gives lactose intolerance activator of the present invention, but be 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 capacity of decomposition that causes such as old or ill or have the inherited genetic factors of lactose intolerance or the Mammals of environmental factors 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 product that contain milk-acid bacteria of the present invention
The present invention also relates to the diet product, these diet product 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 product of the present invention are particularly suitable for improving the diet product 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 stops fully falls ill or the severity of symptom of lactose intolerance is alleviated.
" diet product " among the present invention are unqualified, are not particularly limited for the kind of the diet product that contain milk-acid bacteria of the present invention that comprise beverage, food and functional foodstuff.For example, can exemplify 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 as the beverage that contains milk-acid bacteria of the present invention is 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, grape wine etc.), soda pop and refreshment drink.The preparation 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.
The food that contains milk-acid bacteria of the present invention is not particularly limited, and can be that fresh product also can be a processed food, but particularly preferred 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, contain the diet product of milk-acid bacteria of the present invention 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 emphasized 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 breast, infant formula breast, 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 capacity of decomposition 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, 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 exemplify: 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 making 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 settle down, and lactose capacity of decomposition 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 capacity of decomposition 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 product.
The use level of milk-acid bacteria of the present invention in the diet product is not particularly limited, can be according to each situation and difference.Concrete use level can be considered kind or the required taste or the mouthfeel of diet product, is suitably determined 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 product by the available proper method arbitrarily in this area.For example, milk-acid bacteria of the present invention can be blended directly in the raw material of diet product.Milk-acid bacteria of the present invention can be coated with, coats, permeates or be sprayed on the diet product.Milk-acid bacteria of the present invention can be evenly dispersed in the diet product, also can locally exist.Also can make the preparation of capsule of having added milk-acid bacteria of the present invention etc.Can wrap up milk-acid bacteria of the present invention with edible film or edible Drug coating etc.After adding appropriate excipients etc. in the milk-acid bacteria of the present invention, can be shaped to shapes such as tablet.The diet product 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 product that especially preferably contain the milk fermentation thing that uses milk-acid bacteria preparation of the present invention.In a word, diet product 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 product of the present invention, can use various additives commonly used in the diet product.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 etc.), sanitas (sodium acetate, Sorbic Acid etc.), emulsifying agent (sodium chondroitin sulfate, propylene glycol fatty acid ester etc.), antioxidant (EDTA disodium, vitamins C etc.), pH regulator agent (citric acid etc.), chemistry seasonings (Sodium Inosinate etc.), thickening material (xanthenes glue etc.), swelling agent (lime carbonate etc.), defoamer (calcium phosphate), binding agent (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 product 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 product of the present invention, as milk-acid bacteria of the present invention, preferably contain and be selected from least a of Lactobacillus gasseri OLL2836 strain (preserving number NITE BP-241), Lactobacillus gasseri OLL2948 strain (preserving number NITE BP-242) and mucous membrane Bacterium lacticum OLL2848 strain (preserving number NITE BP-243).In the particularly preferred embodiment, diet product 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 Bacterium lacticum OLL2848 strain (preserving number NITE BP-243).The diet product that this three kinds of milk-acid bacterias combination is contained can also combine with having A type, Type B, the antigenic enteron aisle Saliva Orthana of any blood type of O type, so effective to being tried body widely.
The picked-up or the body that tried that gives diet product of the present invention are not particularly limited, are 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 capacity of decomposition or have the inherited genetic factors of lactose intolerance or the Mammals of environmental factors also preferably as the body that tried that absorbs or give diet product 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] is for cultivation and the preparation of examination milk-acid bacteria
Below employedly the lactobacillus strain from the people accepting Mingzhi Dairy Co., Ltd and provide has been provided, has 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 for the examination milk-acid bacteria, and the inventor etc. uses modification LBS agar, by the isolating totally 104 strain bacterial strains of child's ight soil.These except that the various bacterial strains that belong to lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacterium acidophilum (Lactobacillusacidophilus), newborn Bacterium lacticum (Lactococcus lactis), Lactobacillus gasseri (Lactobacillusgasseri), lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus johnsonii (Lactobacillus johnsonii) and mucous membrane Bacterium lacticum (Lactobacillus mucosae), also comprise the unidentified bacterial strain of bacterial classification for the examination milk-acid bacteria.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 thalline for the examination milk-acid bacteria is 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.Zhi Bei this nutrient solution carries out centrifugal (6,000 * g, 20 minutes, 4 ℃) like this, and the thalline of collecting is cleaned with 0.05M phosphoric acid buffer (pH6.8), is suspended in then in the distilled 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 mensuration system with the mucinous binding ability of enteron aisle by inventor, by above-mentioned bacterial strain for screening enteron aisle adhesivity height (the RU value is more than 100) in the examination lactobacillus strain, employed mensuration system write up is in International Application No. WO 2006/067940.
Specifically, by 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 at 37 ℃ with each bacterial strain subculture three times in the MRS substratum, then with 500 μ L dispensings in the centrifuge tube of 1.5mL capacity.This nutrient solution is carried out centrifugal (6,000rpm, 4 ℃, 10 minutes), remove supernatant, the gained thalline is cleaned twice with PBS damping fluid (pH7.2), be suspended in the distilled water lyophilize then.This thalline is prepared into 0.1mg/mL concentration with HBS-EP damping fluid (0.01MHEPES (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 by 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 by 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 the 4M guanidine hydrochloride solution.Then carry out purifying by 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 the 4M guanidine hydrochloride solution, 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 by phenol sulfuric acid process (absorbancy of 490nm is measured in the reaction back), absorbance measurement protein by 280nm, the result, selection has 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, divides and gets large intestine Saliva Orthana component.The purifying thing that obtains like this is corresponding with the people's in source blood type, makes 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 of each blood type by antigen antibody reaction.
Above-mentioned each HCM that obtains, is undertaken by 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, fixing acetate buffer (10mM, pH4.0) the middle solution of HCM arbitrarily (A-HCM, B-HCM, O-HCM) that adds the above-mentioned purifying of 30 μ L, the preparation mixing solutions used to 120 μ L, flow into sensing chip, make carboxyl covalent attachment on HCM and the sensing chip by the amine linked reaction.And, use 1M ethanolamine hydrochloric salt-NaOH (pH8.5), 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.With importing EDC/NHS is 1000～2000RU with the HCM fixed amount that the difference of adding ethanolamine solutions display data (reportpoint) is afterwards represented before.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 (pH7.4)
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 being undertaken by the BIACORE system, the binding capacity of assay and probe (interaction) is by 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,, deduct value in conjunction with preceding RU value gained by the RU value after the combination and be equivalent to as each milk-acid bacteria of assay and every 1mm as probe according to the combination/dissociation curve that obtains by this analysis
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, from the bacterial strain of 104 strains for the value more than the mucinous binding ability demonstration of screening and at least a enteron aisle 100RU the examination milk-acid bacteria.We can say in the bacterial strain of screening that the enteron aisle adhesivity strengthens (enteron aisle adhesivity height) 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, attempting further, screening has enteron aisle adhesivity height and the active bacterial strain of high lactose lytic enzyme.Here, in the present embodiment, at first carry out the preparation of the ONPGlc-6P that uses in this determination of activity.
1.ONPGlc-6P chemosynthesis
Synthetic 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 in phosphoric acid-beta-glucosidase enzyme (ortho-nitrophenyl base-β-D-glycopyranoside-6-phosphoric acid ester (ONPGlc-6P) of using in the determination of activity of P-β-glc).Specifically, at first 0.9g ortho-nitrophenyl base-β-D-glycopyranoside (ONPGlc, Sigma, St. Louis, the U.S.) is dissolved in advance in cooled on ice, blended contains in the 7.5mL trimethyl phosphite 99 of 54 μ L distilled water and 840 μ L Phosphorus Oxychlorides then.Then this mixed solution was stirred on ice 5 hours, react.The reaction back adds borneol (distilled water), while drip strong aqua pH meter neutralization solution (pH7.0), termination reaction.
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 distilled 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 by rotatory evaporator down repeatedly at 40 ℃, removes pyridine.Then, supply with the preparation ply of paper and analyse (PPC).Promptly, 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 by rise method.Launching needs 2 day time, after launching to finish, makes this filter paper drying in the ventilating chamber, 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 distilled water, stirs down at 4 ℃ and spend the night, carry out the extraction of ONPGlc-6P thus.Remove filter paper after the extraction, concentrate water layer by rotatory evaporator, use then with distilled water as the TOYOPEARL HW40S post of mobile phase (2.6 φ * 90cm, East ソ-, Tokyo), remove by gel-filtration and to mix the byproduct of reaction that exists.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 spraying equably on thin layer plate detected at moisture eliminator (Yamato, the Tokyo) internal heating of heating to 150 ℃ in 3 minutes.Purifying ONPGlc-6P is made in the lyophilize of the ONPGlc-6P component that reclaims.Confirm purity for these purifying product by TLC, by
1H-NMR carries out the affirmation of structure.
[embodiment 4] β-gal, P-β-gal and the active mensuration of P-β-glc
104 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 1.0mL0.05M phosphoric acid buffer (pH6.8), adds 50 μ L toluene-acetone mixed solutions (1:9 (v/v)), vigorous stirring 3 minutes each bacterial strain.In 25 these suspension of μ L, add the 0.05M phosphoric acid buffer (pH6.8) that 100 μ L contain the ortho-nitrophenyl base-β-D-glycopyranoside-6-phosphoric acid ester (ONPGlc-6P) of ortho-nitrophenyl base-β-D-galactopyranoside (ONPGal), ortho-nitrophenyl base-β-D-galactopyranoside-6-phosphoric acid ester (ONPGal-6P) or above-mentioned preparation, reacted 15 minutes down at 37 ℃.The reaction back adds 125 μ L0.5M sodium carbonate solution stopped reactions, removes the thalline composition by 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 the activity value of every 1mg thalline and the culture supernatant 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
According to the enteron aisle adhesivity shown in the foregoing description and β-gal, P-β-gal and the active measurement result of P-β-glc, 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 the proteinic P-β of 46.576 units/mg-gal activity, the OLL2948 strain shows the proteinic P-β of 50.194 units/mg-glc activity, the OLL2848 strain shows the proteinic β of 107.090 units/mg-gal activity, but other lactobacillus strain that these activity values and enteron aisle adhesivity are high compares, and 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, by 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 (Lactobacillusgasseri), and the OLL2848 strain belongs to mucous membrane Bacterium lacticum (Lactobacillusmucosae) (with reference to aftermentioned embodiment).These three kinds of lactobacillus strains are preserved in independent administrative corporation's goods evaluation technique basal disc assessing mechanism special permission microorganism 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: NITE 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 as follows: the OLL2836 strain is NITE BP-241, and the OLL2948 strain is NITE BP-242, and the OLL2848 strain is MTE BP-243.The microbiological property of these bacterial strains has been confirmed as as described in the above-mentioned table 1.
As mentioned above, 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, by 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 determined, is undertaken by 16S rDNA nucleotide sequence analysis.Below, be that example is set forth with the qualification test of carrying out with OLL2848 strain and OLL2948 strain, the OLL2836 strain is also substantially similarly identified.
1. detect the 16SrDNA fragment of screening strain by direct PCR method
Succeeding transfer culture is carried out in OLL2848 strain and OLL2948 strain as mentioned above on the MRS substratum, carry out direct PCR then.To inject 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.Add Taq archaeal dna polymerase (TaKaRa, capital of a country) in this suspension, 10 * PCR damping fluid, dNTP Mix (TaKaRa, capital of a country), one group of primer are made 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 and 1 [72 ℃ 1 minute] are circulated.
2. agarose gel electrophoresis
Then, will carry out electrophoresis by the dna fragmentation of above PCR reaction amplification.Electrophoresis be with 2% sepharose as running gel, electrophoretic buffer uses 1 * tbe buffer liquid (90mMTris-boric acid, 2mM EDTA, pH8.0), carries out under the constant voltage of 100V.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 to soak to carry out in 10 minutes in ethidium bromide (EB) solution (0.5 μ g/mL).
3. by extracting DNA in the sepharose
Carry out UV irradiation by 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 by 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 ℃.Then the magnetic beads that adds 15 μ L in this reaction solution stirs, and at room temperature places then 1 minute.Centrifugal (6,000rpm, 5 seconds, 4 ℃) remove 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 ℃) remove supernatant then.Add the distilled water of 15 μ L and stir, supernatant that then will be by centrifugal (15,000rpm, 1 minute, 4 ℃) recovery is as purify DNA solution.
4.DNA determining 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 determine.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 determined 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 database (DDBJ), carry out homology analysis by 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 Bacterium lacticum 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 Bacterium lacticum, and the OLL2948 strain belongs to Lactobacillus gasseri.
Industrial applicability
Method of the present invention can be used for efficiently screening improving the effective lactic acid of lactose intolerance disease Bacterial strain. The lactic bacteria strain that is obtained by method of the present invention can give the patient easily, and can Have with the active ingredient as the medicine that improves sustainably lactose intolerance disease or functional food Use sharply.
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 patent application of quoting in this specification sheets by 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; Ki tazawa, Haruki
Contriver: Kawai, Yasushi; Itoh, Hiroyuki
<400>1
<210>2
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉kind of artificial sequence: primer
<400>2
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉kind of artificial sequence: primer
<400>3
<210>4
<211>559
<212>DNA
＜213〉mucous membrane Bacterium lacticum
<400>4
<210>5
<211>524
<212>DNA
＜213〉Lactobacillus gasseri
<400>5
Claims (11)
1. screening has the method that lactose intolerance improves the milk-acid bacteria of 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.
2. the described method of claim 1, wherein, 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 is measured in the analysis of use surface plasma body resonant vibration, and the RU value of this binding ability of expression is screened as enteron aisle adhesivity enhanced bacterium for the bacterium more than the 100RU.
3. claim 1 or 2 described methods, wherein, the lactose decomposing enzyme activity is at least a lactase activity of beta-galactosidase enzymes, phosphoric acid-beta-galactosidase enzymes and phosphoric acid-beta-glucosidase enzyme.
4. each described method in the claim 1～3, wherein, the lactobacillus milk-acid bacteria is Lactobacillus gasseri (Lactobacillus gasseri) or mucous membrane Bacterium lacticum (Lactobacillusmucosae).
5. milk-acid bacteria, its by each described method in the claim 1～4 obtain, enteron aisle adhesivity and the active all enhanced milk-acid bacterias of lactose decomposing enzyme.
6. 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 Bacterium lacticum OLL2848 strain (preserving number NITE BP-243).
7. lactose intolerance activator, this activator contains a kind of claim 5 or 6 described milk-acid bacterias at least.
8. the described lactose intolerance activator of claim 7, this activator contains 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 Bacterium lacticum OLL2848 strain (preserving number NITEBP-243) at least.
9. diet product, these diet product contain a kind of claim 5 or 6 described milk-acid bacterias at least.
10. described diet product of claim 9, these diet product 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 Bacterium lacticum OLL2848 strain (preserving number NITE BP-243) at least.
11. claim 9 or 10 described diet product, these diet product are selected from infant food, junior food, nursing women's food, the elderly's food, patient's food, health functional food, dietary supplements, fermented-milk and lactobacillus drink.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP175897/2006 | 2006-06-26 | ||
| JP2006175897 | 2006-06-26 | ||
| PCT/JP2007/062522 WO2008001676A1 (en) | 2006-06-26 | 2007-06-21 | Lactic acid bacterium for amelioration of lactose intolerance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101479382A true CN101479382A (en) | 2009-07-08 |
| CN101479382B CN101479382B (en) | 2012-07-04 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007800237640A Expired - Fee Related CN101479382B (en) | 2006-06-26 | 2007-06-21 | Lactic acid bacterium for amelioration of lactose intolerance |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JP5054687B2 (en) |
| CN (1) | CN101479382B (en) |
| HK (1) | HK1134937A1 (en) |
| TW (1) | TWI383049B (en) |
| WO (1) | WO2008001676A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239963A (en) * | 2010-05-12 | 2011-11-16 | 青岛东海药业有限公司 | Preparation applied to pig and chicken breeding |
| CN102946892A (en) * | 2010-02-05 | 2013-02-27 | 维塔凯尔有限责任公司 | A composition for use in the therapy of lactose intolerance or conditions arising from lactase deficiency |
| CN109730156A (en) * | 2018-12-26 | 2019-05-10 | 李卫平 | A kind of child antidiarrhea nutrition packet and preparation method thereof |
| CN111979145A (en) * | 2020-08-07 | 2020-11-24 | 上海交通大学 | Human-derived lactobacillus mucosae and application thereof |
| CN112694985A (en) * | 2019-10-07 | 2021-04-23 | 大江生医股份有限公司 | Lactobacillus fermentum TCI275, compositions thereof and uses thereof |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013168438A1 (en) * | 2012-05-10 | 2013-11-14 | 国立大学法人岩手大学 | Protein having lactase activity, gene encoding said protein, recombinant vector carrying said gene, transformant, method for producing said protein, and use of said protein |
| 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 |
| JP6603230B2 (en) * | 2014-03-05 | 2019-11-06 | ディーエスエム アイピー アセッツ ビー．ブイ． | Liquid lactase composition |
| TW202142129A (en) | 2020-04-08 | 2021-11-16 | 日商雪印惠乳業股份有限公司 | Composition for improving intestinal bacterial flora |
| WO2024013211A1 (en) | 2022-07-12 | 2024-01-18 | Novozymes A/S | Method for providing relief from lactose induced abdominal discomfort |
Family Cites Families (4)
| 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 |
| WO2006059408A1 (en) * | 2004-12-01 | 2006-06-08 | Meiji Dairies Corporation | Lactic acid bacterium binding to human abo blood types |
-
2007
- 2007-06-21 CN CN2007800237640A patent/CN101479382B/en not_active Expired - Fee Related
- 2007-06-21 WO PCT/JP2007/062522 patent/WO2008001676A1/en active Application Filing
- 2007-06-21 JP JP2008522516A patent/JP5054687B2/en active Active
- 2007-06-26 TW TW096123069A patent/TWI383049B/en not_active IP Right Cessation
-
2009
- 2009-12-23 HK HK09112106.7A patent/HK1134937A1/en not_active IP Right Cessation
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102946892A (en) * | 2010-02-05 | 2013-02-27 | 维塔凯尔有限责任公司 | A composition for use in the therapy of lactose intolerance or conditions arising from lactase deficiency |
| CN107375912A (en) * | 2010-02-05 | 2017-11-24 | 维塔凯尔有限责任公司 | For treat lactose intolerance or because alactasia trigger disease composition |
| CN102239963A (en) * | 2010-05-12 | 2011-11-16 | 青岛东海药业有限公司 | Preparation applied to pig and chicken breeding |
| CN102239963B (en) * | 2010-05-12 | 2015-04-08 | 青岛东海药业有限公司 | Preparation applied to pig and chicken breeding |
| CN109730156A (en) * | 2018-12-26 | 2019-05-10 | 李卫平 | A kind of child antidiarrhea nutrition packet and preparation method thereof |
| CN112694985A (en) * | 2019-10-07 | 2021-04-23 | 大江生医股份有限公司 | Lactobacillus fermentum TCI275, compositions thereof and uses thereof |
| CN111979145A (en) * | 2020-08-07 | 2020-11-24 | 上海交通大学 | Human-derived lactobacillus mucosae and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JPWO2008001676A1 (en) | 2009-11-26 |
| TW200817508A (en) | 2008-04-16 |
| CN101479382B (en) | 2012-07-04 |
| WO2008001676A1 (en) | 2008-01-03 |
| TWI383049B (en) | 2013-01-21 |
| JP5054687B2 (en) | 2012-10-24 |
| HK1134937A1 (en) | 2010-05-20 |
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