CN105671120A - Bifidobacterium having activity of reducing amount of intestinal putrefacted substance - Google Patents

Bifidobacterium having activity of reducing amount of intestinal putrefacted substance Download PDF

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CN105671120A
CN105671120A CN201610085654.8A CN201610085654A CN105671120A CN 105671120 A CN105671120 A CN 105671120A CN 201610085654 A CN201610085654 A CN 201610085654A CN 105671120 A CN105671120 A CN 105671120A
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microorganism
concentration
enteral
corrupt substance
fos
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CN105671120B (en
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天藤公子
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Meiji Co Ltd
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Abstract

It becomes possible to reduce the amount of an intestinal putrefacted substance effectively by the efficient use of a highly safe and useful microorganism. Attention is focused on bifidobacteria which are believed to be predominant generally in a human living body (intestine), and the screening of bifidobacteria is carried out. As a result, a bifidobacterium strain which has a potent indol amount reduction activity and of which the activity of reducing the amount of indoxyl sulfate in serum has been confirmed can be selected.

Description

There is the bacillus bifidus of the minimizing effect of enteral corrupt substance
The divisional application of PCT application that the application is international filing date to be on July 5th, 2011, international application no be PCT/JP2011/065350, the application number entering thenational phase are 201180033393.0, denomination of invention is " bacillus bifidus with the minimizing effect of enteral corrupt substance ".
Technical field
The present application relates to the ameliorative way of the state of intestinal bacterium. More particularly, to reduce the useful microorganism of the indole based compound as corruption abnormal smells from the patient reason that intestinal bacterium causes select and this selects the microorganism of gained.
Background technology
Due to irregular, the pressure etc. of the confusion of dietetic life, life, there is constipation repeatedly, diarrhoea, or the people of the symptom such as abdomen rises, aerogenesis is on the increase. These symptoms are considered to be affected by enteral environment, and in other words, its reason is in that in intestinal flora (group) putrefaction bacteria becomes advantage. Further, intestinal flora is under anaerobic produced the corrupt odour components such as indole or derivatives thereof by tryptophan. The relation of this intestinal flora Yu the generation of spoilage product has been carried out big quantity research (non-patent literature 1).
Additionally, it is believed that indole is produced and by, after intestinal absorption, being metabolised to indoxyl in liver by tryptophan by enteral putrefaction bacteria, and then be combined with sulphuric acid, form indoxyl sulfate. Think during the excretion of Healthy People indoxyl sulfate is extremely urinated, but dialysis patient etc. is then become the cause of disease (non-patent literature 2) of the complication such as uremia.
Removing for the indole etc. as intestinal toxic material, for instance, patent documentation 1 uses bifidobacterium thermophilum's (PVC Off ィ De バ Network テ イ Le system/サ モ Off ィ ラ system) with indole minimizing ability. The extract of Salacia prinoides (Willd.) DC. platymiscium or micropowder are minced and lactic acid bacteria and/or bacillus bifidus for improving enteral environment it addition, patent documentation 2 describes. And then, patent documentation 3 describes the compositions utilized containing bacterium acidi propionici to promote the growth of the bacillus bifidus in intestinal, it is prevented that release foul gas. It addition, there was reported inulin, oligosaccharide contributes to improving intestinal flora (non-patent literature 3).
Prior art literature
Patent documentation
Patent documentation 1: Japanese Unexamined Patent Publication 2006-158216
Patent documentation 2: Japanese Unexamined Patent Publication 2007-031345
Patent documentation 3: Japanese Unexamined Patent Publication 2007-186529
Non-patent literature
Non-patent literature 1: intestinal bacterium learns magazine the 19th volume 169-177 page (2005)
Non-patent literature 2: dialysis can No. 3 312-316 pages (1991) of will 24 volume
Non-patent literature 3:TheJournalofNutrition129(7Suppl): 1438S-1441S(1999).
Summary of the invention
Invention to solve the technical problem that
The corrupt substance that in vivo (enteral) generates of known people can promote cancer occur, aging, it is believed that as long as intravital corrupt substance can be reduced energetically, namely can help to the health of people.Therefore first, the problem in the present application is in that, effectively utilizes the high useful microorganism of safety to efficiently reduce enteral corrupt substance.
Above-mentioned corrupt substance is mainly when big enteral generates, and as an example of the superior microorganism (strain) of big enteral, has lactic acid bacteria, bacillus bifidus (Bifidobacterium). This bacillus bifidus is used to food as the microorganism in vivo playing useful function since ancient times, has the real result of diet experience. Therefore, the 1st problem of the application is in that, by selecting the microorganism (bacterial strain) that can efficiently reduce corrupt substance in vivo (enteral) dominant bacillus bifidus of people.
But, find the material or microorganism that reduce the in vivo corrupt substance that (enteral) generates mainly by external system in the past. Owing to intestinal flora can't be reproduced at present in vitro, thus to whether reflecting that actual enteral situation carries out experiment and has query. Therefore, the 2nd problem of the present application is in that, it is provided that the system of suitable in vitro and in vivo is used as reducing checking or the searching system of the action effect of the material of the corrupt substance that (enteral) generates in vivo or microorganism.
And then, the 3rd problem of the present application is in that, it is provided that dialysis patient etc. has the microorganism of the effect of the concentration reducing the indoxyl sulfate as the complication cause of disease in serum.
For solving the means of technical problem
As the useful microorganism that safety is high, present inventor, based on the screening technique of the present application, separates the bacterial strain effectively reducing enteral corrupt substance, thus completing the present application from lactic acid bacteria and bacillus bifidus.
Checking system as the action effect of the material or microorganism that reduce the corrupt substance that (enteral) generates in vivo, present inventor have studied following method: uses buffer to reproduce the condition that thalline is difficult to breed when screening in vitro, evaluates the minimizing effect (minimizing ability) of corrupt substance. And, present inventor establishes external selection method and/or the evaluation methodology of the microorganism (lactic acid bacteria, bacillus bifidus etc.) reducing the corrupt substance that intestinal bacterium produces, the method is characterized in that, reaction solution is set (design) be lactic acid bacteria, the condition substantially do not bred of bacillus bifidus, and the glucose amount of the energy for the minimum needed for minimizing corrupt substance.
It addition, the checking system of the action effect as the material or microorganism that reduce the corrupt substance that (enteral) generates in vivo, present inventor have studied use mice when screening in vivo, by measuring indoxyl sulfate in serum. And, present inventor establishes internal selection method and/or the evaluation methodology of the microorganism (lactic acid bacteria, bacillus bifidus etc.) of the generation of the indoxyl sulfate reduced in serum, the method is characterized in that, oligofructose (FOS) and/or oligomeric galactose (GOS) and tryptophan (L-Trp) are added in feedstuff (powder feedstuff) and raise mice, evaluate the concentration of indoxyl sulfate in serum.
And then, in order to select the microorganism (bacterial strain) being effectively reduced corrupt substance, first, present inventor is conceived to generally in vivo (enteral) the dominant bacillus bifidus of people, to 32 bacterial strains of bifidobacterium breve (Bifidobacteriumbreve), 92 bacterial strains of bifidobacterium longum (Bifidobacteriumlongum), bifidobacterium adolescentis (Bifidobacteriumadolescenti) 72 bacterial strains total 196 bacterial strain implement screening, it is evaluated in vitro, picks out 9 strains in advance as the high useful microorganism of safety.Then, the effect of corrupt substance, effect is in vivo reduced in order to verify, zoopery is implemented for above-mentioned 9 strains selected, carry out interior evaluating, pick out indole minimizing effect strong and confirm the bifidobacterium breve OLB6129 of minimizing effect of indoxyl sulfate in serum, thus completing the present application.
It addition, in the present application, comprise medicine and food, wherein contain the bacillus bifidus of minimizing effect of the indoxyl sulfate that the above-mentioned indole minimizing effect selected is strong, show in serum as effective ingredient.
This specification comprises the content recorded in the description of Japanese patent application 2010-152962 as the application preference basis and/or accompanying drawing.
Invention effect
The bifidobacterium breve (B.breve) of the present application is derived from the bacterial strain of people, in the system that this bacterial strain self is not bred, it is achieved that substantially reduce the excellent effect of indoxyl sulfate in indole and serum. It addition, the safety of the B.breve being eaten for a long time is high, the bacillus bifidus OLB6129 of the present invention can also the generally applicable or application at field of food, medicine field.
Accompanying drawing explanation
Mice is given the impact of the concentration on the indoxyl sulfate in serum that bifidobacterium breve (viable bacteria) causes by [Fig. 1]
Mice is given the impact of the concentration on the indoxyl sulfate in serum that bifidobacterium longum (viable bacteria) causes by [Fig. 2]
Mouse oral is given B.breveOLB6129(viable bacteria by [Fig. 3]) in, B.breveOLB6129(viable bacteria) the impact of the concentration on the indoxyl sulfate in serum that causes of concentration
[Fig. 4] detect in TOS propanoic acid agar culture medium just in the clump count of B.breveOLB6129 in (Fecal) and cecal content (cecal)
Mouse oral is given B.breveOLB6129(viable bacteria by [Fig. 5]) in, with or without adding the FOS impact of the concentration on the indoxyl sulfate in serum caused
[Fig. 6] detect in TOS propanoic acid agar culture medium just in B.breveOLB6129 in (Fecal) clump count with or without the relation adding FOS.
Detailed description of the invention
1. first
The corrupt substance that in vivo (enteral) generates of known people can promote cancer occur, aging, it is believed that as long as intravital corrupt substance can be reduced energetically, namely can help to the health of people. As the corrupt substance mainly generated at big enteral, it is possible to enumerate phenol, paracresol, indole, scatol, ammonia etc. Among these corrupt substances, particularly indole, scatol etc. are odor pollutants, and additionally it is also possible that health is brought dysgenic composition, thus expectation reduces indole, scatol.
As an example of the superior microorganism (strain) of big enteral, there are lactobacillus, bacillus bifidus (Bifidobacterium). Bacillus bifidus is used to food as the microorganism in vivo playing useful function since ancient times. It addition, bacillus bifidus is considered to have whole intestinal effect, thus be also used to medicines for relieving intestinal disorders etc., it helps the improvement of intestinal flora, it is believed that by improving enteral environment, to suppressing diarrhoea, prevent pachylosis, improve pollinosis, the Taoism of immunity etc. also effective.
2. for verifying, find the appraisement system of the action effect of the microorganism reducing enteral corrupt substance
2-1. reduces the in-vitro screening of the microorganism of enteral corrupt substance or screens in advance
In order to use microorganism, under enteral environment, reduce enteral corrupt substance (generating and produce the material of odor pollutant etc. at enteral), it is important that this specific microorganism is preferential existence at enteral.That is, the character that should possess as this microorganism, it is desirable to for adapting to the patience of the condition of enteral environment.
The screening technique of state (practical situation) of microorganism (thalline), the evaluation methodology existed in enteral environment is met accordingly, it would be desirable to be suitable for. Therefore, when screening reduces material or the microorganism of enteral corrupt substance in vitro, present inventor reproduces, first by buffer, the condition that microorganism is difficult to breed, and have studied the evaluation methodology of the minimizing effect of enteral corrupt substance (minimizing ability). By adopting above-mentioned condition, it is possible to do not breed even if selecting microorganism, it is also possible to remove microorganism corrupt substance, excellent closer to enteral environment when. For example, it is possible to condition as the energy of the minimum needed for setting condition that object microorganism do not breed substantially and reducing corrupt substance, determine the composition of reaction solution, concentration.
More specifically, use level for the medium component as energy source, for instance, the concentration comparison making glucose, lactose, fructose etc. is low as the common condition of culture of microorganism, and as removing the degree of enteral corrupt substance, applicable 0.3～0.7(w/v) %.
Should illustrate, in screening (evaluation is selected) method (in vitro tests), exemplified with the result that the concentration of the reaction solution of the carbon source to glucose etc is studied, even if being other composition, it is also possible to same determine the composition of reaction solution, concentration.
2-2. reduces the internal screening of the microorganism of enteral corrupt substance
In the past, fail to implement zoopery by the minimizing effect of the indoxyl sulfate in internal abundant confirmation serum. The minimizing effect of the indoxyl sulfate in serum, although expecting to play at enteral, even if microorganism (thalline) being given in vivo, it is also considered as arriving that its major part before enteral is likely dead or it likely can not stably maintain (maintenance) arriving after enteral. Therefore, in the present application, microorganism is being given to after in vivo, when expecting to contribute to the raising of its viability, stability etc. and try to be added in feedstuff oligofructose (FOS), can confirm that the minimizing effect of indoxyl sulfate in serum.
It addition, commonly used mice is the healthy individuals in children's age in zoopery, thus intravital corrupt substance can be suppressed to minute quantity, and most cases can lower than detection limit. Therefore, in the present application, in the zoopery using mice, to in vivo be added in feedstuff (powder feedstuff) as the tryptophan (L-Trp) of benzazole precursor, during to attempt the baseline improving the indole in vivo generated, for the baseline of indole, confirm significance rising. That is, become stably to be evaluated definitely as the concentration of the indoxyl sulfate in the serum of the index (mark) of corrupt substance. Further, for specific bacterial strain, it becomes able to the minimizing effect of corrupt substance is evaluated, confirmed to the form of the significant difference to compare with matched group.
More specifically, can be added in feedstuff (powder feedstuff) with the concentration of regulation by oligofructose (FOS) and/or oligomeric galactose (GOS) and tryptophan (L-Trp) to feed common mice (zoopery with), evaluate the concentration of indoxyl sulfate in serum. Now, as the concentration of oligofructose (FOS) and/or the total amount of oligomeric galactose (GOS), it is set as 0.5～20(w/v) %, it is preferable that it is set as 0.5～15(w/v) %, and more preferably it is set as 0.5～10(w/v) %.Should illustrating, concrete experimental result is omitted, but the concentration as oligofructose (FOS) is set as 0.5(w/v) % time, as the viable count in feces, the viability having confirmed B.breveOLB6129 significantly improves. It addition, as the concentration of tryptophan, be set as 1～4(w/v) %, it is preferable that it is set as 1.5～3.5(w/v) %, and more preferably it is set as 2～3(w/v) %. Thus, it is possible to select and/or evaluate or confirm to make the microorganism (lactic acid bacteria, bacillus bifidus etc.), particularly bacillus bifidus (microorganism of Bifidobacterium) that produce to reduce of the indoxyl sulfate in serum in vivo.
3. make the microorganism that enteral corrupt substance reduces
As the microorganism making enteral corrupt substance reduce, in order to select the microorganism (bacterial strain) that can effectively reduce corrupt substance, present inventor is generally to have studied for object in vivo (enteral) dominant lactic acid bacteria of people, bacillus bifidus, particularly bacillus bifidus. Further, as bacillus bifidus, for 32 bacterial strains of B.breve, 92 bacterial strains of B.longum, B.adolescentis total 196 bacterial strain of 72 bacterial strains implement screening, evaluated in vitro. As its result, pick out total 9 bacterial strain of 6 bacterial strains of B.breve and 3 bacterial strains of B.longum.
And then, in vivo reduce the effect of corrupt substance, effect to verify, above-mentioned 9 bacterial strains selected are implemented zoopery, has evaluated in vivo. As its result, pick out the bacterial strain of the minimizing effect of the indole playing enteral.
Should illustrate, the bifidobacterium breve OLB6129(B.breveOLB6129 that screening obtains) it is preserved in the biological sustenance center (contact address: east, つ く ば city, postcode 305-8566 Hitachinaka County, Japan 1-1-1 つ く ば center central the 6th) of Independent Administrative Leged Industrial Technology Complex Inst's special permission on May 26th, 2010, this microorganism carries out preservation with deposit number FERMBP-11257.
4. contain food or the medicine of the microorganism reducing enteral corrupt substance
4-1. contains the food of the microorganism reducing enteral corrupt substance
The microorganism (lactic acid bacteria, bacillus bifidus etc., it is preferred to B.breveOLB6129) reducing enteral corrupt substance of the present application can be added in food. Namely, food as the present application, any food being added with the microorganism reducing enteral corrupt substance can be enumerated, preferably can enumerate the fermented dairy product or leben that are added with the microorganism reducing enteral corrupt substance, reduce the microorganism (B.breveOLB6129) of enteral corrupt substance and the fermented dairy product of oligofructose (FOS) and/or oligomeric galactose (GOS) or leben it is preferred that can enumerate to be added with. Should illustrate, it is known that GOS can carry out utilizing (http://www.nyusankin.or.jp/scientific/hosono_3.html) identically with FOS. It addition, for the addition of oligofructose (FOS) and/or oligomeric galactose (GOS), with the densitometer of total amount, it is possible to be set as 0.5～20(w/v) %, it is preferable that it is set as 0.5～15(w/v) %, and more preferably it is set as 0.5～10(w/v) %.
Additionally, the food of the microorganism (lactic acid bacteria, bacillus bifidus etc., it is preferred to B.breveOLB6129) reducing enteral corrupt substance containing the present application can as renal dialysis patients for preventing or to improve the health-care food (functional food) of dialysis complication applicable.
4-2.
The medicine of the microorganism (lactic acid bacteria, bacillus bifidus etc., it is preferred to B.breveOLB6129) reducing enteral corrupt substance containing the present application can be suitable for as the preventive of the dialysis complication reducing agent or renal dialysis patients of enteral corrupt substance or improving agent.And then, the medicine of the present application is alternatively arranged as the improving agent that abdomen rises and is suitable for.
The medicine of the present application can be made as the dosage form that powder, granule, tablet, capsule, solution etc. are suitable. The medicine of the present application can contain oligofructose (FOS) and/or oligomeric galactose (GOS). Should illustrating, with the densitometer of total amount, the content of oligofructose (FOS) and/or oligomeric galactose (GOS) can set that to be 0.5～20(w/v) %, it is preferable that it is set as 0.5～15(w/v) %, and more preferably it is set as 0.5～10(w/v) %. In formulation, it is possible to add excipient, binding agent, disintegrating agent, lubricant etc. for formulation and conventional adjuvant. As excipient, for instance have starch, lactose, white sugar, methylcellulose, carboxymethyl cellulose, sodium alginate, calcium hydrogen phosphate, synthetic aluminium silicate, microcrystalline Cellulose, polyvinyl pyrrolidone (PVP), hydroxypropyl starch (HPS) etc. It addition, as binding agent, for instance there are aqueous solution or their hydrous ethanol solution etc. of starch, microcrystalline Cellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone (PVP), gum arabic powder, gelatin, glucose, white sugar etc. Further, as disintegrating agent, for instance have starch, carboxymethyl cellulose, carboxymethylcellulose calcium, microcrystalline Cellulose, hydroxypropyl starch, calcium phosphate etc. And then, as lubricant, for instance have Brazil wax, light anhydrous silicic acid, synthetic aluminium silicate, natural aluminium silicate, synthetic magnesium silicate, hydrogenated oil and fat, hydrogenated vegetable oil derivant (SterotexHM), Oleum sesami, Cera Flava, titanium oxide, Aluminium Hydroxide stearic acid, calcium stearate, magnesium stearate, Talcum, calcium hydrogen phosphate and sodium lauryl sulphate etc.
The medicine of the present application it is desirable to be orally administered to, for bacillus bifidus, owing to safety is determined, thus arbitrary amount can be given, such as, its administered dose can be set to every 1 day give bacillus bifidus 1,000,000,000～500,000,000,000, it is preferred to 10,000,000,000～500,000,000,000.
Embodiment
［ reference example 1 ］ has the condition of the selection method " in vitro tests " of the microorganism of the minimizing effect of corrupt substance and sets
Firstly, for the composition of reaction solution, substantially do not breed, play the minimizing effect of indole (index of enteral corrupt substance) by bacillus bifidus and the composition of reaction solution in the way of evaluating this effect clearly, can be determined.
In the reaction solution used in this experiment, for instance, glucose is set as 1.0(w/v) % time, bacillus bifidus can excessive proliferation, it is impossible to be sufficiently reproduced the environment of enteral. Namely, owing to the microorganisms such as bacillus bifidus (thalline) are not bred substantially at enteral, thus in order to evaluate the minimizing effect of the corrupt substance that bacillus bifidus etc. realizes when close to enteral environment, it should set the condition that bacillus bifidus is not bred substantially, to reproduce above-mentioned environment. Therefore, glucose is set as such as 0.7(w/v) below %, it is preferable that it is set as 0.6(w/v) below %, and more preferably it is set as 0.55(w/v) below %.
It addition, for the reaction solution used in this experiment, it should set bacillus bifidus and substantially do not breed and the condition reducing required minimum energy that provides corrupt substance, it is thus regarded that original cooperation as substrate by medium component is inappropriate.
On the other hand, the reaction solution for using in this experiment, for instance, glucose is set as 0.1(w/v) % time, the activity (activity) of bacillus bifidus reduces, it is difficult to the minimizing effect of performance indole.So, although the minimizing effect etc. of indole is cashed out difference by each strain of bacillus bifidus originally, but this difference becomes to be difficult to embody, it is difficult to evaluate its effect clearly. Therefore, glucose is set as, for instance, 0.3(w/v) more than %, it is preferable that 0.4(w/v) more than %, more preferably 0.45(w/v) more than %. It addition, also determine the composition of reaction solution in the same manner for other composition.
Composition as reaction solution, it is possible to according to each 20 μ g of phenol paracresol indole scatol, Bis-Tris0.1M, magnesium sulfate (MgSO4) 0.75mM, glucose 0.5(w/v) % uses, pH is about 6.5.
What ［ embodiment 1 ］ had a microorganism of the minimizing effect of corrupt substance selects " in vitro tests "
For 34 bacterial strains of B.breve, 93 bacterial strains of B.longum, B.adolescentis total 199 bacterial strain of 72 bacterial strains, when carrying out quiescent culture, 2 bacterial strains of B.breve, B.longum 1 bacterial strain in, it is extremely slow to grow. Therefore, using above-mentioned 3 bacterial strains as experimental subject, finally for 32 bacterial strains of B.breve, 92 bacterial strains of B.longum, B.adolescentis total 196 bacterial strain of 72 bacterial strains carried out screening (evaluation is selected).
Above-mentioned bacterial strains (viable bacteria) each 1mg is suspended in reference example 1 determine reaction solution (pH:6.5, phenol paracresol indole scatol: each 20 μ g, Bis-Tris:0.1M, magnesium sulfate (MgSO4): 0.75mM, glucose: 0.5(w/v) %) in 2ml. Further, by this suspension (reaction solution) after 37 DEG C of standing 24h react, centrifugation, remove bacillus bifidus (thalline), the pH of its supernatant is adjusted to 7.
Then, this supernatant diethyl ether is divided 2 extractions, after this extracting solution is complemented to 5ml, by gas chromatography (GC) to this measured in solution (detection) indole, phenol, paracresol, scatol concentration.
Should illustrating, the analysis condition of gas chromatography (GC) is as described below. Namely, it is equipped with the detector of flame ionization detector: GC-2014 (Shimadzu), post (size): TC-1 (GLSciencesInc.), I.D.=0.25mm, L=30m, dF=0.25mm, column temperature: 60-170 DEG C (raising with 5 DEG C/min), 170-250 DEG C of (raising with 20 DEG C/min), carrier gas: nitrogen, flow velocity: 71.3ml/min, injector temperature: 240 DEG C, detector temperature: 250 DEG C, volume injected: 1 μ l.
The results are shown in 32 bacterial strains of table 1(B.breve), 92 bacterial strains of table 2(B.longum), 72 bacterial strains of table 3(B.adolescentis).
Should illustrating, the bacterial strain that arrangement numbering starts with " JCM " is preserved in following preservation mechanism.
Preservation mechanism name: Physical Chemistry Inst's バ イ オ リ ソ ス セ Application タ microbial material exploitation room (contact address: postcode 351-0198 Japan Yu Xian He Guang city 2-1, telephone number 048-467-9560).
[table 1]
[table 2]
[table 3]
In the above results, the bacterial strain that minimizing concentration is more than 13mg/g-cell of the indole relative to microorganism (thalline) 1g is selected, total 9 bacterial strain of 6 bacterial strains of what result met is B.breve and 3 bacterial strains of B.longum. Should illustrating, the minimizing concentration of indole is that more than 13mg/g-cell refers to that the slip of indole is more than 65%.
What ［ embodiment 2 ］ had a microorganism of the minimizing effect of corrupt substance selects " in vivo test (zoopery) "
Undertaken screening (evaluation is selected) by zoopery (in vivo test) by 9 bacterial strains selected in above-described embodiment 1.
After buying Balb/c mice, so as to freely absorb feedstuff (powder CRF-1/FOS(and the pure pharmaceutical worker's industry (strain) of light): 10(w/v) %, L-Trp: 2.5(w/v) %) 1 week, tame. After terminating during this domestication, the bacterial strain (viable bacteria) of regulation is suspended in normal saline, gives 2 weeks according to the frequency of 1 day 1 time continuously at morning. Now, normal saline give capacity, each bacterial strain give capacity, each bacterial strain give concentration respectively 1010Cfu/ml, 0.2ml/ pcs/day, 2x109Cfu/ pcs/day. Whole blood, the concentration of the indoxyl sulfate in mensuration serum is carried out adopting when this gives period (2 weeks) end.
Should illustrating, the bacterial strain (viable bacteria) giving mice is to select (screening) in test in vitro. Specifically, 6 bacterial strains (MEP22023004, OLB6129, MEP22023007, MEP22023012, MEP22023015, MEP22023016) of B.breve and 3 bacterial strains (MEP22023039, MEP22023061, MEP22023078) of B.longum are picked. Further, the normal saline of the capacity such as use as a control group, compares with the group minimizing effect to corrupt substance that gives of regulation bacterial strain (viable bacteria). The results are shown in Fig. 1 (6 bacterial strains of B.breve), Fig. 2 (3 bacterial strains of B.longum).
In the above results, mice is given with viable bacteria by 3 bacterial strains (Fig. 2) of 6 bacterial strains (Fig. 1) of B.breve and B.longum, the concentration of the indoxyl sulfate in the serum of mensuration mice when this gives period end, result gives in group at B.breveOLB6129, confirms its concentration and substantially reduces compared with matched group. That is, for B.breveOLB6129(FERMBP-11257) give group, compared with matched group, the concentration of the indoxyl sulfate in serum has confirmed significant minimizing. By this result, imply that B.breveOLB6129 in vivo can reduce corrupt substance.
The impact on the minimizing effect of corrupt substance of the concentration of the B.breveOLB6129 that live body is given by ［ embodiment 3 ］
［ experimental procedure ］
After Balb/c mice (SLC, ♀, 6 week old) is tamed one week, as shown in table 4, it being divided into 4 groups based on body weight, to each group, by the B.breveOLB6129(viable bacteria of normal saline Yu normal saline) suspension is orally administered to 2 weeks at morning continuously. Domestication chien shih is all organized and is freely absorbed feedstuff (powder CRF-1/FOS(and the pure pharmaceutical worker's industry (strain) of light): 10(w/v) %, L-Trp: 2.5(w/v) %).
[table 4]
(1) behind 2 weeks that give period of B.breveOLB6129, by the quiet blood sampling of tail, the concentration of indoxyl sulfate in serum is measured. After serum ultra-pure water (Milli-Q) is diluted to 100 times, filter with SYRINGELESSFILTERDEVICE0.2 μm (Whatman), according to existing report (Takayamaetal., AmJKidneyDis.2003Mar; 41(3Suppl1): S142-5), it is measured by high performance liquid chromatography (HPLC).
(2) B.breveOLB6129 give period terminate time gather just, gather cecal content at when dissected, measure each contained B.breveOLB6129(administration bacterium) bacterium number.
［ experimental result ］
B.breveOLB6129 give period terminate time, confirm the concentration of indoxyl sulfate in serum, for the minimizing effect of the concentration of indoxyl sulfate, 2x108Cfu/ pcs/day give group (III) and 2x109Cfu/ pcs/day give in group (IV), with normal saline give group (comparison) (I) compared with there is significant difference, but 2x107Cfu/ pcs/day give group (II) cfu/ only with normal saline give group (comparison) (I) compared with, not there is significant difference (Fig. 3).
It addition, in just with the B.breveOLB6129(administration bacterium in cecal content) detect (Fig. 4) in the way of consumption interdependence.
Live body is given the impact on the minimizing effect of corrupt substance of the presence or absence of FOS by ［ embodiment 4 ］
［ experimental procedure ］
By Balb/c mice (SLC, ♀, 6 week old) tame one week after, as shown in table 5, it is divided into 4 groups based on body weight, for each group, by the B.breveOLB6129(viable bacteria of the normal saline and normal saline that are not added with FOS) suspension, the normal saline that with the addition of FOS and normal saline B.breveOLB6129(viable bacteria) suspension be orally administered to 2 weeks at morning continuously. It is that all group freely absorbs feedstuff (powder CRF-1/L-tryptophan: 2.5(w/v) % during domestication). Should illustrate, for B.breveOLB6129(viable bacteria) concentration, be set as in embodiment 3 about corrupt substance minimizing effect show necessity minimum consumption namely, 2x108Cfu/ pcs/day.
[table 5]
(1) behind 2 weeks that give period of FOS, by the quiet blood sampling of tail, the concentration of indoxyl sulfate in serum is measured. After serum ultra-pure water (Milli-Q) is diluted to 100 times, filter with SYRINGELESSFILTERDEVICE0.2 μm (Whatman), according to existing report (Takayamaetal., AmJKidneyDis.2003Mar; 41(3Suppl1): S142-5), it is measured by high performance liquid chromatography (HPLC).
(2) FOS give period terminate time, gather B.breveOLB6129 give group just, measure by intravital B.breveOLB6129(administration bacterium) bacterium number.
［ experimental result ］
FOS gives period when terminating, and confirms the concentration of indoxyl sulfate in serum, only B.breveOLB6129 give in group, the minimizing effect of the concentration of indoxyl sulfate is little (Fig. 5). It addition, the minimizing effect of the concentration for indoxyl sulfate, give group for only FOS, with normal saline give group (I) compared with, it does not have significant difference (Fig. 5). On the other hand, for the minimizing effect of the concentration of indoxyl sulfate, give in group at B.breveOLB6129 and FOS, with normal saline give group (I) etc. compared with, there is significant difference (Fig. 5). Should illustrate, 2 groups (OLB6129+) giving group that give group and B.breveOLB6129 and FOS for only B.breveOLB6129, measure just in B.breveOLB6129(administration bacterium) bacterium number, result FOS give group being observed, viable count significantly increases (Fig. 6).
[deposit number]
FERM　BP-11257
This specification is quoted whole issue publication, patents and patent applications is fully incorporated in this specification as reference.

Claims (5)

1. oligofructose (FOS) and/or oligomeric galactose (GOS) and tryptophan (L-Trp) in preparation containing the application reduced in the medicine of microorganism of enteral corrupt substance or food,
Described application comprises screening technique, described method is characterised by, oligofructose (FOS) and/or oligomeric galactose (GOS) and tryptophan (L-Trp) are added in the feedstuff of mice, raise mice, evaluate the concentration of indoxyl sulfate in serum.
2. the application described in claim 1, wherein, making the concentration of oligofructose (FOS) and/or the concentration of oligomeric galactose (GOS) is 0.5～20(w/v) %, and to make the concentration of tryptophan be 1～4(w/v) %.
3. the application described in claim 1 or 2, it is characterized in that, implement to reduce the in-vitro screening method of the microorganism of enteral corrupt substance in advance, described screening technique is formed by comprising following method: is added on the concentration of the glucose of the energy of minimum required for the minimizing of enteral corrupt substance when microorganism is not bred substantially to reactant liquor, and evaluates the minimizing effect of the enteral corrupt substance added to described reactant liquor.
4. the application described in claim 3, wherein, the concentration making glucose is 0.3～0.7(w/v) %.
5. the application described in claim 3, wherein, making microorganism is lactic acid bacteria and/or bacillus bifidus.
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