WO2014171478A1 - ラクトバチルス属菌含有組成物 - Google Patents
ラクトバチルス属菌含有組成物 Download PDFInfo
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- WO2014171478A1 WO2014171478A1 PCT/JP2014/060812 JP2014060812W WO2014171478A1 WO 2014171478 A1 WO2014171478 A1 WO 2014171478A1 JP 2014060812 W JP2014060812 W JP 2014060812W WO 2014171478 A1 WO2014171478 A1 WO 2014171478A1
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- Prior art keywords
- composition according
- intestinal tract
- lactic acid
- lactobacillus
- fat
- Prior art date
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
- A23L2/382—Other non-alcoholic beverages fermented
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
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- A23V2400/167—Pentosus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Definitions
- the present invention relates to a Lactobacillus-containing composition. More specifically, the present invention relates to a composition containing a novel Lactobacillus pentosus.
- Some lactic acid bacteria and bifidobacteria have excellent physiological activities such as intestinal regulation and immunostimulatory effects, and are used in various applications depending on the characteristics of the bacterial species.
- research has been conducted on the diet effect by ingesting these bacteria into the body, and many reports have been given.
- Patent Document 1 reports that Lactobacillus rhamnosus ATCC 53103 strain degrades lipid (triacylglycerol) causing obesity and suppresses absorption into the body.
- L. is a kind of plant lactic acid bacteria. It is also known that brevis KB290 reaches the intestine alive and is excellent in intestinal survival rate and intestinal reachability (however, the number of excreted bacteria is smaller than the number of ingested bacteria) (see Non-Patent Document 1) ).
- Non-Patent Document 2 below indicates that Lactobacillus acidophilus L-92 is excellent in intestinal reachability of the bacterial strain by recovering 93% of the number of ingested bacteria from feces. L.
- Non-Patent Document 4 describes animalis ssp. When lactis DN-173 010 was administered to adults, about 20% of DN-173 010 was detected in feces compared to the number of ingested bacteria.
- An object of the present invention is to provide a composition containing lactic acid bacteria exhibiting intestinal growth.
- the present invention relates to a composition containing Lactobacillus pentosus TUA4337L strain (Accession No. NITE BP-1479) having the ability to grow in the intestinal tract.
- the composition of the present invention When the composition of the present invention is ingested, the lactic acid bacteria having a proliferative ability in the intestinal tract are proliferated, thereby enhancing the physiological activity of the bacterial cells and, consequently, the excellent effect of obtaining a diet effect.
- FIG. 1 is a diagram showing the results of screening with artificial intestinal fluid.
- FIG. 2 is a diagram showing the transition of weight gain. The “*” mark in the figure indicates that there is a significant difference (p ⁇ 0.05) with respect to the high fat diet group.
- FIG. 3 is a diagram showing the amount of triglyceride in serum. The “*” mark in the figure indicates that there is a significant difference (p ⁇ 0.05) with respect to the high fat diet group.
- composition of the present invention is characterized by containing a lactic acid bacterium of Lactobacillus pentosus TUA4337L strain (hereinafter also referred to as the lactic acid bacterium of the present invention) having the ability to grow in the intestinal tract.
- a lactic acid bacterium of Lactobacillus pentosus TUA4337L strain hereinafter also referred to as the lactic acid bacterium of the present invention
- the lactic acid bacterium of the present invention is a Lactobacillus pentosaus TUA4337L strain, and has the ability to grow in the intestinal tract.
- “having the ability to proliferate in the intestinal tract” or “proliferate in the intestinal tract” means that after reaching the intestinal tract, it proliferates in the small intestine and / or the large intestine, preferably in the small intestine.
- the degree of proliferative ability can be evaluated as “proliferating ability” when the value is 10 times or more of OD 660 at the time of inoculation when cultured in artificial intestinal fluid at 37 ° C. for 6 hours. .
- the present inventors investigated the growth ability of about 480 types of lactic acid bacteria possessed by the present inventors in artificial intestinal fluid, and administered a suspension of bacteria belonging to Lactobacillus pentosas selected from them to animals.
- the Lactobacillus pentosus TUA4337L strain was found to have a significantly increased number of bacteria discharged compared to the number of administered bacteria, and the present invention was completed.
- Lactobacillus pentosasus TUA4337L strain is identified as NRIC 0883 and accession number NITE BP-1479, with an international administration date of December 10, 2012, the National Institute of Technology and Evaluation, Japan Patent Evaluation Center for Microorganisms (Chiba Prefecture, Japan) It has been deposited with Kisarazu City Kazusa Kamashishi 2-5-8).
- Lactobacillus pentosus TUA4337L strain is abbreviated as TUA4337L strain.
- Tables 1 and 2 show the mycological properties of the TUA4337L strain.
- the sugar assimilation properties in Table 2 are the results of measurement using a bacterial identification kit API 50CH (BIOMETRIEUX).
- “+” means a sugar that is assimilated
- “ ⁇ ” means a sugar that is not assimilated.
- the TUA4337L strain which will be described in detail in Examples below, has a feature that the number of bacteria discharged is larger than the number of ingested bacteria, that is, has intestinal growth.
- the intestinal proliferative property is, for example, preferably 10 times or more, more preferably 15 times or more, after culturing at 37 ° C. in artificial intestinal fluid for 6 hours, based on the number of bacteria at the start of the culture.
- the number of bacteria is preferably 20 times or more, and more preferably 25 times or more.
- the recA gene sequence (SEQ ID NO: 1) decoded from DNA extracted from the TUA4337L strain has 99% homology with the recA gene sequence of Lactobacillus ospentosus IG1 strain.
- the homology is determined by, for example, using a search program BLAST using an algorithm developed by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990)). Is shown.
- the medium for culturing the TUA4337L strain is not particularly limited, and includes a normal medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients, and the like.
- culture in an agar medium or a liquid medium is also possible.
- the culture temperature is preferably 10 to 45 ° C., more preferably 15 to 42 ° C., further preferably 28 to 38 ° C., further preferably 35 to 37 ° C.
- the proliferative pH is preferably pH 3.0 to 12. 5, More preferably, the pH is 3.5 to 12.0.
- composition of the present invention contains the Lactobacillus pentosus TUA4337L strain having the ability to grow in the intestinal tract as described above in various forms.
- Lactobacillus pentosasus TUA4337L strain contained in the composition of the present invention examples include lactic acid bacteria (live bacteria and dead bacteria) themselves, lactic acid bacteria-containing materials, lactic acid bacteria-treated products, and the like.
- Viable bacteria can be obtained from lactic acid bacteria-containing materials such as the lactic acid bacteria culture solution.
- Dead bacteria can be obtained, for example, by subjecting live bacteria to heating, ultraviolet irradiation, formalin treatment, acid treatment, and the like.
- the obtained live bacteria and dead bacteria can be further processed by grinding or crushing.
- the lactic acid bacteria in each of the above forms are preferably live bacteria from the viewpoint of exhibiting an effect of sufficiently proliferating in the intestinal tract, but dead bacteria may be mixed.
- Examples of the lactic acid bacteria include live cells, wet bacteria, and dry bacteria.
- Examples of the lactic acid bacterium-containing material include lactic acid bacterium suspension, lactic acid bacterium culture (including microbial cells, culture supernatant, and medium components), and lactic acid bacterium culture solution (obtained by removing solid content from the bacterium culture). it can.
- Examples of the lactic acid bacterium treated product include a ground product, a crushed product, a liquid product (extracted liquid, etc.), a concentrate, a pasted product, a dried product (spray dried product, freeze-dried product, vacuum dried product, drum dried product, etc. ), Dilutions and the like.
- the Lactobacillus pentosus TUA4337L strain in the present invention can be used alone or in combination of two or more forms as long as it has the ability to grow in the intestinal tract.
- the total content in the composition of the present invention is not particularly limited, it is usually preferably from 0.00001 to 99.9% (g / g), and particularly preferably about 0.0001% to 50% (g / g).
- the number of cells is preferably in the range of 1.0 ⁇ 10 2 to 1.0 ⁇ 10 12 cells / g, and in the range of 1.0 ⁇ 10 6 to 1.0 ⁇ 10 12 cells / g. More preferably, it is within.
- the above “pieces / g” can be expressed as “CFU / g” in living bacteria.
- the lactic acid bacteria of the present invention may be used in combination with a strain having an action other than growing in the intestinal tract.
- composition of the present invention contains Lactobacillus pentosasus TUA4337L strain having the ability to grow in the intestinal tract, carriers, bases, and / or additives usually used in the food field, the pharmaceutical field, etc. Can be contained within a range not impairing the effects of the present invention.
- various components such as known sweeteners, acidulants, vitamins, excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, stable Agents and the like.
- composition of the present invention contains known ingredients such as cosmetic ingredients and lifestyle-related disease prevention / amelioration ingredients in combination of one or more in order to add other useful effects as necessary. be able to.
- the form of the composition of the present invention is not particularly limited as long as it is a form in which the Lactobacillus pentosasus TUA4337L strain having the ability to grow in the intestinal tract can be ingested into the body.
- tablet forms that can be ingested as supplements are exemplified. Specific examples include various forms such as tablets, capsules, drinks, seasonings, processed foods, desserts, and confectionery. Among these forms, it is preferably provided as a fermented food.
- Fermented food is a general term for food fermented using plant lactic acid bacteria, and includes beverages.
- the type of fermented food is not particularly limited.
- fermented milk lactic acid bacteria beverages
- fermented soy milk pickles, kimchi, fruit wine, miso, soy sauce and other fermented fruits and vegetables
- fermented foods such as fermented fruit juice yogurt fermented with fruit juice.
- a beverage or food containing the lactic acid bacterium of the present invention that is, a beverage or food containing Lactobacillus pentosus TUA4337L strain that has the ability to grow in the intestinal tract has the ability to grow in the intestine when eaten. Compared with eating lactic acid bacteria, it exhibits a high fat absorption inhibitory action, and since its action persists, as a health functional food or health food having a continuous fat absorption inhibitory action, for example, body weight It may be possible to provide a display indicating that it is used for suppressing increase or for weight reduction, for preventing obesity or for improving obesity, and for dieting.
- the health functional food means the health functional food defined by the Ministry of Health, Labor and Welfare, and includes the nutrition functional food and the food for specified health.
- the health functional food and the health food may be either food or beverage. .
- composition of the present invention can be prepared according to a known method in the food field, the pharmaceutical field or the like depending on the form.
- the composition of the present invention is appropriately set according to the form, purpose of intake and the age, weight, and symptoms of the person who takes the composition, but for example, the amount of lactic acid bacteria is 1.0 ⁇ 10 6 per day. It is preferable to take the above amount / kg body weight orally in 1 to several times.
- the dry weight is preferably 0.00001 to 1 g, more preferably 0.0001 to 0.2 g, and still more preferably 0.0003 to about 50 kg per adult body weight. 0.002 g.
- the composition of the present invention has the characteristics that the lactic acid bacterium of the present invention suppresses fat absorption, and has the ability to grow in the intestinal tract.
- intake means “intake” and / or “administration”.
- the ingestion subject in this specification is preferably a human who needs a fat absorption inhibitory action, but may be a pet animal or the like.
- this invention also provides the fat absorption inhibitor which consists of a Lactobacillus pentosas which has the growth ability in an intestinal tract in order to suppress absorption from the intestinal tract of fat derived from a meal.
- the present invention provides a method for suppressing fat absorption, characterized by using an effective amount of the composition containing the Lactobacillus pentosasus TUA4337L strain for an individual who needs to suppress fat absorption. .
- the individual who needs to suppress fat absorption is not particularly limited as long as the individual exhibits a disease that exhibits a therapeutic effect by suppressing fat absorption.
- individuals having diseases such as obesity or diabetes caused by obesity, hyperlipidemia, hypertension, arteriosclerosis are exemplified.
- diseases such as obesity or diabetes caused by obesity, hyperlipidemia, hypertension, arteriosclerosis are exemplified.
- arteriosclerosis For the purpose of preventing or ameliorating the disease, for example, those who are concerned about body weight, those who are concerned about blood glucose level, those who are concerned about blood pressure, and the like are also included in the individual.
- the effective amount is an amount that suppresses fat absorption when the TUA4337L strain is administered to the individual as compared to an individual that is not administered.
- the specific effective amount is appropriately set according to the administration form, administration method, purpose of use, individual age, weight, symptoms, etc., and is not constant.
- the term “administration” is used to include all modes of administration, ingestion, taking, and drinking.
- Example 1 ⁇ Screening using proliferation ability in artificial intestinal fluid as index>
- lactic acid bacteria possessed by the present inventors about 480 strains (including the JCM strain), which are mainly plant lactic acid bacteria, were evaluated for the proliferation ability in the artificial intestinal fluid.
- each lactic acid bacterium was inoculated from a glycerol stock into MRS medium (Difco Laboratories) (10 mL) at 1 v / v%, and cultured at 35 ° C. for 16 to 17 hours.
- MRS medium Difco Laboratories
- OD 660 absorbance at 660 nm
- 100 ⁇ L of each culture solution prepared in MRS medium so that OD 660 of the culture solution was 10 was obtained.
- FIG. ⁇ Artificial intestinal fluid (pH 6.45)> MRS medium 9mL 10 w / v% bile (Wako Pure Chemical Industries, Ltd.) solution 1 mL 1w / v% pancreatin (from Porcine: SIGMA) 100 ⁇ L
- the bile solution and pancreatin solution used were sterilized by treatment with a 0.22 ⁇ m filter (PVDF membrane, manufactured by Millipore).
- Lactobacillus pentosas and Lactobacillus plantarum tend to have a high growth rate.
- Lactobacillus pentosas TUA4337L strain has a particularly high growth rate and is excellent in growth in the intestinal tract.
- DNA was extracted using a kit (QIAamp DNA Stool Mini Kit: Qiagen) (cell disruption was performed using Glass beads (150 to 212 ⁇ m: SIGMA) 300 mg, phenol / Chloroform / isoamylcohol (25: 24: 1) 300 ⁇ L and buffer ASL (reagent in the kit) 900 ⁇ L were added, centrifuged at 3000 rpm for 1 min on MULTI-BEADS SHOCKER MB-200 (YASUI KIKAI), and left on ice for 1 min.
- kit QIAamp DNA Stool Mini Kit: Qiagen
- primer1 5′-GCAAGTCGAACGAACTCTGGTATT-3 ′ (SEQ ID NO: 2)
- primer2 5′-CGGACCATGCGGTCCAA-3 ′ (SEQ ID NO: 3)
- PCR was carried out using 7500 Real Time PCR System (Applied Biosystems), 95 ° C for 30 seconds, 95 ° C for 5 seconds, and 60 ° C for 34 seconds for a total of 60 cycles. The number of copies per 1 g of intestinal contents is determined from the strength, the total amount of intestinal contents and the dilution factor.
- the number of 16S rDNA copies per cell is obtained and converted to the number of bacteria. In mice that have not been administered lactic acid bacteria, it is confirmed that neither Lactobacillus pentosas nor Lactobacillus plantarum is detected by the real-time PCR.
- each group in Table 5 was allowed to freely ingest each solid diet.
- the high-fat diet + live bacteria group was prepared in the same manner as in Example 2, and the high-fat diet + dead bacteria group was prepared as follows. An amount of 100 million cells was administered.
- PBS (-) 250 ⁇ L without lactic acid bacteria was administered to the normal diet group, and 250 ⁇ L of liquid feed without lactic acid bacteria was administered to the high fat diet group.
- ⁇ Preparation of administration sample (sample containing dead bacteria)> [1] Inoculation of 1% v / v% from glycerol stock of TUA4337L strain into MRS medium (30 mL) [2] Culture (35 ° C., 20 hr) [3] Centrifugation (8000 rpm, 5 min) of the culture solution and removal of the supernatant, and then suspending the suspension of [4] [3] in 30 mL of PBS ( ⁇ ) (8000 rpm, 5 min) and removal of the supernatant Then, resuspend in 5 mL of PBS ( ⁇ ) [5] Count the number of bacteria with a microscope [6] Separate the solution of 20 billion cells into a 15 mL centrifuge tube, centrifuge (8000 rpm, 5 min), remove the supernatant, 5 mL of artificial gastric juice (125 mM NaCl, 7 mM KCl, pH 1.0) was added, and after stirring,
- Example 4 Fluorescence absorption inhibitory effect>
- the same content as in Example 3 was continued for 2 weeks.
- olive oil (nacalai quest) was administered (5 mL / kg), and after 3 hours, dissection was performed and serum was collected from the vena cava.
- Triglyceride (TG) in serum was measured using Triglyceride E-Test Wako (Wako Pure Chemical Industries, Ltd.). The results are shown in FIG.
- the comparison between groups performed the significant difference determination by the t test by the significance level 0.05.
- a pharmaceutical product (tablet) containing the TUA4337L strain is produced by the method described below. 66.7 g of dry pulverized product of TUA4337L strain is mixed with 232.0 g of lactose and 1.3 g of magnesium stearate, and tableted with a single tableting machine to produce a tablet having a diameter of 10 mm and a weight of 300 mg.
- a lactic acid bacteria beverage is prepared by mixing the raw materials shown in Table 6 using TUA4337L strain.
- Peach juice is inoculated with 2% by weight of TUA4337L and cultured at 30 ° C. for 38 hours to produce peach fermentation juice.
- carrot juice is fermented to produce a carrot juice.
- composition of the present invention contains lactic acid bacteria having the ability to grow in the intestinal tract, when ingested in the body, the lactic acid bacteria reach the intestinal tract and proliferate, thereby suppressing fat absorption and weight gain. Can be suppressed, and is preferably used for the purpose of a diet effect.
- Sequence number 1 of a sequence table is the base sequence of recA of Lactobacillus pentosaus TUA4337L.
- Sequence number 2 of a sequence table is a base sequence of a Lactobacillus pentosasus / plantarum specific primer.
- Sequence number 3 of a sequence table is a base sequence of a Lactobacillus pentosasus / plantarum specific primer.
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Abstract
Description
本発明者らが保有する乳酸菌のうち、主に植物性乳酸菌である約480株(JCM株を含む)について、人工腸液における増殖能を評価した。
<人工腸液(pH6.45)>
MRS培地 9mL
10w/v%胆汁(和光純薬工業株式会社)溶液 1mL
1w/v%パンクレアチン(from Porcine:SIGMA) 100μL
なお、胆汁溶液及びパンクレアチン溶液は0.22μmのフィルター(PVDF membrane、Millipore社製)で処理することにより無菌にしたものを用いた。
高脂肪食を自由摂取させたマウスに以下のように調製したTUA4337L株を投与して、***される菌数を定量した。具体的には、以下のように調製した投与サンプルを用いて、乳酸菌約10億cells(1.0×109cells)分(菌体懸濁液250μLに相当)をC57BL/6Jマウス(10週齢、雄)に午前10時ごろに1回投与した(n=5)。
<投与サンプル(生菌含有サンプル)の調製>
〔1〕TUA4337L株のグリセロールストックから、MRS培地(30mL)へ1v/v%植菌
〔2〕培養(35℃、20hr)
〔3〕培養液を遠心(8000rpm、5min)して上清除去後、PBS(-)30mLで懸濁
〔4〕〔3〕の懸濁液を遠心(8000rpm、5min)して上清除去後、PBS(-)5mLで再懸濁
〔5〕顕微鏡で菌数カウント
〔6〕15mLの遠心管に200億cells分の溶液を分取し、遠心(8000rpm、5min)して上清除去後、液状飼料(高脂肪食 60kcal%FAT:Research Diet)5mLに懸濁して菌体懸濁液を調製(液状飼料はPBS(-)で調製)
<リアルタイムPCRによる菌数測定法>
〔1〕糞便100mg(湿重量)に対してPBS(-)1mLを添加後、スパチュラーを用いて解砕
〔2〕糞便100mg分をエッペンチューブ(登録商標)に採取後、遠心(15000rpm、5min)して上清除去後、PBS(-)1mLで懸濁(遠心から懸濁までの操作を2回繰り返す)
〔3〕〔2〕の懸濁液から上清除去後、キット(QIAamp DNA Stool Mini Kit:キアゲン)を用いてDNAを抽出
(細胞破砕は、Glass beads(150~212μm:SIGMA)300mg、phenol/chloroform/isoamylalcohol(25:24:1)300μL、及びbuffer ASL(キット中の試薬)900μLを添加し、MULTI-BEADS SHOCKER MB-200(YASUI KIKAI)で3000rpm、1min遠心後、氷上1min静置の操作を3回繰り返すことで行う)
〔4〕下記に示す条件のリアルタイムPCRにより腸管内容物中の乳酸菌を定量
((リアルタイムPCR条件))
(1) SYBR Premix Ex Taq ll(Takara Bio)10μL、each primer(10μM)0.8μL、ROX reference DyeII 0.4μL、滅菌水6μL、DNA溶液2μLを混合してPCR反応液を調製する。プライマーはラクトバチルス・ペントーサス及びラクトバチルス・プランタラムの16S rDNAを特異的に検出する以下のプライマーを用いる(ラクトバチルス・ペントーサスとラクトバチルス・プランタラムの16S rDNAの配列は100%同一である)。
primer1:5’-GCAAGTCGAACGAACTCTGGTATT-3’(配列番号2)
primer2:5’-CGGACCATGCGGTCCAA-3’(配列番号3)
(2) PCRは7500 Real Time PCR System(Applied Biosystems)を用いて、95℃ 30秒の後、95℃ 5秒、60℃ 34秒を1サイクルとして、合計60サイクル反応を行い、得られた蛍光強度と腸管内容物合計量と希釈倍率から腸管内容物1g当りのコピー数を求める。
(3) 別途1cell当たりの16S rDNAのコピー数を求め、菌数に換算する。なお、乳酸菌を投与していないマウスにおいては、前記リアルタイムPCRによりラクトバチルス・ペントーサス及びラクトバチルス・プランタラムのいずれも検出されないことを確認する。
C57BL/6Jマウス(8週齢、雄)を、通常食群、高脂肪食群、高脂肪食+生菌群、高脂肪食+死菌群の4群に群分けし(各n=10)、それぞれに以下の表5に示す食餌を32日間続け、体重を毎日測定して平均値を算出した。平均値の推移を図2に示す。なお、群間比較は、有意水準0.05によるt検定を用いて行った。
〔1〕TUA4337L株のグリセロールストックから、MRS培地(30mL)へ1v/v%植菌
〔2〕培養(35℃、20hr)
〔3〕培養液を遠心(8000rpm、5min)して上清除去後、PBS(-)30mLで懸濁
〔4〕〔3〕の懸濁液を遠心(8000rpm、5min)して上清除去後、PBS(-)5mLで再懸濁
〔5〕顕微鏡で菌数カウント
〔6〕15mLの遠心管に200億cells分の溶液を分取し、遠心(8000rpm、5min)して上清除去後、人工胃液(125mM NaCl、7mM KCl、pH1.0)5mLを添加して攪拌後60min放置
〔7〕〔6〕の溶液を遠心(8000rpm、5min)して上清除去後、液状飼料(60kcal%FAT)5mLで懸濁して菌体懸濁液を調製
実施例3における通常食群、高脂肪食群、高脂肪食+生菌群の群構成で(各n=12)、それぞれに実施例3と同じ内容の食餌を2週間続けた。その後、一晩絶食させた後、オリーブオイル(nacalai tesque)を投与し(5mL/kg)、さらに3時間後に解剖して大静脈から血清を採取した。血清中のトリグリセライド(TG)をトリグリセライド E-テストワコー(和光純薬工業株式会社)を用いて測定した。結果を図3に示す。なお、群間比較は、有意水準0.05によるt検定による有意差判定を行った。
以下に示す方法により、TUA4337L株を含有する医薬品(錠剤)を製造する。
TUA4337L株の乾燥粉砕物66.7gを、乳糖232.0gおよびステアリン酸マグネシウム1.3gとともに混合し、単発式打錠機にて打錠することにより、直径10mm、重量300mgの錠剤を製造する。
牛乳、脱脂粉乳、水を混合したミックスを調製し、加熱殺菌した後、40℃前後まで冷却し、スターターとしてTUA4337L株を接種したのち、発酵室にて静置発酵させる。ここで、静置発酵の温度は、適宜選択できる。また、発酵開始時における溶存酸素濃度を抑えるため、窒素などの不活性ガスによる置換処理を行ってもよい。このようにして得られたTUA4337L発酵乳を、市販の牛乳に添加し3日間静置しヨーグルトを調製する。
TUA4337L株を用いて表6に示した原料を混合して乳酸菌飲料を調製する。
モモ果汁に、TUA4337Lを2重量%量を接種し、30℃で38時間培養してモモ発酵果汁を製造する。また、同様にニンジン汁を発酵させてニンジン発酵汁を製造する。
配列表の配列番号2は、ラクトバチルス・ペントーサス/プランタラム特異的プライマーの塩基配列である。
配列表の配列番号3は、ラクトバチルス・ペントーサス/プランタラム特異的プライマーの塩基配列である。
Claims (16)
- 腸管内での増殖能を有するラクトバチルス・ペントーサス TUA4337L株(受託番号NITE BP-1479)を含有する組成物。
- 腸管からの脂肪吸収抑制作用を有する請求項1記載の組成物。
- 高脂肪食による腸管での脂肪吸収を抑制する請求項1又は2記載の組成物。
- 食した場合、ラクトバチルス・ペントーサス TUA4337L株が腸管内で増殖し、それにより増殖能のない乳酸菌を食した場合と比べて高い脂肪吸収抑制作用を発揮する、請求項1~3いずれか記載の組成物。
- 食した場合、ラクトバチルス・ペントーサス TUA4337L株が腸管内で増殖し、それにより増殖能のない乳酸菌を食した場合と比べて脂肪吸収抑制作用が持続する、請求項1~4いずれか記載の組成物。
- 体重増加の抑制用又は体重低減用に供される、請求項1~5いずれか記載の組成物。
- 肥満の防止用又は肥満改善用に供される、請求項1~6いずれか記載の組成物。
- 高脂肪食と共に、あるいは高脂肪食を食する前に供される、請求項1~7いずれか記載の組成物。
- 飲料又は食品である、請求項1~8いずれか記載の組成物。
- ヨーグルトである、請求項1~9いずれか記載の組成物。
- 食事由来の脂肪の腸管からの吸収抑制剤である、請求項1~8いずれか記載の組成物。
- 1日1回、朝食時に食する、請求項1~11いずれか記載の組成物。
- ダイエット用である、請求項1~12いずれか記載の組成物。
- 脂肪吸収を抑制させる使用のための、ラクトバチルス・ペントーサス TUA4337L株(受託番号NITE BP-1479)を含有する組成物。
- 脂肪吸収を抑制させるための、ラクトバチルス・ペントーサス TUA4337L株(受託番号NITE BP-1479)を含有する組成物の使用。
- ラクトバチルス・ペントーサス TUA4337L株(受託番号NITE BP-1479)の有効量を含む組成物を脂肪吸収の抑制を必要とする個体に投与する工程を含む、脂肪吸収の抑制方法。
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Application Number | Priority Date | Filing Date | Title |
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KR1020157032780A KR20150143803A (ko) | 2013-04-17 | 2014-04-16 | 락토바실러스속 균 함유 조성물 |
JP2015512501A JP5891337B2 (ja) | 2013-04-17 | 2014-04-16 | ラクトバチルス属菌含有組成物 |
NZ712603A NZ712603A (en) | 2013-04-17 | 2014-04-16 | Composition containing bacterium belonging to genus lactobacillus |
EP14785100.0A EP3018199B1 (en) | 2013-04-17 | 2014-04-16 | Composition containing bacterium belonging to genus lactobacillus |
MYPI2015703670A MY183171A (en) | 2013-04-17 | 2014-04-16 | Composition containing bacterium belonging to genus lactobacillus |
CA2908032A CA2908032A1 (en) | 2013-04-17 | 2014-04-16 | Composition containing bacterium belonging to genus lactobacillus |
AU2014254813A AU2014254813B2 (en) | 2013-04-17 | 2014-04-16 | Composition containing bacterium belonging to genus Lactobacillus |
ES14785100.0T ES2684074T3 (es) | 2013-04-17 | 2014-04-16 | Composición que contiene una bacteria que pertenece al género Lactobacillus |
CN201480021905.5A CN105121627B (zh) | 2013-04-17 | 2014-04-16 | 含有乳杆菌属菌的组合物 |
US14/784,064 US9603880B2 (en) | 2013-04-17 | 2014-04-16 | Composition containing bacterium belonging to genus lactobacillus |
HK16100873.4A HK1213011A1 (zh) | 2013-04-17 | 2016-01-26 | 含有乳桿菌屬菌的組合物 |
US15/358,280 US10159269B2 (en) | 2013-04-17 | 2016-11-22 | Composition containing bacterium belonging to genus Lactobacillus |
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CA (1) | CA2908032A1 (ja) |
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JP2017007947A (ja) * | 2015-06-16 | 2017-01-12 | サントリーホールディングス株式会社 | 体温低下抑制剤 |
JP2018153182A (ja) * | 2017-03-20 | 2018-10-04 | 大江生醫股▲ふん▼有限公司TCI Co.Ltd | ラクトバチルスプランタルムtci378、およびそれを用いた脂肪の減少および胃腸機能の改善における使用 |
JP2019062747A (ja) * | 2017-09-28 | 2019-04-25 | サントリーホールディングス株式会社 | 乳酸菌入り飲料 |
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EP2987853B1 (en) * | 2013-04-17 | 2017-05-31 | Suntory Holdings Limited | Bacterium belonging to genus lactobacillus |
NZ712603A (en) | 2013-04-17 | 2020-02-28 | Suntory Holdings Ltd | Composition containing bacterium belonging to genus lactobacillus |
CN107929329B (zh) * | 2017-07-07 | 2023-06-06 | 西北民族大学 | 一种消栓降脂益生复合菌中药颗粒及其制备方法 |
CN109207389B (zh) * | 2017-07-07 | 2023-06-06 | 西北民族大学 | 一种消栓降脂益生复合菌中药口服液及其制备方法 |
TWI739078B (zh) * | 2018-04-25 | 2021-09-11 | 日商曾根農場股份有限公司 | 脂肪累積抑制用組成物 |
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JP2017007947A (ja) * | 2015-06-16 | 2017-01-12 | サントリーホールディングス株式会社 | 体温低下抑制剤 |
JP2018153182A (ja) * | 2017-03-20 | 2018-10-04 | 大江生醫股▲ふん▼有限公司TCI Co.Ltd | ラクトバチルスプランタルムtci378、およびそれを用いた脂肪の減少および胃腸機能の改善における使用 |
US11273189B2 (en) | 2017-03-20 | 2022-03-15 | Tci Co., Ltd | Lactobacillus plantarum TCI378 and its uses in losing fat and improving gastrointestinal functions |
JP2019062747A (ja) * | 2017-09-28 | 2019-04-25 | サントリーホールディングス株式会社 | 乳酸菌入り飲料 |
JP7046549B2 (ja) | 2017-09-28 | 2022-04-04 | サントリーホールディングス株式会社 | 乳酸菌入り飲料 |
Also Published As
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JP5891337B2 (ja) | 2016-03-22 |
CN105121627A (zh) | 2015-12-02 |
AU2014254813B2 (en) | 2019-08-01 |
CN105121627B (zh) | 2017-12-15 |
US20170112168A1 (en) | 2017-04-27 |
EP3018199B1 (en) | 2018-05-30 |
US20160058807A1 (en) | 2016-03-03 |
NZ712603A (en) | 2020-02-28 |
US9603880B2 (en) | 2017-03-28 |
JPWO2014171478A1 (ja) | 2017-02-23 |
ES2684074T3 (es) | 2018-10-01 |
CA2908032A1 (en) | 2014-10-23 |
EP3018199A1 (en) | 2016-05-11 |
US10159269B2 (en) | 2018-12-25 |
AU2014254813A1 (en) | 2015-10-22 |
HK1213011A1 (zh) | 2016-06-24 |
MY183171A (en) | 2021-02-18 |
KR20150143803A (ko) | 2015-12-23 |
EP3018199A4 (en) | 2016-11-30 |
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