WO2019054396A1 - Agent permettant d'inhiber le nombre de bactéries du genre fusobacterium et/ou de bactéries du genre sutterella - Google Patents

Agent permettant d'inhiber le nombre de bactéries du genre fusobacterium et/ou de bactéries du genre sutterella Download PDF

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WO2019054396A1
WO2019054396A1 PCT/JP2018/033742 JP2018033742W WO2019054396A1 WO 2019054396 A1 WO2019054396 A1 WO 2019054396A1 JP 2018033742 W JP2018033742 W JP 2018033742W WO 2019054396 A1 WO2019054396 A1 WO 2019054396A1
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bacteria
fusobacterium
kestose
genus
months
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PCT/JP2018/033742
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English (en)
Japanese (ja)
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実可子 篠原
吉弘 門田
巧 栃尾
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物産フードサイエンス株式会社
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Priority to JP2019542255A priority Critical patent/JP7262391B2/ja
Publication of WO2019054396A1 publication Critical patent/WO2019054396A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • 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
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the present invention is an agent for suppressing the number of Fusobacterium bacteria and / or bacteria of the genus Stelerus, which comprises 1-kestose as an active ingredient, a food composition for suppressing the number of bacteria, a feed for animals for suppressing the number of bacteria and a method for suppressing the number of bacteria
  • the present invention relates to a method of preventing or treating hemorrhagic diarrhea and use of 1-kestose for producing a medicament for preventing or treating acute hemorrhagic diarrhea.
  • Fusobacterium (Fusobacterium) is an anaerobic gram-negative bacillus and has been reported to inhabit the digestive organs of animals such as human intestine and oral cavity.
  • Sterella bacteria (Sutterella) are also anaerobic or microaerophilic gram-negative short bacilli and are a component species of the intestinal flora of animals such as humans.
  • Fusobacterium bacteria and Stellara bacteria are associated with various diseases and health conditions, for example, prominent in feces of dogs suffering from acute hemorrhagic diarrhea (Acute hemorrhagic diarrihea; AHD). The increase is suggested to be involved in such diseases (Non-patent Document 1; Table 3 and FIG. 3 etc.).
  • AHD in dogs is characterized by symptoms such as bloody vomiting and diarrhea, and is a digestive disease that can be concentrated and become serious.
  • antibiotics have been administered for the purpose of suspicion of Clostridial infection and for the prevention of sepsis (non-patented).
  • Literature 2 discloses the intestinal bacterial flora is destroyed or resistant bacteria are generated.
  • the present inventors have made it a task to suppress the number of Fusobacterium bacteria and bacteria of Stellara bacteria without causing the above-mentioned risks associated with the use of antibiotics.
  • Another object of the present invention is to contribute to the prevention and treatment of diseases such as AHD by suppressing the number of such bacteria. That is, the present invention provides a substance and method capable of effectively suppressing the number of Fusobacterium bacteria and Sterella bacteria, and a substance and method which can contribute to the prevention and treatment of diseases such as AHD. With the goal.
  • the agent for suppressing the number of Fusobacterium bacteria and / or Stelerus bacteria according to the present invention comprises 1-kestose as an active ingredient.
  • the agent for suppressing the number of bacteria according to the present invention can be suitably used to suppress the number of Fusobacterium bacteria and / or bacteria of Stella bacteria in dogs.
  • the bacteria count inhibitor according to the present invention can be suitably used for the prevention or treatment of acute hemorrhagic diarrhea.
  • the food composition for suppressing the number of Fusobacterium bacteria and / or Stelerus bacteria according to the present invention contains 1-kestose as an active ingredient.
  • the animal feed for reducing the number of Fusobacterium bacteria and / or Stelerus bacteria according to the present invention contains 1-kestose as an active ingredient.
  • the method for suppressing the number of Fusobacterium bacteria and / or Stelerus bacteria according to the present invention is a Fusobacterium bacteria and / or Stellara bacteria in the human or animal body by allowing the human or animal to take in 1-kestose. It has the process of suppressing the number of bacteria.
  • the method for preventing or treating AHD according to the present invention is a method for preventing or treating AHD, by causing a human or animal suffering from or capable of suffering from AHD to ingest 1-kestose into the body of the human or animal. Suppressing the number of Fusobacterium bacteria and / or Stelerus bacteria in
  • the use of 1-kestose according to the present invention is the use of 1-kestose for producing a medicament for preventing or treating AHD.
  • the present invention it is possible to effectively suppress the number of at least one of Fusobacterium bacteria and / or Stellara bacteria in human or animal organisms.
  • the suppression of the number of bacteria can contribute to the prevention and treatment of diseases such as AHD.
  • 1-kestose is a kind of oligosaccharide which is contained in vegetables and grains such as onion, garlic, barley, rye, etc., and it is a substance with eating experience since ancient times, mutagenicity test, acute The safety is extremely high as toxicity is not observed in any of the toxicity test, subchronic toxicity test and chronic toxicity test (Food and Development, Vol. 49, No. 12, p. 9, 2014).
  • 1-kestose is highly soluble in water and has a good sweet taste similar to sugar, so it can be taken on a daily basis as it is or as a seasoning such as a sweetener etc. It can be easily incorporated into pharmaceuticals, animal feeds and the like.
  • Fusobacterium bacteria and Stellara spp. Can be conveniently and effectively taken without any risk (the destruction of the intestinal bacterial flora and the development of resistant bacteria) caused by the use of the above-mentioned antibiotics.
  • the number of at least one of the bacteria can be suppressed.
  • the term "Fusobacterium” refers to a bacterium belonging to the genus Fusobacterium.
  • the bacteria belonging to the genus Fusobacterium for example, Fusobacterium mortiferum, Fusobacterium necrophorum, Fusobacterium gondinoformans, Fusobacterium russii, Fusobacterium perfoetens, Fusobacterium simum, Fusobacterium simiae, Fusobacterium nuclearium,
  • Sterella bacteria refers to bacteria belonging to the genus Stellara.
  • bacteria belonging to the genus Stellara include Sutterella wadsworthensis, Sutterella parvirubra, Sutterella stercoricanis and the like.
  • 1-kestose is a trisaccharide oligosaccharide consisting of one molecule of glucose and two molecules of fructose.
  • 1-kestose can be produced by using sucrose as a substrate and performing an enzyme reaction with an enzyme as disclosed in JP-A-58-201980. Specifically, first, ⁇ -fructofuranosidase is added to a sucrose solution, and the mixture is allowed to stand at 37 ° C. to 50 ° C. for about 20 hours to perform an enzyme reaction to obtain a 1-kestose-containing reaction solution.
  • 1-kestose-containing reaction solution By subjecting this 1-kestose-containing reaction solution to a chromatographic separation method as disclosed in JP-A-2000-232878, 1-kestose and other sugars (glucose, fructose, sucrose, tetrasaccharide or more) can be obtained.
  • the oligosaccharide is separated and purified to obtain a high purity 1-kestose solution.
  • the high purity 1-kestose solution is concentrated and then crystallized by a crystallization method as disclosed in Japanese Examined Patent Publication No. 6-70075 to obtain 1-kestose as crystals.
  • 1-kestose is contained in commercially available fructooligosaccharides, it may be used as it is or after separation and purification of 1-kestose from fructooligosaccharides by the method described above. That is, as the 1-kestose of the present invention, a 1-kestose-containing composition such as an oligosaccharide containing 1-kestose may be used.
  • the purity of 1-kestose is preferably 80% or more, more preferably 85% or more, and still more preferably 90% or more.
  • the "purity" of 1-kestose refers to the mass% of 1-kestose when the total mass of sugar is 100.
  • 1-kestose is used by being ingested by humans or animals.
  • an intake amount (dosage amount) of 1-kestose for example, 0.04 g / kg body weight or more per day can be mentioned.
  • the amount of intake concerned may be divided into several times and ingested not only once a day.
  • any method may be used as long as it reaches the intestine which is a habitat of Fusobacterium bacteria and Stelerus bacteria.
  • a method of orally ingesting 1-kestose into a human or an animal as it is, or in the form of food or drink, medicine, or animal feed specifically, there can be mentioned a method of orally ingesting 1-kestose into a human or an animal as it is, or in the form of food or drink, medicine, or animal feed.
  • the method of administering 1-kestose directly from the anus or via a inserted tube 1-kestose is added to the enteral nutrient, and this is inserted into the digestive tract such as stomach or small intestine, etc.
  • the method of administering by enteral nutrition etc. can be mentioned.
  • AHD can be prevented or treated by allowing humans or animals to ingest 1-kestose to suppress the number of at least one of Fusobacterium bacteria and / or Stellara bacteria in the body or intestine.
  • 1-kestose can be used to manufacture a pharmaceutical for the prevention or treatment of AHD.
  • “suppressing the number of bacteria” refers to suppressing an increase in the number of bacteria in any cell, tissue or organ of a living body.
  • the number of bacteria of the bacteria in the intestine can be obtained by measuring the number of bacteria of the bacteria in feces. Can be checked whether or not Specifically, for example, as shown in Example 1 (5) described later, 16S rDNA is subjected to real-time PCR using primers specific to the bacteria, using feces before and after intake of 1-kestose as a sample. Count the number of copies. Primers specific to the bacteria can be designed based on the known base sequences of the bacteria.
  • specific primers can be designed based on the 16S rDNA sequences shown in SEQ ID NO: 1-4 for Fusobacterium bacteria and in SEQ ID NO: 5 for Stellaella bacteria.
  • real-time PCR can be performed using a commercially available kit for quantifying the bacteria.
  • the 16S rDNA copy number can be used as an indicator of the number of bacteria. Therefore, as a result of measuring the 16S rDNA copy number of Fusobacterium or Sterella bacteria, if the 16S rDNA copy number in feces after intake of 1-kestose is smaller than before intake, intake of 1-kestose suppresses the number of bacteria.
  • the number of bacteria in feces before and after 1-kestose intake is about those who are in the condition that the number of Fusobacterium bacteria and bacteria of Sterella bacteria increase with the passage of time, such as those suffering from acute hemorrhagic diarrhea (AHD). If the degree of increase is small, even if it is the same degree (constant) or increased, it can be judged that the number of bacteria is suppressed by the intake of 1-kestose.
  • Examples of the form of the microbe number inhibitor of the present invention and the preventive or therapeutic drug for AHD include forms consisting only of 1-kestose which is an active ingredient, as well as forms of pharmaceuticals, food additives, supplements and the like.
  • its dosage form is, for example, powder, tablet, sugar coating, capsule, granule, dry syrup, liquid, syrup, drop, drink, etc. Or a liquid dosage form can be mentioned.
  • the pharmaceutical preparations, food additives and supplements of each of the above dosage forms can be produced by methods known to those skilled in the art.
  • 800 g of 1-kestose and 200 g of lactose are mixed well, and then 300 mL of 90% ethanol is added to wet it. Subsequently, the wet powder is granulated and then air-dried at 60 ° C. for 16 hours, and then sized to obtain 1000 g of a powder of appropriate fineness (800 mg / 1 g of 1-kestose content).
  • the food composition for controlling the number of bacteria according to the present invention may be in the form of ordinary food such as confectionery, beverages, processed foods, health foods, infant foods, etc. besides those consisting of only 1-kestose which is an active ingredient. Can be mentioned. When it is in the form of food or drink, it can be manufactured by adding an active ingredient in a normal manufacturing process. Since the sweetness of 1-kestose is 30, and its taste, physical properties, and processability are close to those of sucrose, some or all of the sugar is replaced with 1-kestose, etc. It can be handled in the same manner as sugar to produce various foods and beverages.
  • the form of animal feed for suppressing number of bacteria of the present invention is also composed of only 1-kestose which is an active ingredient, dairy cattle feed, feed for beef cattle, livestock feed such as pig feed, poultry feed, etc., dog feed
  • the form of normal animal feed such as pet feed (pet food) such as cat feed, can be mentioned. When it is in the form of a normal animal feed, it can be produced by adding an active ingredient in the production process.
  • Example 1 Changes in the number of Fusobacterium spp. And Sterella spp.
  • 1-kestose intake (1) Preparation of a tablet mainly composed of 1-kestose 1-kestose (purity 98% by mass or more; product food science) 800 g and stearin 8 g of magnesium acid is thoroughly mixed and then used in a continuous tableting machine (RT-S-9, Kikusui Seisakusho Ltd.) for tableting to give 2000 tablets (1-kestose content: 396 mg / 1) with a diameter of 8 mm and a weight of 400 mg. Got a lock).
  • a continuous tableting machine R-S-9, Kikusui Seisakusho Ltd.
  • DNA was extracted from this suspension using Magtration System 12 GC (Precision System Science) and GC series MagDEA DNA 200 (Precision System Science). The concentration of DNA was measured using a spectrophotometer ND-1000 (NanDrop Technologies), adjusted to 10 ng / ⁇ L, and used as total fecal DNA.
  • sequences with high homology are grouped as one group, and this is classified into one bacterial species (or one genus) Treated as.
  • the sequence with the highest frequency of appearance in the group was taken as a representative sequence, and the homology analysis with the sequence database was performed to identify the bacterial species.
  • the abundance ratio was calculated as a percentage of the number of leads in each group to the total number of leads.
  • the chimera sequence check is ⁇ Robert C. Edgar et al., BIOIN FORMATICS, Vol. 27, No. 16, pp. 2194-2200, June 2011>
  • the homology analysis is ⁇ Chika Kasai et al., BMC Gastroenterology, 15 In the volume, article 100, August 2015>.
  • the abundance ratio of the 16S rDNA of Fusobacterium bacteria was 7.18 at the start of the test, while 1.53 after 1 month after the start of the test and 1.98 after 2 months.
  • One month after the end of 44, 1-kestose intake (3 months later) was 2.41.
  • the abundance ratio of 16S rDNA of Fusobacterium mortiferum was 5.97 at the beginning of the test, whereas it was 1.64 after 1 month, 1.55 after 2 months, 1.85 after 3 months Met.
  • the abundance ratio of 16S rDNA of Fusobacterium varium was 0.55 at the start of the test, whereas it was 0.09 after 1 month, 0.07 after 2 months and 0.23 after 3 months Met.
  • the abundance ratio of 16S rDNA of Fusobacterium perfoetens was 0.46 at the beginning of the test, whereas it was 0.03 after 1 month, 0.06 after 2 months and 0.08 after 3 months Met.
  • the abundance ratio of 16S rDNA of Fusobacterium necrogenes was 0.13 at the beginning of the test, whereas it was 0.02 after 1 month, 0.04 after 2 months, 0.01 after 3 months Met.
  • the abundance ratio of 16S rDNA of Fusobacterium bacteria starts the test at any time point one month after the start of the test, two months after and one month after the end of the intake of 1-kestose (three months later) It was noticeably small compared to the time. From this result, it was revealed that intake of 1-kestose reduces the abundance ratio of 16S rDNA of Fusobacterium bacteria in feces.
  • the abundance ratio of 16S rDNA of Sterella bacteria was 0.22 at the beginning of the test, but 0.10 after 1 month, 0.09 after 2 months, and the end of 1-kestose intake One month after (3 months after) it was 0.02.
  • the abundance ratio of 16S rDNA of Sutterella stercoricanis was 0.31 at the start of the test, while it was 0.11 after 1 and 2 months and 0.02 after 3 months.
  • the abundance ratio of 16S rDNA of Sterella bacteria starts the test at any time point one month after the start of the test, two months after and one month after the end of the intake of 1-kestose (three months later) It was noticeably small compared to the time. From this result, it was revealed that intake of 1-kestose reduces the abundance ratio of 16S rDNA of Sterella bacteria in feces.
  • real-time PCR was performed in the same manner using Sutterella Detection Kit (Techno Surga Lab) in place of Fusobacterium Detection Kit to quantify the 16S rDNA copy number of Sterella bacteria per gram of feces. Furthermore, real-time PCR is performed in the same manner using a universal primer (forward primer; CCTACGGGAGGCAGCAG (SEQ ID NO: 6), reverse primer: ATTACCGCGGCTGCTGG (SEQ ID NO: 7)) to quantify 16S rDNA copy number of total eubacteria contained in 1 g of feces. did.
  • a universal primer forward primer; CCTACGGGAGGCAGCAG (SEQ ID NO: 6), reverse primer: ATTACCGCGGCTGCTGG (SEQ ID NO: 7)
  • the calibration curve for quantifying Fusobacterium genus bacteria and Sterella genus bacteria was produced based on the result of having performed real-time PCR on the same conditions using the standard sample contained in the said kit.
  • the calibration curve for quantifying whole eubacteria is E. coli.
  • the obtained 16S rDNA copy number was calculated as the median at each collection time of feces.
  • the 16S rDNA copy number (median) of whole eubacteria is shown in the line graph of FIG. 1
  • the 16S rDNA copy number (median) of Fusobacterium bacteria is shown in the line graph of FIG.
  • the values are shown in the line graph of FIG. Assuming that the 16S rDNA copy number at the start of the test is 100%, the 16S rDNA copy number is expressed as a percentage at one month, two months after the start of the test and one month after the end of intake (three months). It was the rate (%).
  • the 16S rDNA copy number of total eubacteria was 100%, and the 16S rDNA copy number of each of Fusobacterium bacteria and Sterella bacteria was expressed as a percentage, and this was taken as the occupancy rate (%).
  • the results for whole eubacteria are shown in Table 2, the results for Fusobacterium bacteria are shown in Table 3, and the results for Sterella bacteria are shown in Table 4.
  • the 16S rDNA copy number of whole eubacteria is 1 month after the start of the test, 2 months after and 1 month after the end of the intake of 1-kestose (3 months after), It increased with the passage of time.
  • the 16S rDNA copy number of Fusobacterium bacteria decreased with the lapse of time from the start of the test.
  • the 16S rDNA copy number of Sterella bacteria also decreased with the passage of time from the start of the test, as shown in FIG. 3 and Table 4.
  • the rate of change of Fusobacterium bacteria was 33% at one month after the start of the test, 40% at two months, and 32% at three months. That is, the number of 16S rDNA copies of the Fusobacterium bacteria is lower than that at the start of the test at any one month after the start of the test, two months after the start of the test, and one month after the end of intake of 1-kestose (three months). It became remarkably small to about 1/3.
  • the occupancy rate of the Fusobacterium bacteria was 4.76% at the start of the test, but 1.20% after one month from the start of the test, 1.35% after two months, after three months The rate was 0.82%. That is, the percentage of Fusobacterium bacteria in total eubacterial bacteria at one month after the start of the test, two months after the start of the test and one month after the end of the intake of 1-kestose (after three months) In comparison, they were significantly reduced to about 1 ⁇ 4, 1 ⁇ 3 and 1 ⁇ 6, respectively.
  • the rate of change of Sterella bacteria was 59% one month after the start of the test, 50% two months later, and 17% three months later. That is, the number of 16S rDNA copies of the Sterella bacteria is 1 month after the start of the test, 2 months after the start of the test, and 1 month after the end of the intake of 1-kestose (3 months after). It becomes significantly smaller from about 1/2 to less than 1/5.
  • the occupancy rate of Sterella bacteria was 0.13% at the start of the test, while 0.06% at one month after the start of the test, 0.04% at two months after the test
  • the result was 0.01%. That is, in any one month after the start of the test, two months after, and one month after the end of intake of 1-kestose (three months after), the ratio of the bacteria of the bacteria belonging to all eubacterial bacteria is the same as at the start of the test. In comparison, they were significantly reduced to 1/4, 1/6 and 1/24, respectively.

Abstract

Le problème décrit par la présente invention est de fournir un agent permettant d'inhiber le nombre de bactéries du genre Fusobacterium et/ou de bactéries du genre Sutterella, l'agent étant susceptible d'inhiber efficacement le nombre de bactéries du genre Fusobacterium et de bactéries du genre Sutterella et susceptible de contribuer à la prévention et au traitement de maladies telles que la diarrhée hémorragique aiguë. La solution selon l'invention porte sur un agent permettant d'inhiber le nombre de bactéries du genre Fusobacterium et/ou de bactéries du genre Sutterella comprenant le 1-kestose utilisé comme principe actif. La présente invention permet d'obtenir un agent permettant d'inhiber le nombre de bactéries du genre Fusobacterium et/ou de bactéries du genre Sutterella qui peut inhiber efficacement le nombre de bactéries du genre Fusobacterium et de bactéries du genre Sutterella, est très sûr, et peut être mélangé facilement avec divers aliments et produits pharmaceutiques ou pris quotidiennement de manière pratique.
PCT/JP2018/033742 2017-09-13 2018-09-12 Agent permettant d'inhiber le nombre de bactéries du genre fusobacterium et/ou de bactéries du genre sutterella WO2019054396A1 (fr)

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Cited By (1)

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JP2021102568A (ja) * 2019-12-25 2021-07-15 物産フードサイエンス株式会社 フリシェラ属細菌の菌数抑制剤

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021102568A (ja) * 2019-12-25 2021-07-15 物産フードサイエンス株式会社 フリシェラ属細菌の菌数抑制剤

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