JP6557876B2 - Probiotics or prebiotics and their production methods, microbial preparations, health foods and pharmaceuticals - Google Patents

Description

The present invention relates to a thermophilic bacterium utilizing a thermophilic bacterium that exhibits different functions depending on the constitution of the animal, and a mixed solution containing the thermophilic bacterium, depending on the genetic background of the animal and the constitution of the intestinal bacterial flora. It pays attention to the point which has the function which brings a different physiological reaction, and relates to the formulation and its manufacturing method using the function.

  Probiotics and prebiotics are known as preparations for adjusting the intestinal function of animals. As these techniques, techniques using microorganisms that mainly live at normal temperature, such as lactic acid bacteria, yeasts, Bacillus subtilis, and the like are widely used (Patent Documents 1 and 2). For example, Patent Document 1 discloses a method for producing non-fermenting lactic acid bacteria using bifidobacteria and Lactobacillus acidophilus, and Patent Document 2 discloses a method for controlling adiponectin secretion, which is one of lactic acid bacteria, Lactobacillus gasseri ( There is a preparation that utilizes the culture supernatant of Lactobacillus gasseri) SBT2055 (FERM BP-10953). In Patent Document 3, the growth of Bifidobacterium is promoted, the release of granulocyte colony-stimulating factor is promoted, the differentiation of T helper cell type I is promoted, and / or the differentiation of T helper cell type II is promoted. Kibanash slang seeds and polysaccharides extract and pharmaceutical composition for suppressing cerevisiae, and methods for preparing the same have been published, promote the growth of Bifidobacterium, promote the release of granulocyte colony-stimulating factor, Techniques relating to the extract of Kavanasuslan species / polysaccharides and pharmaceutical compositions for promoting differentiation of T helper cell type I and / or suppressing differentiation of T helper cell type II have been introduced.

  In addition, as a non-patent document, a global study is known as a study for identifying a bacterial species and a bacterial group involved in the control of immune system cells among the species of Clostridium (Non-patent document 1). . Furthermore, a very important study that searched for effective genes of segmented filamentous bacteria (SFB) (Non-patent Document 2), a specific bacterium that regulates the immune system, or bifidobacteria with a protective function against O157 (non- Patent Document 3) is known.

  However, these techniques do not take into account the control of the gut microbiota by type in consideration of the genetic background of the host. In recent years, there have been reports of research data that show that both diet and sex differences affect the composition of the gut microbiota (Non-patent Document 4), and the diversity of gut microbiota It has been pointed out that it is important for human metabolome countermeasures (Non-patent Document 5). In the future, it is assumed that it is necessary to design appropriate probiotic combinations based on the genetic background of the host.

 On the other hand, the inventors have succeeded in probiotic technology that affects the living body of animals by utilizing thermophilic bacteria, which is one of the extreme environmental microorganisms that are difficult to grow in the normal temperature range (patents). Documents 4 and 5, Non-patent document 5)

Japanese Patent No. 4898859 Patent No. 5225652 Japanese Patent No. 5395733 Patent No. 5578375 Japanese Patent No. 5041228

Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y, Nishikawa H, Fukuda S, Saito T, Narushima S, Hase K, Kim S, Fritz JV, Wilmes P, Ueha S, Matsushima K, Ohno H, Olle B , Sakaguchi S, Taniguchi T, Morita H, Hattori M, Honda K *. "Treg induction by a rationally selected mixture of Clos tridia strains from the human microbiota." Nature 500: 232-236. (2013) Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T, Karaoz U, Wei D, Goldfarb KC, Santee CA, Lynch SV, Tanoue T, Imaoka A, Itoh K, Takeda K, Umesaki Y, Honda K *, Littman DR *. "Induction of intestinal Th17 cells by segmented filamentous bacteria." Cell. 139: 485-98. (2009) Fukuda S, Toh H, Hase K, Oshima K, Nakanishi Y, Yoshimura K, Tobe T, Clarke JM, Topping DL, Suzuki T, Taylor TD, Itoh K, Kikuchi J, Morita H, Hattori M, Ohno H. Bifidobacteria can protect from enteropathogenic infection through production of acetate.Nature. 2011 Jan 27; 469 (7331): 543-7. Bolnick DI, Snowberg LK, Hirsch PE, Lauber CL, Org E, Parks B, Lusis AJ, Knight R, Caporaso JG, Svanb ä ck R Individual diet has sex-dependent effects on vertebrate gut microbiota. Nat Commun. 2014 Jul 29; 5: 4500 doi: 10.1038 / ncomms5500. Le Chatelier E, Nielsen T, Qin J, Prifti E, Hildebrand F, Falony G, Almeida M, Arumugam M, Batto JM, Kennedy S, Leonard P, Li J, Burgdorf K, Grarup N, Jorgensen T, Brandslund I, Nielsen HB, Juncker AS, Bertalan M, Levenez F, Pons N, Rasmussen S, Sunagawa S, Tap J, Tims S, Zoetendal EG, Brunak S, Clement K, Dore J, Kleerebezem M, Kristiansen K, Renault P, Sicheritz-Ponten T, de Vos WM, Zucker JD, Raes J, Hansen T; MetaHIT consortium, Bork P, Wang J, Ehrlich SD, Pedersen O. Richness of human gut microbiome correlates with metabolic markers.Nature 500: 541-549. (2013) Miyamoto H, Seta M, Horiuchi S, Iwasawa Y, Naito T, Nishida A, Miyamoto H, Matsushita T, Itoh K, Kodama H (2013) Potential probiotic the rmophiles isolated from mice after compost ingestion.Journal of Applied Microbiology, 114 ( 4): 1147-1157

  Until now, the concept of administering different probiotics depending on the animal constitution has not been established. However, it is known that not only the genetic background but also the bacterial flora that forms the intestine varies greatly depending on the animal species, and there are probiotics or prebiotics that can be used after taking these into account. May be needed.

The constitution differs depending on the genetic background of the animal, and the microbial structure of the intestinal flora unique to the host and the behavior of the concentration of biomolecules differ depending on the constitution. In addition, antibiotics may be used as a control of the intestinal microflora, but the diversity of the intestinal microflora is lost, and there is a possibility of harmful effects in the intestinal environment. In such cases, it is necessary to administer appropriate probiotics.
The present invention is to provide probiotics or prebiotics that solve the above-mentioned problems.

  Utilize thermophilic probiotics that function according to characteristics in animals that tend to gain weight and animals that are less likely to gain weight. This modifies the population of the host intestinal microflora and appropriately controls the behavior of physiologically functional molecules in the liver. Utilize thermophilic probiotics that function even when characteristics of animal species or intestinal aging are observed. This modifies the host gut microbiota population and controls its behavior appropriately. For this purpose, BP-863, which is one of the genus Bacillus, is used. BP-863 has been deposited internationally by the inventor with the Product Evaluation Technology Infrastructure as of February 8, 2010 (Accession No. BP-863).

  Further, the present invention includes a method for producing a preparation that can function as a probiotic for each constitution or a prebiotic even when a mixed microorganism is used.

The invention of claim 1 includes an animal comprising P01931 (International Deposit Number BP-1931), MK-01A (International Deposit Number BP-02066), or MK-03A (International Deposit Number BP-02067) In order to increase the diversity of the intestinal flora of the microbial preparations.

The invention of claim 2 is a method for increasing the diversity of intestinal flora of non-human animals using the microorganism preparation of claim 1.

Invention of Claim 3 exists in the place made into the health food for increasing the diversity of the intestinal microflora of the animal containing the microbe formulation of Claim 1.

The invention of claim 4 is to provide a medicament for increasing the diversity of the intestinal flora of an animal comprising the microorganism preparation of claim 1.

  In the contents of this application, it is assumed that the ripple effect is enormous because it is possible to operate custom-made probiotics or prebiotics in consideration of the genetic background of the host animal. That is, if it is a human, the correspondence according to a person type, an area, a dietary life, and a disease is assumed. In animals, it is clear that pets, livestock, poultry, etc. have different intestinal flora and constitutions, and it is possible to operate probiotics or prebiotics according to the purpose by taking these into consideration. . Further, according to the present invention, in the genetic background of animals, the concentration of biomolecules contributing to the microbial structure and physiological function of the intestinal flora at normal temperature in the host, depending on the characteristics of the constitution that tends to gain weight or the constitution that is difficult to gain weight Can be controlled. Furthermore, it will be possible to develop and disseminate probiotics that act efficiently according to animal constitution. Furthermore, it controls the increase or decrease in the diversity of intestinal flora according to the constitution of the animal's genetic background and the microbial structure of the intestinal flora at room temperature specific to the host, resulting in a reduction in opportunistic infections in particular. In addition, the population of useful bacteria can be maintained.

By using feed containing BP-863 in chickens, pigs, and dogs, we can significantly increase the diversity of gut microbiota, especially in opportunistic infections. For example, the population of Enteroccocus genus in chickens, Streptococcus genus in pigs, and Clostridium genus increased in older dogs is reduced, and species-specific useful bacteria that tend to decrease in each animal species are administered in combination. Solve traditional problems. The constitution differs depending on the genetic background of the animal, and the behavior of the microbial structure of the intestinal flora unique to the host and the concentration of biomolecules may differ depending on the constitution. In such cases, it is necessary to administer appropriate probiotics, but research on probiotics by constitution that adjust such constitution-dependent characteristics is a future issue. In addition, antibiotics may be used as a control of the intestinal microflora, but the diversity of the intestinal microflora is lost, and there is a possibility of harmful effects in the intestinal environment. In such cases, it is necessary to administer appropriate probiotics.

Probiotic concept by constitution Thermophilic bacteria used for probiotics by constitution and their standard strains Regulation of liver glycolysis using constitutional probiotics Regulation of liver β-oxidation and TCA cycle using probiotics by constitution Regulation of hepatic urea cycle using probiotics by constitution Conceptual diagram of intestinal flora control without relying on antibiotics Increasing rate of piglets orally fed BP-863 Diversity of fecal flora in piglets fed BP-863 orally Behavior of opportunistic infections in piglet feces Diversity of bacterial flora in chicken dung behavior of opportunistic infections

  Next, although embodiment of this invention is described, this invention is not limited to these embodiment.

  The microorganism group used in the present invention includes thermophilic microorganisms of a plurality of biological species. Specific biological species include Bacillus coagulans, Bacillus thermoamylovorans, and closely related species. Among them, the microorganism group used in the present invention is the microorganism having the accession number: NITE P-01931 and / or the accession number: NITE BP-1051 and / or the accession number: NITE BP-863 and / or the product evaluation technical infrastructure. (NITE) Mixed Microorganism MK-01 rejected due to being a mixed microorganism (No Acceptance Notification No. 2014-0319) and / or Product Evaluation Technology Infrastructure (NITE) rejected as a mixed microorganism The mixed microorganism MK-03 (contract refusal notice No. 2014-0321) is preferable. Among the closely related species of Bacillus thermoamylovorans, among them, the microorganism group used in the present invention is preferably a microorganism having the deposit number: NITE BP-863.

  Examples of microorganisms that can be mixed in the microorganism material used in the present invention include the genera Lactobacillus and Bifidobacterium, and thermophilic microorganisms Bacillus, Lysinibacillus, Virgibacillus, Anoxybacillus, and Paenibacillus. Furthermore, even if it coexists with Thermophiles inoculum MIROKU H2K including the Meinoothermus genus, Vulcanithermus genus, Thermus genus, Oceanobacillus genus of the Deinococcus-Thermus genus, it exhibits appropriate physiological activity. These microbe group Thermophiles inoculum MIROKU H2K has been stored in Sanriku Co., Ltd. (Kitsuki City, Oita Prefecture) because it was rejected by the National Institute for Product Evaluation Technology due to complex bacteria and difficulty in culturing. In addition, as such a group of microorganisms that can coexist, the accession number PTA-1773 entrusted to ATCC can also be used. In addition, the deposit number: NITE BP-1051 deposited as a mixed microorganism with the Product Evaluation Technology Infrastructure (NITE) can also be used.

  The thermophilic inoculum PTA-1773 has been internationally deposited by the inventor in the ATCC (American Type Culture Collection, 10801 University Boulevard Manassas, Virginia 20110-2209 USA) on May 1, 2000 (Accession Number: PTA- 1773)

  In addition, it is preferable that the preparation of the present invention contains about 10 / g to about 10 9 microorganisms / g of microbial groups or the amount of functional components derived from the microbial groups for the above strains.

  Utilizing the above microbial populations, we have found that the same thermophile shows different physiological responses to phylogenetically different mice. When cells are administered to mice that are easily fattened and mice that are less likely to gain weight, the populations of Bacteroides, Clostridium, and Lactobacillus in the intestinal flora are controlled, and the concentration of functional molecules in the liver is controlled. However, even in the same strain, there are strains showing the same tendency for the effect on the mice of the easily fattened strain and the mice of the strain not easily fattened, and strains showing the opposite effect. Utilizing these properties, it can be used as probiotics for different constitutions to solve conventional problems.

  In addition, by utilizing the above microorganism group and utilizing feed containing BP-863 for chickens, pigs, and dogs, the diversity of gut microbiota is significantly increased, and in particular, opportunistic infections are reduced. It is. For example, the population of Enteroccocus in chickens, Streptococcus in pigs, and the Clostridium cluster XI population that increases with aging are reduced. Utilizing these properties, it will be used as probiotics that can control the intestinal flora without relying on antibiotics, and solve conventional problems.

(Example 1)
The test was conducted with the composition shown in Table 1 for the rearing test under a high fat diet.

  BALB / c and C57BL / 6 (male, 3 weeks old) were introduced, and after preliminary breeding for 5 days, the experiment was started. (1) High fat diet (lard) breeding group (control group) (symbol mA for BALB / c) (symbol mE for C57BL / 6), (2) high fat diet (lard) + thermophilic MK01A solution added to drinking water Group (symbol mB for BALB / c) (symbol mF for C57BL / 6), (3) high fat diet (Lard) + thermophilic P01931 solution drinking water group (symbol mC for BALB / c) (symbol for C57BL / 6 Symbol mG), (4) High fat diet (Lard) + thermophilic MK03A solution (symbol mD for BALB / c) (symbol mH for C57BL / 6) and 4 groups per 4 strains of mice in the drinking water group. Prepared. One group was reared in one cage with 5 animals. Normal feed is formulated feed (MF manufactured by Oriental Yeast Co., Ltd.), and high-fat food is prepared by KTV Oriental Co., Ltd. (Tosu City, Saga Prefecture). The proportion of lipid is 24% (of which 20% is lard) It was adjusted. For drinking water, tap water was freely ingested, and a solution of each thermophile was added to 1.0% drinking water except for the control group. The food was allowed to eat freely within a daily intake limit of 25 grams. After rearing for 2 months, the body weight was measured, blood and the like were collected and dissected, and then the liver and feces were collected and subjected to metabolome analysis and flora analysis. These strains are shown in FIG. Genetically, the 16S rDNA sequence was 100% identical to the standard strain Bacillus coagulans (ATCC), but was morphologically different.

  As a result of the bacterial flora analysis, in BALB / c, as shown in Tables 2 and 3, changes in the flora in feces were confirmed in the 4 groups. In particular, it was confirmed that populations of the genus Clostridium and Lactobacillus increased in the mD group, which had a low tendency to gain weight.

  As a result of the microbial flora analysis, as shown in Tables 4 and 5 in C57BL / 6, changes in the microbial flora in the feces were confirmed in the 4 groups. In particular, it was confirmed that populations of the genus Clostridium and Lactobacillus increased in the mG group, which had a low tendency to gain weight.

  At this time, when the diversity analysis of the flora was confirmed by two kinds of methods, as shown in Tables 6 and 7, the diversity tended to increase in the strain administration group, and there was no difference in that respect. .

Next, liver metabolome analysis was performed by CE-MS. As shown in Table 8, the concentrations of physiologically functional molecules were different in each group. Moreover, it was not always the same depending on the strain.
Regarding glycolysis, as shown in FIG. 3, the suppression tendency was confirmed for F6P and G6P regardless of the mouse strain, but for the TCA cycle, only the mD group administered with the MK03A strain showed an increasing tendency. Although confirmed, the others remained unchanged.

  Next, BALB / c, C57BL / 6 (male, 3 weeks old) and (male, 8 weeks old) were introduced, and after preliminary breeding for 5 days, the experiment was started. Three groups were prepared: (1) normal breeding group (control group), (2) BP-863 added group, and (3) mixed solution drinking water added group. One group consisted of 5 animals and was kept in one cage. For drinking water, tap water was freely consumed, and feed was freely consumed. After rearing for 3 months, the body weight was measured, blood and the like were collected and dissected, and then the liver was collected and subjected to metabolomic analysis.

  As a result of blood analysis, the leptin concentration in serum was higher in C57BL / 6 than in BALB / c, but the latter was more likely to gain weight.

  As a result of liver metabolome analysis, as shown in Tables 9 and 10, the same tendency was not always observed depending on the age of administration start and the mouse strain.

In summary, these strains induce physiological responses similar to those under high fat diet conditions in mice with a tendency to gain weight (C57BL / 6) and mice with a tendency to gain weight (BALB / c). It has been found. As shown in Table 1, with the increase of Bacteroides gate in the intestinal bacterial flora, it is possible to induce almost similar physiological responses such as suppression of liver glycolysis, activation of TCA cycle, activation of urea cycle, etc. found. However, an increase in free amino acids was confirmed only in mice of strains that were not easily fattened. However, the reaction was markedly different in the 3-week-old mice that were new to birth, and slightly different in the 8-week-old mice.
It can be said that the technique of the present invention has novelty over the prior art.

  (Example 2) For piglets whose mother pigs were the same and were born about 30 days after birth, 10 squared BP-863 per kg of feed was mixed with the feed and fed for 3 weeks.

  As a result, it was confirmed that the weight gain rate of the piglets tended to increase by about 5% (see FIG. 7). At this time, the total DNA of porcine feces was extracted and comprehensively analyzed for next generation sequencer using bacterial 16SrDNA. That is, as a result of 3000-lead analysis of bacterial flora in swine feces using bacterial next generation sequencer for bacterial 16SrDNA, changes in the flora between pigs fed diets containing BP-863 and the non-administered group Was confirmed, and opportunistic infections were significantly reduced. On the other hand, out of 3000 leads, the number of bacterial species detected was significantly increased in the BP-863 feeding group. In this analysis, the methods described in DNA Res. Jun; 20 (3): 241-253, 2013 and DNA Res. Feb; 21 (1): 15-25, 2014 were used.

  Specifically, as shown in FIG. 8, as a result of UniFrac analysis, the microbial flora of the BP-863 non-administered group and the administered group were significantly different. In addition, it was revealed that the number of units (OTU) with different sequences detected was increasing as the number of BP-863 administration groups increased. Furthermore, as a result of detailed analysis, the number of closely related strains of the genus Streptococcus, which are assumed to be opportunistic infections, was significantly reduced. Moreover, the tendency for the microbial species which belong to cluster XI among Clostridium genus to be decreased was confirmed. As Clostoridium cluster XI, Clostridium mayombei and the like were mentioned. Clostoridium cluster XI is known to be one of the bacteria expected to increase during obesity in experiments using mice (Nature Jul 4; 499 (7456): 97-101,2013) .

 Furthermore, including other experimental systems, Streptococcus genus decreasing in pigs was assumed to include Streptococcus alactoriticus, Streptococcus galactotiticus, Streptococcus orisuis, Streptococcus hyointestinalis, and the like.

  At this time, among the lactic acid bacteria, the genus Lactobacillus amylovorus and Bifidobacterium tended to increase.

In general, an increase in the bacterial species of Clostridium genus in the intestine was observed with aging, but the tendency of Clostridium XI to decrease in the intestinal flora of animals administered BP-863 was also confirmed in old dogs It was done.

(Example 3) For chicks of egg-laying hens, feed containing BP-863 and feed not containing BP-863 were fed for 18 weeks, and the bacterial flora in feces was analyzed by the same method as in [0050].

  Analysis of the flora in chicken feces using a next-generation sequencer for bacterial 16SrDNA analysis of 1800 reads confirmed changes in the flora between chickens fed with BP-863 and the non-administered group In addition, the number of Enterococuus species known as vancomycin-resistant bacteria decreased significantly. On the other hand, among the 1800 reads, the number of bacterial species detected was significantly increased in the BP-863 feeding group. Specifically, as shown in FIG. 10, a significant difference in the bacterial flora was confirmed between the BP-863 administration group and the non-administration group. In addition, the number of units with different sequences (OTUs) was found to have increased diversity as the number of BP-863 administration groups increased. Among them, Enterococcus gallinarum among the genus Enterococcus was remarkably decreased. At this time, an increasing tendency of Lactobacillus amylovorus was confirmed.

  To summarize these results, BP-863 has a tendency to control the flora, especially to increase the diversity of the flora, while increasing the number of specific useful bacteria and of certain opportunistic infections. It became clear to promote the decrease. These trends indicate that the administration of antibiotics kills many intestinal bacterial flora, thus losing the diversity of the flora, and therefore the present invention technique can be said to have novelty over the prior art. . As described above, in recent years, it has become clear that the diversity of intestinal flora is indispensable for obesity control and prevention of various diseases, and the effectiveness of the present invention was considered to be high. According to the present invention, it is possible to reduce specific opportunistic infections in a manner that does not rely on antibiotics, and at the same time, to provide a variety of gut microbiota and prevent various diseases regardless of animal species. Is expected to be.

NITE p-01931

NITE BP-02066

NITE BP-2067

NITE BP-863

NITE BP-1051

ATCC PTA-1773

Consignment Rejection Notification No. 2014-0319

Consignment Rejection Notification No. 2014-0321

Thermophiles inoculum MIROKU M2K strain

Claims (4)

Variety of intestinal flora of animals characterized by containing P01931 (International Deposit Number BP-1931), MK-01A (International Deposit Number BP-02066), or MK-03A (International Deposit Number BP-02067) microbial agent for increasing the resistance.   A method for increasing the diversity of intestinal flora of non-human animals using the microbial preparation according to claim 1. A health food for increasing the diversity of intestinal flora of an animal comprising the microbial preparation according to claim 1. A pharmaceutical product for increasing the diversity of intestinal flora of animals comprising the microbial preparation according to claim 1.