Method for producing a composition containing cycloastragenol

Abstract

To provide a method for efficiently producing cycloastragenol from astragaloside IV by using microorganisms.SOLUTION: Astragaloside IV is efficiently transformed into cycloastragenol by going through a first process of allowing a lactic acid bacterium and / or its fungus body processed product to act on astragaloside IV, and a second process of allowing a bifidobacterium and / or its fungus body processed product to act on a product obtained in the first process.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for efficiently producing cycloastragenol from astragaloside IV using microorganisms.

Background Art

[0002] Astragaloside IV contained in Astragalus membranaceus Bge. etc. has been reported to be effective in the treatment of diabetic nephropathy and neurodegenerative diseases such as Parkinson's disease (see, for example, Non-Patent Documents 1 and 2). However, when astragaloside IV is orally ingested, it has the drawback of low bioavailability.

[0003] On the other hand, cycloastragenol, which is one of the metabolites (aglycones) of astragaloside IV, is known to have a bioavailability about 10 times higher than that of astragaloside IV. It is considered that directly orally ingesting cycloastragenol, which is a metabolite of astragaloside IV, can efficiently exert the same medicinal effect as astragaloside IV in vivo.

[0004] Conventionally, it has been reported that cycloastragenol can be produced by allowing Bacillus sp. strain LG-502 to act on astragaloside IV (see Non-Patent Document 3). However, simply culturing once using one kind of microorganism as in Non-Patent Document 3 cannot efficiently produce cycloastragenol. Against the background of such conventional technologies, establishment of a method for efficiently producing cycloastragenol from astragaloside IV has been eagerly desired.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a method for efficiently producing cycloastragenol from astragaloside IV using microorganisms.

Means for Solving the Problems

[0007] The present inventor has conducted intensive studies to solve the above problems, and as a result, has found that by subjecting astragaloside IV to a first step of reacting it with lactic acid bacteria and / or a processed product of the bacterial cells thereof, and a second step of reacting the product obtained in the first step with bifidobacteria and / or a processed product of the bacterial cells thereof, astragaloside IV can be efficiently converted into cycloastragenol. The present invention has been completed by further studies based on such findings.

[0008] That is, the present invention provides an invention in the following aspects. Item 1. A method for producing cycloastragenol, comprising the following first step and second step: A first step of reacting astragaloside IV with lactic acid bacteria and / or a processed product of the bacterial cells thereof, and A second step of reacting the product obtained in the first step with bifidobacteria and / or a processed product of the bacterial cells thereof. Item 2. The production method according to Item 1, wherein the lactic acid bacteria are at least one lactic acid bacterium selected from the group consisting of the genus Weissella, the genus Pediococcus, the genus Lactobacillus, and the genus Leuconostoc. Item 3. The production method according to Item 1 or 2, wherein the lactic acid bacteria are at least one lactic acid bacterium selected from the group consisting of Weissella confusa, Weissella cibaria, Pediococcus pentosaceus, Lactobacillus sakei, and Leuconostoc carnosum. Item 4. The production method according to any one of Items 1 to 3, wherein the bifidobacteria are Bifidobacterium pseudocatenulatum and / or Bifidobacterium angulatum. Item 5. The production method according to any one of Items 1 to 4, further comprising a step of isolating cycloastragenol from the cycloastragenol-containing product obtained in the second step. Item 6. A cycloastragenol-containing product obtained by the production method according to any one of Items 1 to 4. Item 7. A food comprising the cycloastragenol-containing product according to Item 6, an extract thereof, a concentrate thereof, or a dried product thereof.

Effects of the Invention

[0009] According to the present invention, it becomes possible to efficiently produce cycloastragenol from astragaloside IV using microorganisms.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0011] 1. Method for producing cycloastragenol The production method of the present invention is a method for producing cycloastragenol, which comprises a first step of allowing lactic acid bacteria and / or a processed product of the bacterial cells thereof to act on astragaloside IV, and a second step of allowing Bifidobacterium and / or a processed product of the bacterial cells thereof to act on the product obtained in the first step. Hereinafter, the production method of the present invention will be described in detail.

[0012] [Cycloastragenol] In the production method of the present invention, cycloastragenol, which is the target product to be produced, is a compound represented by the following general formula (1). Cycloastragenol is the aglycone of astragaloside IV and is a compound known to have significantly higher bioavailability upon oral ingestion compared to astragaloside IV. [Chemical formula]

[0013] [First step] The first step is a step of allowing lactic acid bacteria to act on astragaloside IV.

[0014] · Astragaloside IV In the first step, astragaloside IV is used as a substrate. Astragaloside IV is a kind of lanostane tetracyclic triterpenoid saponin and is a compound represented by the following general formula (2). [Chemical formula]

[0015] In the first step, a purified product or a roughly purified product of astragaloside IV may be used, or a plant extract containing astragaloside IV or the plant raw material itself containing astragaloside IV may be used.

[0016] Since astragaloside IV is contained in mugwort, an astragaloside IV-containing extract obtained by solvent extraction of mugwort, a pulverized product of mugwort, etc. can be used as a substrate.

[0017] · Lactic acid bacteria and / or processed products thereof The type of lactic acid bacteria used in the first step is not particularly limited. For example, lactic acid bacteria belonging to the genus Weissella, Lactobacillus, Tetragenococcus, Leuconostoc, Pediococcus, Ruminococcus, Streptococcus, Lactococcus, Enterococcus, etc. can be mentioned.

[0018] Specific examples of Weissella lactic acid bacteria include Weissella confusa, Weissella cibaria, Weissella paramesenteroides, etc.

[0019] As lactic acid bacteria of the genus Lactobacillus, specifically, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus rapi, Lactobacillus paralimentarius, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus pobuzihii, Lactobacillus mali, Lactobacillus hammesii, Lactobacillus versmoldensis, Lactobacillus paraplantarum, Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus coryniformis, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus crustorum, Lactobacillus pentosus, Lactobacillus buchneri, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus bifidus, etc. can be mentioned.

[0020] As lactic acid bacteria of the genus Tetragenococcus, specifically, Tetragenococcus halophilus, Tetragenococcus koreensis, etc. can be mentioned.

[0021] Examples of lactic acid bacteria belonging to the genus Leuconostoc include, specifically, Leuconostoc citreum, Leuconostoc mesenteroides, Leuconostoc lactis, Leuconostoc carnosum, Leuconostoc dextranicum, and the like.

[0022] Examples of lactic acid bacteria belonging to the genus Pediococcus include, specifically, Pediococcus argentinicus, Pediococcus pentosaceus, Pediococcus ethanolidurans, Pediococcus parvulus, Pediococcus acidilactici, and the like.

[0023] Examples of lactic acid bacteria belonging to the genus Luminococcus include, specifically, Luminococcus productus, and the like.

[0024] Examples of lactic acid bacteria belonging to the genus Streptococcus include, specifically, Streptococcus faecalis R, Streptococcus citrovorum, Streptococcus glycerinaceus, Streptococcus salivarius, and the like.

[0025] Examples of lactic acid bacteria belonging to the genus Lactococcus include, specifically, Lactococcus lactis, and the like.

[0026] Examples of lactic acid bacteria belonging to the genus Enterococcus include, specifically, Enterococcus thailandicus, Enterococcus casseliflavus, and the like.

[0027] From the viewpoint of manufacturing cycloastragenol more efficiently, as the lactic acid bacteria used in the first step, those having an action of releasing the glucose residue of astragaloside IV (hereinafter referred to as M2 metabolic action) can be preferably used. In particular, those having a higher M2 metabolic action than the action of releasing the xylose residue of astragaloside IV (hereinafter referred to as M1 metabolic action) are particularly preferred.

[0028] The M1 metabolic action and the M2 metabolic action can be determined by co-incubating astragaloside IV and lactic acid bacteria and measuring the production amounts of the compound represented by the following general formula (3) and the compound represented by the following general formula (4) in the obtained product. The compound represented by the following general formula (3) is the xylose glycoside of cycloastragenol from which the glucose residue has been eliminated from astragaloside IV, and the compound represented by the following general formula (4) is the glucose glycoside of cycloastragenol from which the xylose residue has been eliminated from astragaloside IV. That is, when co-incubating astragaloside IV and lactic acid bacteria and the presence of the compound represented by the following general formula (3) is recognized in the obtained product, it is determined that the lactic acid bacteria have the M2 metabolic action. Also, when the amount of the compound represented by the following general formula (3) in the obtained product is larger than the amount of the compound represented by the following general formula (4), it is determined that the M2 metabolic action is higher than the M1 metabolic action.

Chemical formula

[0029] Based on the disclosure of this specification, those skilled in the art can measure the M1 metabolic action and the M2 metabolic action for known Bifidobacterium or isolated lactic acid bacteria, and appropriately select lactic acid bacteria having the M2 metabolic action and lactic acid bacteria having a higher M2 metabolic action than the M1 metabolic action.

[0030] As strains of lactic acid bacteria with higher M2 metabolism than M1 metabolism, specifically, Weissella confusa (RD012161, JCM1093, RD012604, etc.), Weissella cibaria (RD012578, RD011819, etc.), Weissella parapenteloides (RD012576, etc.), and other lactic acid bacteria belonging to the genus Weissella;Lactic acid bacteria of the genus Lactobacillus such as Lactobacillus casei (NBRC 15883, etc.), Lactobacillus acidophilus (NBRC13951, NBRC102164, etc.), Lactobacillus alimentarius (RD055252, DSM20249, RD055258, etc.), Lactobacillus brevis (RD012087, ATCC14869, RD055271, RD011723, RD055257, RD012582, NBRC3345, etc.), Lactobacillus sakei (NBRC15893, RD011715, RD011713, RD012596, DSM20017, RD012090, etc.), Lactobacillus rapi (RD012520, etc.), Lactobacillus paraplantarum (NBRC 107152, etc.), Lactobacillus paracasei (RD055262, JCM8130, RD055272, RD011733, RD011743, RD055282, RD011746, RD055276, NBRC15889, etc.), Lactobacillus delbrueckii (NBRC102622, etc.), Lactobacillus plantarum (NBRC101973, NBRC106468, NBRC15891, etc.), Lactobacillus buchneri (RD011765, NBRC103219, RD012653, etc.), Lactobacillus mali (NBRC102159, etc.), Lactobacillus hammesii (RD011266, etc.), Lactobacillus versmoldensis (RD011768, etc.), Lactobacillus paraplantarum (RD055275, RD055269, etc.), Lactobacillus parabuchneri (NBRC110708, etc.), Lactobacillus fermentum (NBRC15885, etc.), Lactobacillus colliniformis (RD011738, etc.), Lactobacillus crustorum (RD055318, RD011724, etc.), Lactobacillus pentosus (RD011734, JCM1558, RD055278, RD011825, RD011748, RD055274, RD011741, etc.), Lactobacillus buchneri (NBRC107764, etc.), Lactobacillus lactis (NBRC107866, etc.);Tetragenococcus halophilus (RD012572, IAM1676, NBRC12172, RD011830, etc.), Tetragenococcus koreensis (RD012590, etc.) lactic acid bacteria of the genus Tetragenococcus; Leuconostoc citreum (RD012577, ATCC49370, NBRC102476, etc.), Leuconostoc mesenteroides (RD011712, NBRC 3349, RD011716, NBRC100496, RD011740, ATCC8293, RD011798, RD012107, etc.), Leuconostoc lactis (RD012583, JCM6123, etc.), Leuconostoc carnosum (RD012174, etc.) lactic acid bacteria of the genus Leuconostoc; Pediococcus argentinicus (NBRC 107827, etc.), Pediococcus pentosaceus (NBRC3182, RD012175, DSM20336, RD012523, RD012594, etc.), Pediococcus ethanolidurans (RD011265, etc.), Pediococcus parvulus (RD011824, JCM5889, etc.), Pediococcus acidilactici (NBRC109619, etc.) lactic acid bacteria of the genus Pediococcus; Streptococcus salivarius (RD012113, ATCC7073, etc.) lactic acid bacteria of the genus Streptococcus; Lactococcus lactis (RD012601, etc.) lactic acid bacteria of the genus Lactococcus; Enterococcus thailandicus (RD012568, RD012543, etc.), Enterococcus casseliflavus (NBRC100478, etc.) lactic acid bacteria of the genus Enterococcus, etc. are mentioned. In this specification, strains with "RD" and "NBRC" attached before the strain number are strains stored and available for distribution at NBRC (NITE Biological Resource Center), strains with "ATCC" attached before the strain number are strains stored and available for distribution at ATCC (American Type Culture Collection), and strains with "JCM" attached before the strain number are strains stored and available for distribution at JCM (Japan Collection of Microorganism).;

[0031] Preferable examples of lactic acid bacteria used in the first step include lactic acid bacteria belonging to the genus Weissella, Pediococcus, Lactobacillus, Leuconostoc; more preferably Weissella confusa, Weissella cibaria, Pediococcus pentosaceus, Lactobacillus sakei, Leuconostoc carnosum; even more preferably Weissella confusa (RD012161), Weissella cibaria (RD012578), Pediococcus pentosaceus (NBRC3182), Lactobacillus sakei (RD011715).

[0032] In the first step, one type of lactic acid bacteria may be used alone, or two or more types of lactic acid bacteria may be used in combination.

[0033] In the first step, the lactic acid bacteria may be used as live cells, but as long as the enzymes contained in the lactic acid bacteria maintain their activity, cell-treated products of the lactic acid bacteria can also be used. Examples of cell-treated products of lactic acid bacteria include cell lysates of lactic acid bacteria, dried products of lactic acid bacteria (freeze-dried products, spray-dried products, etc.).

[0034] · Conditions for allowing lactic acid bacteria and / or processed products thereof to act on astragaloside IV To allow lactobacilli and / or their cell-treated products to act on astragaloside IV, astragaloside IV and lactobacilli and / or their cell-treated products may be coexisted and incubated in a medium.

[0035] Examples of media for coexisting astragaloside IV with lactobacilli and / or their cell-treated products include buffer solutions, culture media, etc.

[0036] Specific examples of buffer solutions include phosphate buffer solution, citrate buffer solution, Tris buffer solution, acetate buffer solution, borate buffer solution, etc. In addition, the buffer solution may contain components necessary for the growth of lactic acid bacteria and bifidobacteria as needed. Examples of such components include nitrogen sources such as yeast extract, polypeptone, meat extract; carbon sources such as glucose, sucrose; inorganic salts such as phosphates, carbonates, sulfates; vitamins; amino acids, etc.

[0037] The medium may be either a solid medium or a liquid medium.

[0038] Examples of the pH of the medium include, for example, pH 2 to 9, preferably pH 3 to 8, and more preferably 4 to 7.5.

[0039] Examples of the content of astragaloside IV in the medium include, for example, 0.01% by weight or more, preferably 0.05 to 20% by weight, and more preferably 0.1 to 1% by weight.

[0040] Examples of the amount of lactic acid bacteria and / or their cell-treated products added per 1 g of the medium include, for example, 1×10 4 cfu / g or more, preferably 1×10 6 cfu / g or more, and more preferably 1×10 7 ~1×10 9 cfu / g, and even more preferably 5×10 7 ~1×10 8 cfu / g.

[0041] Examples of the temperature conditions for incubation in a state where astragaloside IV coexists with lactic acid bacteria and / or their cell-treated products in the medium include, for example, 20 to 40°C, preferably 25 to 37°C.

[0042] Examples of the incubation time in a state where astragaloside IV coexists with lactic acid bacteria and / or their cell-treated products in the medium include, for example, 12 to 200 hours, preferably 24 to 96 hours, and more preferably 60 to 85 hours.

[0043] The atmosphere for incubation in a state where astragaloside IV coexists with lactic acid bacteria and / or their cell-treated products in the medium may be either aerobic conditions or anaerobic conditions. For example, when using viable lactic acid bacteria, aerobic conditions are preferred.

[0044] The product obtained by allowing lactobacilli and / or their cell-treated products to act on astragaloside IV in this manner is subjected to the second step described below.

[0045] [Second Step] The second step is a step of allowing Bifidobacterium and / or its cell-treated products to act on the product obtained in the first step. The product obtained by the second step contains cycloastragenol.

[0046] · Bifidobacterium and / or processed products thereof The type of Bifidobacterium used in the second step is not particularly limited. For example, Bifidobacterium pseudocatenulatum, Bifidobacterium angulatum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium catenulatum, Bifidobacterium longum, Bifidobacterium dentium, Bifidobacterium scardovii, Bifidobacterium breve, Bifidobacterium uniformis, etc. can be mentioned.

[0047] From the viewpoint of producing cycloastragenol more efficiently, as the Bifidobacterium used in the second step, those having an M1 metabolic action (the action of releasing the xylose residue of astragaloside IV) can be preferably used. In particular, those having an M1 metabolic action higher than the M2 metabolic action (the action of releasing the glucose residue of astragaloside IV) are particularly preferred.

[0048] The M1 metabolism and the M2 metabolism can be determined by co-incubating astragaloside IV and Bifidobacterium, and measuring the production amounts of the compound represented by the general formula (3) and the compound represented by the general formula (4) in the obtained product. That is, when co-incubating astragaloside IV and Bifidobacterium, and the presence of the compound represented by the following general formula (4) is recognized in the obtained product, it is determined that the Bifidobacterium has the M1 metabolism. Also, when the amount of the compound represented by the following general formula (4) in the obtained product is larger than the amount of the compound represented by the following general formula (3), it is determined that the M1 metabolism is higher than the M2 metabolism.

[0049] Based on the disclosure of this specification, those skilled in the art can measure the M1 metabolism and the M2 metabolism for known Bifidobacterium or isolated Bifidobacterium, and appropriately select Bifidobacterium with the M1 metabolism and Bifidobacterium with the M1 metabolism higher than the M2 metabolism.

[0050] Specific examples of the Bifidobacterium strains with the M1 metabolism higher than the M2 metabolism include Bifidobacterium pseudocatenulatum (JCM1200, JCM7041, etc.), Bifidobacterium angulatum (JCM7096, etc.), Bifidobacterium adolescentis (JCM7046, etc.), Bifidobacterium bifidum (JCM7004, etc.), Bifidobacterium catenulatum (JCM1194, etc.), Bifidobacterium longum (JCM1222, etc.), Bifidobacterium dentium (JCM1195, etc.), Bifidobacterium scardovii (JCM12489, etc.), Bifidobacterium breve (JCM7020, JCM1273, JCM1192, etc.), Bifidobacterium uniformis (JCM7020, etc.).

[0051] Preferable examples of the Bifidobacterium used in the second step include Bifidobacterium pseudocatenulatum and Bifidobacterium angulatum; more preferably, Bifidobacterium pseudocatenulatum (JCM1200, JCM7041) and Bifidobacterium angulatum (JCM7096).

[0052] In the second step, one type of Bifidobacterium may be used alone, or two or more types of Bifidobacterium may be used in combination.

[0053] In the second step, the Bifidobacterium may be used as live cells, but as long as the enzymes contained in the Bifidobacterium maintain their activity, processed products of the Bifidobacterium cells can also be used. Examples of the processed products of the Bifidobacterium cells include disrupted cells of the Bifidobacterium, dried products of lactic acid bacteria (freeze-dried products, spray-dried products, etc.).

[0054] · Conditions for allowing Bifidobacterium and / or processed products thereof to act on the product obtained in the first step To allow the Bifidobacterium and / or its processed product to act on the product obtained in the first step, the Bifidobacterium and / or its processed product may be added to the product and incubated.

[0055] The Bifidobacterium and / or its processed product may be directly added to the product obtained in the first step, but before adding the Bifidobacterium and / or its processed product, heat sterilization, concentration, dilution, pH adjustment, addition of nutrient components, etc. may be performed as necessary.

[0056] The amount of the Bifidobacterium and / or its processed product added to the product obtained in the first step is, for example, 1×10 4 cfu / g or more, preferably 1×10 6 cfu / g or more, more preferably 1×10 7 ~1×10 9 cfu / g, still more preferably 5×10 7 ~1×10 8 cfu / g.

[0057] As temperature conditions for adding and incubating Bifidobacterium and / or its processed bacterial cells to the product obtained in the first step, for example, 20 to 40°C, preferably 25 to 37°C can be mentioned.

[0058] As the time for adding and incubating Bifidobacterium and / or its processed bacterial cells to the product obtained in the first step, for example, 12 to 100 hours, preferably 24 to 96 hours, more preferably 60 to 85 hours can be mentioned.

[0059] The atmosphere for adding and incubating Bifidobacterium and / or its processed bacterial cells to the product obtained in the first step may be either aerobic conditions or anaerobic conditions.

[0060] By allowing Bifidobacterium to act on the product obtained in the first step, a cycloastragenol-containing product in which cycloastragenol is efficiently produced and accumulated can be obtained.

[0061] 2. Cycloastragenol-containing substance The cycloastragenol-containing product obtained by the manufacturing method can be used as it is, or, if necessary, can be subjected to treatments such as concentration, extraction, drying, etc., and used in foods, pharmaceuticals, etc.

[0062] Also, by isolating cycloastragenol from the cycloastragenol-containing product obtained by the manufacturing method, a purified product or a roughly purified product of cycloastragenol can be obtained and used in foods, pharmaceuticals, reagents, etc.

Examples

[0063] Examples are shown below to more specifically explain the present invention, but the present invention is not limited thereto.

[0064] 1. Preparation of cell pellets The cell pellets of lactic acid bacteria 1 to 4 and Bifidobacterium a to c shown in Table 1 were prepared by the following method. Each cell pellet was prepared immediately before use.

[0065]

Table 1

[0066] 50 μL of the glycerol stock of each cell was cultured in 15 mL of GAM medium diluted 2-fold from the predetermined concentration under anaerobic conditions at 37 °C for 24 hours with a shaking speed of 130 rpm.

[0067] The culture solution was centrifuged at 1500 g for 20 minutes, the supernatant was decanted, and a cell pellet was obtained. The obtained cell pellet was washed with 1 mL of 0.85% sodium chloride solution, the washing solution was transferred to a centrifuge tube, and centrifuged again at 20000 g for 20 minutes to obtain a cell pellet again.

[0068] 2. Production of cycloastragenol 2-1. Examples 1 to 6 (acting in the order of lactic acid bacteria, Bifidobacterium) <First step> 0.7 mL of a potassium phosphate buffer at pH 6.5 and a concentration of 0.1 M was mixed with 13.3 μL of a DMSO solution containing 15 mg / mL of astragaloside IV, and each lactic acid bacteria pellet shown in Table 2 was added to the mixed solution. Astragaloside IV and lactic acid bacteria were incubated in the buffer at 37 °C for 72 hours under aerobic conditions at a shaking speed of 1300 rpm to obtain a product.

[0069] <Second step> To the obtained product, each Bifidobacterium pellet shown in Table 2 was added, and incubated at 37 °C for 72 hours under static conditions without aeration to obtain a product.

[0070]

Table 2

[0071] 2-2. Comparative Examples 1 to 7 (acting with lactic acid bacteria or Bifidobacterium alone) Mix 0.7 mL of potassium phosphate buffer at pH 6.5 and a concentration of 0.1 M with 13.3 μL of a DMSO solution containing 15 mg / mL of astragaloside IV. Add the pellets of each lactic acid bacterium or Bifidobacterium shown in Table 3 to the mixture, and incubate astragaloside IV and the lactic acid bacterium or Bifidobacterium in the buffer under aerobic conditions at a shaking speed of 1300 rpm at 37 °C for 72 hours to obtain a product.

[0072]

Table 3

[0073] 2-3. Comparative Examples 8 to 13 (acting with lactic acid bacteria and Bifidobacterium simultaneously) Mix 0.7 mL of potassium phosphate buffer at pH 6.5 and a concentration of 0.1 M with 13.3 μL of a DMSO solution containing 15 mg / mL of astragaloside IV. Add a combination of the lactic acid bacterium pellets and Bifidobacterium pellets shown in Table 4 to the mixture, and incubate astragaloside IV, the lactic acid bacterium, and Bifidobacterium in the buffer under aerobic conditions at a shaking speed of 1300 rpm at 37 °C for 72 hours to obtain a product.

[0074]

Table 4

[0075] 2-4. Comparative Examples 14 to 24 (acting in the order of Bifidobacterium, lactic acid bacteria) <Step 1> Mix 0.7 mL of potassium phosphate buffer at pH 6.5 and a concentration of 0.1 M with 13.3 μL of a DMSO solution containing 15 mg / mL of astragaloside IV. Add the pellets of each Bifidobacterium shown in Table 5 to the mixture, and incubate astragaloside IV and Bifidobacterium in the buffer under aerobic conditions at a shaking speed of 1300 rpm at 37 °C for 72 hours to obtain a product.

[0076] <Step 2> Add the pellets of each lactic acid bacterium shown in Table 4 to the obtained product, and incubate at 37 °C for 72 hours under aerobic conditions at a shaking speed of 1300 rpm to obtain a product.

[0077]

Table 5

[0078] 3. Composition analysis of the product The products obtained after the second step of Examples 1 to 6 and Comparative Examples 14 to 24, and the products obtained in Comparative Examples 1 to 13 were freeze-dried. After adding 1 mL of methanol to the total amount of the obtained freeze-dried product and mixing well, the liquid fraction was recovered by solid-liquid separation. The liquid fraction was subjected to HPLC, and the contents of astragaloside IV (general formula (1)), cycloastragenol (general formula (2)), xylose glycoside of cycloastragenol (general formula (3)), and glucose glycoside of cycloastragenol (general formula (4)) were measured. The HPLC measurement conditions are as follows.

[0079] <HPLC Measurement Conditions> Instrument configuration: Injector; Shimadzu Corporation Auto Sampler SIL-10AF Pump, etc.; Shimadzu Corporation Online Degasser DGU-12A Shimadzu Corporation Pump LC-10ADVP Shimadzu Corporation Column Oven CTO-10AVP Shimadzu Corporation Controller SCL-10AVP Detector; Shimadzu Corporation Ultraviolet Detector SPD-10AVP Data processing device; Shimadzu Corporation LCsolution Column: 5C18-AR-2 (Nacalai Tesque; 4.6 ID x 150 mm) Column temperature: 35 °C Measurement wavelength: 203 nm Flow rate: 1.5 mL / min Mobile phase: A; Purified water B; Acetonitrile Analysis conditions: 0 min to 1.5 min; A 70%, B 30% 1.5 to 8 points; A 70%, B 30% - A 20%, B 80% 8 to 8.01 points; A 20%, B 80% - A 0%, B 100% 8.01 to 12.5 points; A 0%, B 100% 12.5 to 12.51 points; A 0%, B 100% - A 20%, B 80% 12.51 to 13.01 points; A 20%, B 80% - A 70%, B 30% 13.01 to 18 points; A 70%, B 30%

[0080] 4. Results The obtained results are shown in FIGS. 1 to 4. When bifidobacteria were allowed to act after lactobacilli were allowed to act on astragaloside IV, a significantly larger amount of cycloastragenol production was observed (Examples 1 to 6). In contrast, when lactobacilli or bifidobacteria were allowed to act alone on astragaloside IV, only a trace amount of cycloastragenol production was observed (Comparative Examples 1 to 7). Also, when lactobacilli and bifidobacteria were allowed to act simultaneously on astragaloside IV, almost no cycloastragenol production was observed (Comparative Examples 8 to 13). Furthermore, even when lactobacilli were allowed to act after bifidobacteria were allowed to act on astragaloside IV, the amount of cycloastragenol production was small (Comparative Examples 14 to 24).

[0081] From the above results, it became clear that in order to efficiently produce cycloastragenol from astragaloside IV, it is effective to allow microorganisms to act on astragaloside IV in two steps in the order of lactobacilli and bifidobacteria.

[0082] Reference Test Example 1 Each of the following lactobacillus pellets was prepared under the conditions described in "1. Preparation of cell pellets" in the column of Test Example 1 above. <List of lactobacilli used> Weissella confusa: JCM1093 strain, RD012604 strain Weissella cibaria: Strain RD011819 Weissella paramesenteroides: Strain RD012576 Lactobacillus casei: Strain NBRC 15883 Lactobacillus acidophilus: Strains NBRC13951, NBRC102164 Lactobacillus alimentarius: Strains RD055252, DSM20249, RD055258 Lactobacillus brevis: Strains RD012087, ATCC14869, RD055271, RD011723, RD055257, RD012582, NBRC3345 Lactobacillus sakei: Strains NBRC15893, RD011713, RD012596, DSM20017, RD012090 Lactobacillus rapi: Strain RD012520 Lactobacillus paralimentarius: Strain NBRC107152 Lactobacillus paracasei: Strains RD055262, JCM8130, RD055272, RD011733, RD011743, RD055282, RD011746, RD055276, NBRC1588 Lactobacillus delbrueckii: Strain NBRC102622 Lactobacillus plantarum: Strains NBRC101973, NBRC106468, NBRC15891 Lactobacillus parabuchneri: Strains RD011765, NBRC103219, RD012653 Lactobacillus mali: Strain NBRC102159 Lactobacillus hamnesii: Strain RD011266 Lactobacillus versmoldensis: Strain RD011768 Lactobacillus paraplantarum: Strains RD055275, RD055269 Lactobacillus parabuffelii: Strain NBRC110708 Lactobacillus fermentum: Strain NBRC15885 Lactobacillus colliniformis: Strain RD011738 Lactobacillus crustorum: Strains RD055318 and RD011724 Lactobacillus pentosus: Strains RD011734, JCM1558, RD055278, RD011825, RD011748, RD055274, RD011741 Lactobacillus buchneri: Strain NBRC107764 Lactobacillus lactis: Strain NBRC107866 Tetragenococcus halophilus: Strains RD012572, IAM1676, NBRC12172, RD011830 Tetragenococcus koreensis: Strain RD012590 Leuconostoc citreum: Strains RD012577, ATCC49370, NBRC102476 Leuconostoc mesenteroides: Strains RD011712, NBRC3349, RD011716, NBRC100496, RD011740, ATCC8293, RD011798, RD012107 Leuconostoc lactis: Strains RD012583, JCM6123 Leuconostoc carnosum: Strain RD012174 Pediococcus argentinicus: Strain NBRC107827 Pediococcus pentosaceus: Strains RD012175, DSM20336, RD012523, RD012594 Pediococcus ethanolidurans: Strain RD011265 Pediococcus parvulus: Strains RD011824, JCM5889 Pediococcus acidilactici: Strain NBRC109619 Streptococcus salivarius: Strains RD012113, ATCC7073 Lactococcus lactis: Strain RD012601 Enterococcus thailandicus: Strains RD012568, RD012543 Enterococcus casseliflavus: NBRC100478 strain

[0083] 0.7 mL of potassium phosphate buffer with pH 6.5 and a concentration of 0.1 M was mixed with 13.3 μL of a DMSO solution containing 15 mg / mL of astragaloside IV. Each lactic acid bacteria pellet was added to the mixed solution, and astragaloside IV and lactic acid bacteria were incubated in the buffer under aerobic conditions at 37 °C with a shaking speed of 1300 rpm for 72 hours to obtain a product. Regarding the obtained product, the contents of astragaloside IV, cycloastragenol, the xylose glycoside of cycloastragenol, and the glucose glycoside of cycloastragenol were measured under the conditions described in "3. Composition analysis of the product" in the column of Test Example 1 above.

[0084] As a result, it was confirmed that in each of the above lactic acid bacteria, the content of the xylose glycoside of cycloastragenol was higher than the content of the glucose glycoside of cycloastragenol, and the M2 metabolic action was higher than the M1 metabolic action.

[0085] Reference Test Example 2 Each of the following Bifidobacterium pellets was prepared under the conditions described in "1. Preparation of cell pellets" in the column of Test Example 1 above. <List of each Bifidobacterium used> Bifidobacterium adolescentis: JCM7046 strain Bifidobacterium bifidum: JCM7004 strain Bifidobacterium catenulatum: JCM1194 strain Bifidobacterium longum: JCM1222 strain Bifidobacterium dentium: JCM1195 strain Bifidobacterium scardovii: JCM12489 strain Bifidobacterium breve: JCM7020 strain, JCM1273 strain, JCM119 strain Bifidobacterium uniformis: JCM7020 strain

[0086] 0.7 mL of a potassium phosphate buffer at pH 6.5 and a concentration of 0.1 M was mixed with 13.3 μL of a DMSO solution containing 15 mg / mL of astragaloside IV. Each Bifidobacterium pellet was added to the mixture, and astragaloside IV and Bifidobacterium were allowed to stand and incubate at 37 °C for 72 hours under non-aerated conditions in the buffer to obtain a product. For the obtained product, the contents of astragaloside IV, cycloastragenol, the xylose glycoside of cycloastragenol, and the glucose glycoside of cycloastragenol were measured under the conditions described in "3. Composition Analysis of the Product" in the column of Test Example 1 above.

[0087] As a result, it was confirmed that in each of the above Bifidobacterium, the content of the glucose glycoside of cycloastragenol was higher than the content of the xylose glycoside of cycloastragenol, and the M1 metabolic action was higher than the M2 metabolic action.

Claims

1. A method for producing cycloastragenol, comprising the following first step and second step: A first step of allowing lactic acid bacteria having an action of releasing the glucose residue of astragaloside IV and / or a treated product of the bacterial cells thereof to act on astragaloside IV, and A second step of allowing bifidobacteria having an action of releasing the xylose residue of astragaloside IV and / or a treated product of the bacterial cells thereof to act on the product obtained in the first step.

2. The production method according to claim 1, wherein the lactic acid bacteria are at least one lactic acid bacterium selected from the group consisting of the genus Weissella, the genus Pediococcus, the genus Lactobacillus, and the genus Leuconostoc.

3. The production method according to claim 1 or 2, wherein the lactic acid bacteria are at least one lactic acid bacterium selected from the group consisting of Weissella confusa, Weissella cibaria, Pediococcus pentosaceus, Lactobacillus sakei, and Leuconostoc carnosum.

4. The production method according to any one of claims 1 to 3, wherein the bifidobacteria are Bifidobacterium pseudocatenulatum and / or Bifidobacterium angulatum.

5. The production method according to any one of claims 1 to 4, further comprising a step of isolating cycloastragenol from the cycloastragenol-containing product obtained in the second step.

6. A cycloastragenol-containing product obtained through the following first step and second step: A first step of allowing lactic acid bacteria having an action of releasing the glucose residue of astragaloside IV and / or a treated product of the bacterial cells thereof to act on astragaloside IV, and A second step of allowing bifidobacteria having an action of releasing the xylose residue of astragaloside IV and / or a treated product of the bacterial cells thereof to act on the product obtained in the first step to produce a cycloastragenol-containing product.

7. A food product comprising the cycloastragenol-containing substance, its concentrate, or its dried product according to claim 6.

Images