Health food ingredients

Abstract

To develop a health food for more sufficiently exhibiting promotion effect of health maintenance required for a functionality display food or the like, and to more efficiently manufacturer such a health food.SOLUTION: A health food material contains, as an essential component, an out-of-bacterial cell high polymer substance produced by a Paenibacillus polymyxa AK-T1-11 strain (received No: NITE ABP-03577) cultured in a fermentation medium containing cane sugar as a carbon source and yeast extract as a nitrogen source, in which a medium material is a saccharified starch.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a health food material used in functional foods and the like that are preferred for promoting health, and a method for producing the same. [Background technology]

[0002] Enzamin (registered trademark) is a health food that promotes human health. It is a fermented metabolite extract that is thought to enhance metabolic activity so that the body can efficiently produce various components necessary for the body, as well as to enhance immune function by transforming T lymphocytes into blastocytes. The method for producing it is also disclosed in a patent publication (Patent Document 1).

[0003] In the above-mentioned production method, a saccharified product obtained by hydrolyzing starch, including corn starch, with amylase is used as a medium base material, to which yeast extract is added as a nitrogen source to prepare a fermentation medium. This medium is inoculated with Paenibacillus polymyxa Akazawa strain, which was called Bacillus subtilis AK when the above-mentioned patent document was first filed and is now called Akazawa (AK) bacteria, and after long-term cultivation, the resulting liquid component is separated and used as a food concentrate, which can be used to produce health foods, health nutritional foods, and cosmetics (Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 3902015 Publication [Patent Document 2] Japanese Patent Application Laid-Open No. 2012-143187 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the above-mentioned method for producing health foods, indicator components useful for health foods were not identified, so there was still room for further improvement in the strain and culture conditions to produce specific components more efficiently.

[0006] Many of the AK bacteria, which are traditionally considered to be natto bacteria or Bacillus bacteria, have been selected through subculture and long-term dry storage, and bacterial groups that produce large amounts of physiologically active substances have been selected. However, it was not clear at what fermentation stage and in what amounts the effective specific functional components were produced.

[0007] Therefore, cultivation was merely based on trial and error, relying on past experience, and it was necessary to determine the changes in concentration of specific components over time as indicators, and to be able to produce health foods more efficiently under optimal cultivation conditions.

[0008] Therefore, the object of this invention is to solve the above-mentioned problems, identify a strain that produces useful specific indicator components more efficiently, and by culturing it under specified culture conditions, create a health food that more fully exhibits the health-promoting effect required of functional food products, etc., and to more efficiently manufacture such excellent health foods. [Means for solving the problem]

[0009] In order to solve the above problems, in this invention, a conventional strain of Paenibacillus polymyxa was improved, and further research was conducted, including on the culture conditions, to produce a health food material containing, as an essential ingredient, an extracellular polymeric substance produced by Paenibacillus polymyxa AK-T1-11 strain (Accession Number: NITE ABP-03577) cultured in a fermentation medium whose medium base consists of a starch hydrolyzate, sucrose, or a mixture of these, and which contains water-soluble components of yeast as a nitrogen source.

[0010] The above-mentioned health food material is obtained by culturing the Paenibacillus polymyxa AK-T1-11 strain in a specified medium, which efficiently produces extracellular polymeric substances that are indicator components useful for human health. Therefore, health food materials can be obtained that use the specified extracellular polymeric substances produced by these strains as essential ingredients.

[0011] The extracellular polymeric substances include polysaccharides called exopolysaccharides (EPS), which include one or more polysaccharides selected from levan, levan oligosaccharides, and β-glucan, and a representative example of the β-glucan is curdlan.

[0012] By incorporating the required amount of such health food ingredients into food, it is possible to create health foods that more fully exhibit the health-promoting effects required of functional foods, etc.

[0013] In order to produce a health food using such a health food material, a method for producing a health food material can be adopted in which a starch saccharified product, sucrose, or a mixture thereof is used as a medium base material, yeast extract is added to this as a nitrogen source to prepare a fermentation medium, Paenibacillus polymyxa AK-T1-11 strain is inoculated into this and cultured, and the liquid component containing the produced extracellular polymeric substance is separated and used as a food stock solution.

[0014] According to the above-mentioned production method, a specific strain of bacteria efficiently utilizes sugars and nitrogen sources in a specific medium, producing fermentation products such as exopolysaccharides (EPS) such as levan and β-glucan, which have various activities, oligosaccharides, lipids, amino acids, bacterial cell wall components, and nucleic acids.

[0015] Incidentally, levan and its oligosaccharides are sugars that are beneficial for health promotion and can be used in functional foods, pharmaceutical products, and feed. Because they are highly hydrophilic, their use in cosmetics is also being studied.

[0016] β-Glucan is a substance present in the cell walls of bacteria, a type of insoluble dietary fiber, and has the effect of lowering blood cholesterol levels. For example, curdlan, which contains only β-1,3-bonds, is also used as a food additive as a thickening polysaccharide.

[0017] In addition, beta-glucan activates immune cells and enhances immunity by binding to foreign substance receptors on the cell membranes of innate immune cells such as macrophages and natural killer (NK) cells.

[0018] The starch saccharification product is preferably a low-molecular-weight starch saccharification product obtained by hydrolyzing starch, including corn starch, with amylase, and sucrose can be used in addition to or instead of the starch saccharification product. The use of sucrose as described above can significantly increase the amount of exopolysaccharide produced.

[0019] It is also preferable that the fermentation medium is adjusted to a pH of 7.0 or less. To this end, by adding an appropriate amount of a pH buffer, the transition to an acidic range during cultivation can be delayed, thereby ensuring sufficient cultivation time in the first stage and enabling more efficient production of the desired final product.

[0020] Preferably, the first stage of the culture is performed at pH 5.5 to 7.2 and 28 to 32°C for one week or more, and the second stage is performed at pH 4.0 to 6.0 and 8 to less than 13°C for one week or more.

[0021] The two-stage cultivation method described above can increase the production of specific types of active ingredients, especially EPS, which further enhances the amount and function of active ingredients produced by conventional cultivation methods. [Effects of the Invention]

[0022] The health food material of the present invention contains, as an essential ingredient, a specific extracellular polymeric substance produced when a specific strain of Paenibacillus polymyxa is cultured in a specific medium, and therefore has the advantage of being able to create a health food that more fully exhibits the health-promoting effects required of functional food products, etc.

[0023] In addition, the method for producing health food materials of this invention has the advantage that it selects strains of fermenting fungi that produce useful indicator components as efficiently as possible and adjusts them to specified culture conditions, thereby efficiently producing health food materials that more fully exhibit the health-promoting effects required for functional food products, etc. [Brief explanation of the drawings]

[0024] [Figure 1] Chromatogram showing the relationship between retention time and detection intensity of polysaccharides of known molecular weights by HPLC [Figure 2] A molecular weight calibration curve showing the relationship between the log molecular weight (MW) and retention time of polysaccharides of known molecular weights by HPLC. [Figure 3] Chromatogram showing the molecular weight distribution of the liquid component after cultivation in Example 1, showing the relationship between HPLC retention time and log molecular weight (MW). [Figure 4] Chromatogram showing the molecular weight distribution of the liquid component after cultivation in Example 2, showing the relationship between HPLC retention time and log molecular weight (MW). [Figure 5] Chromatogram showing the molecular weight distribution of the liquid component after cultivation in Example 3, showing the relationship between HPLC retention time and log molecular weight (MW). [Figure 6] Chromatogram showing the molecular weight distribution of the liquid component after cultivation in Example 4, showing the relationship between HPLC retention time and log molecular weight (MW). DETAILED DESCRIPTION OF THE INVENTION

[0025] The health food material according to an embodiment of the present invention contains, as an essential ingredient, extracellular polymeric substances produced by a predetermined strain of Paenibacillus polymyxa cultured in a predetermined medium, and can be produced as follows: a starch saccharification product, sucrose, or a mixture thereof is used as a medium base; a water-soluble component of yeast is added to this as a nitrogen source to prepare a fermentation medium; the Paenibacillus polymyxa AK-T1-11 strain is inoculated into the medium and cultured; and the liquid component containing the produced extracellular polymeric substances is separated and used as a food concentrate.

[0026] The starch saccharification product of the culture medium base material is obtained by hydrolyzing starch derived from plants such as grains, potatoes, plant roots and stems, and beans with the digestive enzyme amylase in the final step of the process of decomposing starch, and is preferably a low-molecular-weight starch saccharification product obtained by hydrolyzing starch containing corn starch with amylase.

[0027] Sucrose (sucrose) is a disaccharide obtained from many plants as a carbon source, and it is preferable to use granulated sugar containing sucrose as the main component. In this invention, sucrose alone can be used as a carbon source.

[0028] The water-soluble components of yeast are extracts from yeast, also known as yeast extract or yeast extract, and are obtained by autolysis or acid hydrolysis of the yeast cells themselves. They are well known as components of culture media for microbial culture, containing amino acids, nucleic acids, minerals, vitamins, etc.

[0029] It is also preferable to add a pH buffer to the fermentation medium to adjust the pH to 7.0 or less. For example, when an easily metabolized component such as glucose is used as the carbon source, acid tends to accumulate in the medium, and a drop in pH may damage the fungi involved in the fermentation.

[0030] Examples of pH buffers include dipotassium hydrogen phosphate, disodium hydrogen phosphate, etc. In addition, in order to appropriately adjust the osmotic pressure inside and outside the cell, an appropriate amount of sodium chloride, calcium chloride, etc. may be added as needed.

[0031] The fermentation bacterium to be inoculated into the fermentation medium containing the above-mentioned essential components is the Paenibacillus polymyxa AK-T1-11 strain, and one or more mutant strains having slightly different characteristics such as preferred growth conditions and the ability to produce extracellular polymeric substances may also be included to the extent that the object of the present invention is not impaired.

[0032] Based on the latest biochemical classification by API50CH and the results of the complete bacterial 16S rRNA base sequence, the Paenibacillus polymyxa strain AK-T1-11 has been identified as Paenibacillus polymyxa of the genus Paenibacillus, which was transferred from the genus Bacillus in the 1993 reclassification of bacterial species.

[0033] This Paenibacillus polymyxa AK-T1-11 strain grows efficiently in a medium that uses a saccharified product of starch, including corn starch, hydrolyzed with amylase, or sucrose, or a mixture of these as a substrate, to which a small amount of water-soluble yeast components, such as yeast extract, is added as a nitrogen source.Unlike ordinary bacteria, it can fix nitrogen from the atmosphere, so it grows easily even in a medium that contains only a small amount of nitrogen source.

[0034] This is effective in preventing putrid odors and the production of allergens. Furthermore, because they form spores that are resistant to high heat and ultraviolet light and can survive in harsh environments and conditions, such as high and low temperatures, they are easy to store, and this property can be used to prevent the loss of bacterial activity caused by bacteriophage infection. Conversely, it is also easy to obtain mutant strains that specifically produce large amounts of various physiologically active substances.

[0035] The mycological properties of the above strain are as follows: <Paenibacillus polymyxa strain AK-T1-11> (a) Morphological properties (1) Cell shape and size: Bacillus 0.6-0.8 x 3.0-50 μm, chains, short to simple (2) Cellular polymorphism: None (3) Motility: Yes (peritrich flagella) (4) Presence or absence of spores: Present, oval, near the center of the bacterial body, bacterial body swelling, present (b) Culture properties (1) Nutrition agar plate culture: Round, viscous colonies (2) Meat juice liquid culture: bacterial cells aggregate at the top (c) Biochemical properties (1) Gram reaction: variable (2) Nitrate reduction: positive (3) VP test: positive (4) Urease: Negative (5) Indole formation: negative (6) Hydrogen sulfide production: negative (7) Citric acid utilization: positive (8) Catalase: Positive (9) Oxidase: Negative (10) Growth range: pH 5.5-7.6, temperature 10°C-45°C

[0036] This Paenibacillus polymyxa AK-T1-11 strain was internationally deposited with the National Institute of Technology and Evaluation's Patent Microorganisms Depositary (NPMD) on December 24, 2021 (date of receipt) under receipt number NITE ABP-03577.

[0037] The preferred first-stage culture conditions for this invention vary depending on the strain, but are pH 5.5-7.2, 28-32°C, and a period of at least one week but less than two months. Under more acidic conditions than those mentioned above and below the specified temperature, the strain used in this invention is unlikely to efficiently assimilate sugars and nitrogen sources, even if cultured for two months or longer, and the expected effects of the resulting food product will not be fully achieved. Furthermore, under alkaline conditions exceeding the pH range mentioned above and at temperatures exceeding the specified temperature, culture is insufficient, and the bacteria will not efficiently assimilate sugars and nitrogen sources, and the resulting functional food product will similarly fail to fully achieve the expected effects.

[0038] The low-temperature culture conditions for the second stage preferably employed in this invention are pH 4.0 to 6.0, a culture temperature of 13 to 17°C, preferably 8°C or higher and lower than 13°C, and a culture period of 1 week or longer and lower than 4 months.

[0039] Under conditions of a stronger acidity than those mentioned above and below the specified temperature, even if the culture is continued for more than four months, it is estimated that the fermentation products, such as amino acids, lipopolysaccharides, and lipids, which have various activities, will not be sufficiently reduced in molecular weight, and the resulting food will not fully achieve the desired effects.

[0040] Furthermore, even if the bacteria are cultured under neutral or alkaline conditions beyond the above-mentioned acidic range and at temperatures higher than the specified temperature, it is estimated that the activity will decrease, and the resulting functional food will not fully achieve the desired effects, as described above.

[0041] Thus, the culture method preferably employed in the present invention involves culturing at pH 5.5 to 7.2 and 28 to 32°C for at least one week, preferably less than two months, as the first stage, and then culturing at pH 4.0 to 6.0 and 8 to less than 13°C for at least one week, preferably less than four months, as the second stage.

[0042] A continuous centrifuge is used to separate the liquid components produced, and the resulting food-grade concentrate can be used as is, but can also be concentrated or diluted as necessary before use.

[0043] The resulting food-grade concentrate contains β-glucans such as levan and curdlan, and oligosaccharides produced by fermentation with the bacteria. This can be confirmed by measuring the molecular weight distribution using a high-performance liquid chromatograph (HPLC) equipped with a size-exclusion chromatography column.

[0044] In addition to such qualitative analysis, quantitative analysis can be performed by treating the sample with ethanol precipitation, then hydrolyzing it with dilute sulfuric acid to break it down into its constituent sugars, and then quantifying the constituent sugars using HPLC. [Example]

[0045] <Preparation of medium> Fermentation media were prepared using the blending ratios shown in Table 1 (all parts shown below are parts by mass) according to the following procedure.

[0046] [Table 1]

[0047] [Examples 1 and 2] To 1.5 parts of cornstarch, 0.05 parts of calcium chloride, 0.1 parts of salt (roasted salt, sodium chloride), and 18.3 parts of purified water were added and heated with steam while stirring. The mixture was heated to 91°C to gelatinize the cornstarch. Purified water was further added while stirring, and when the temperature was lowered to around 60°C, 0.03 parts of amylase was added and saccharification was carried out while stirring.

[0048] After saccharification, 1 or 11 parts of sucrose (granulated sugar), 1 part of glucose (grape sugar), 0.3 parts of yeast extract (domestically produced), and 0.05 parts of disodium hydrogen phosphate were added and stirred to dissolve, and then a basket containing 1 part of rice syrup was placed in the tank and dissolved.

[0049] After dissolving the rice syrup, purified water was added up to 100 parts, and 0.2 parts of 10% sodium hydroxide was added to adjust the pH.

[0050] The pH and Brix were measured to confirm that they were within the standard range, and if they were within the standard range (pH 7.2-7.6, Brix 4.5-4.8), the mixture was dispensed into culture vessels, sterilized by high-pressure steam (121°C, 15 minutes), and then rapidly cooled with water. The pH standard range was set on the assumption that the medium pH would quickly reach pH 5.5-7.2 after cultivation.

[0051] [Examples 3 and 4] To 10 or 15 parts of sucrose (granulated sugar), 0.2 parts of yeast extract (manufactured by BBL), 0.55 parts of dipotassium hydrogen phosphate, and 0.02 parts of magnesium sulfate were added and dissolved by stirring. Next, while adding up to 100 parts of purified water, 0.2 parts of 10% sodium hydroxide was added to adjust the pH of the medium.

[0052] The pH and Brix were measured and confirmed to be within the above standard criteria. If they were within the reference values (pH 7 or less, Brix 4.5 - 4.8), they were dispensed into the culture tank, subjected to high-pressure steam sterilization (121°C, 15 minutes), and then rapidly cooled by water cooling.

[0053] <Inoculation and Cultivation of AK Bacteria> The AK-T1-11 strain, which had been adsorbed onto beads for strain preservation in spore form and stored at -80°C, was heat-treated at 85°C for 10 minutes (a treatment to promote spore germination) immediately before inoculating it into the above medium, and then put into 200 mL of enrichment medium in a 500 mL Erlenmeyer flask, one bead at a time, and cultured overnight at 30°C and 150 rpm in a rotary shaker incubator.

[0054] Next, the culture broth after the shaking culture was immediately aseptically inoculated into a stainless-steel culture tank containing 30 L of fresh liquid medium, 100 mL each, and then left standing in a 30°C constant-temperature room for the first stage of cultivation for 1 week to 2 months.

[0055] The culture tank after the predetermined cultivation period had elapsed was moved to a constant-temperature room at 8 - 10°C, and the second-stage low-temperature cultivation was carried out for 2 - 4 months.

[0056] <Measurement of Molecular Weight Distribution> HPLC was performed on the culture broth after the above low-temperature cultivation. At that time, a liquid delivery pump (LC-20AD), a system controller (CBM-20ALite), a degassing unit (DGU-20A3R), a column thermostat (CTO-20A), a UV-VIS detector (SPD-20A), a differential refractive index detector (RID-20A), and an LC-workstation (Lab solution single) of Shimadzu products were used.

[0057] To measure the molecular weight distribution of sugars, size exclusion chromatography was performed using a Shodex Sugar KS-803 (φ8mm × 300mm) and KS-804 (φ8mm × 300mm) connected columns. The column was kept at a constant temperature of 80°C, the mobile phase was water, the flow rate was 1.25mL / min, and 20μL was injected using a differential refractometer detector.

[0058] The molecular weights of the unknown components were determined by measuring components with known molecular weights in advance and creating a molecular weight calibration curve calculated from the relationship between retention time (elution time) and molecular weight. The components with known molecular weights were nine components: glucose (molecular weight: MW 180), sucrose (MW 342), and pullulan (MW 6,100, MW 9,600, MW 22,000, MW 47,100, MW 107,000, MW 194,000, MW 337,000), a type of polysaccharide consisting only of glucose. The chromatogram measured at a concentration of 1,000 mg / L is shown in Figure 1.

[0059] The molecular weight calibration curve equation was calculated by the least squares method using the retention times of the nine components as explanatory variables and the logarithmic molecular weight values ​​as response variables. The molecular weight calibration curve is shown in Figure 2. The molecular weight calibration curve equation was calculated in the same manner as above and is shown as the following equation.

[0060] Molecular weight calibration curve equation: y=-0.4636x+9.2797 Contribution ratio: 0.9964

[0061] The culture medium of Example 1-4 was collected after the start of the culture, and the molecular weight distribution was measured by the HPLC method described above. The results are shown in Figures 3-6. Furthermore, polysaccharides contained in the culture solution after the low-temperature culture were separated by ethanol precipitation, hydrolyzed into monosaccharides, and then measured by HPLC, where fructose was detected. From this, it was determined that the polysaccharides contained in the Enzamin stock solution were levan, which is a polymer formed by fructose linked by glycosidic bonds. Furthermore, the water-insoluble polysaccharides contained in the culture broth sediment after the low-temperature culture were separated and decomposed into monosaccharides by hydrolysis, and the monosaccharides were measured by HPLC. As a result, it was determined that the polysaccharides were curdlan, a β-1,3-glucan in which glucose units are linked by glycosidic bonds.

[0062] In this way, two types of polysaccharides were identified, and as is clear from the results shown in Figures 3 to 6, it was confirmed that after cultivation, disaccharides (sucrose) were decomposed into monosaccharides, and new oligosaccharides and two types of polysaccharides (levan and curdlan) were produced.

[0063] Sodium hydroxide was then added to adjust the pH to within the range of 7.3 to 7.8, and the resulting mixture was placed in a culture can and sterilized at 120°C for 20 minutes under high pressure. After cooling, Paenibacillus polymyxa (the conventionally known Bacillus subtilis AK strain: FERM P-18291) was inoculated and fermented for 60 days at a pH of 4.5 to 6.5 in a thermostatic chamber at 30±2°C. The culture solution was then aged for 180 days at a pH of 4.0 to 6.0 in a thermostatic chamber at 15±2°C to clarify the culture solution. The resulting solution was filtered to obtain 54.3 parts of a liquid stock solution for a health and nutritional food (hereinafter referred to as the culture filtrate).

[0064] The culture filtrate was subjected to size exclusion chromatography (SEC) using a Tosoh column (TSKgel G2500PWXL) and a liquid high-performance chromatograph (Shodex: GPC SYSTEM-21) with a mobile phase of a 55:45:0.1 mixture of water, acetonitrile, and trifluoroacetic acid. The detector sensitivity (ultraviolet spectrophotometer: mV) was compared with the elution time of a standard sample with a known molecular weight to analyze the molecular weight distribution. The percentage of the area of ​​the molecular weight fractions in the total area is shown in Table 2.

[0065] [Table 2]

[0066] As is clear from the results shown in Table 2, the comparative example used the conventionally known Paenibacillus polymyxa (Bacillus subtilis AK strain: FERM P-18291), and production of oligosaccharides with molecular weights of 500 or less was observed, but production of polysaccharides with molecular weights of 10,000 or more was not significant.

[0067] In contrast, in the above-mentioned Examples 1 to 4, since the Paenibacillus polymyxa AK-T1-11 strain was used, significant production of polysaccharides was observed, and the production of levan at a molecular weight log (MW) of approximately 5.5 and curdlan at a molecular weight log (MW) of approximately 6.0 was observed.

[0068] The production amounts of such levan and curdlan were found to increase significantly with increasing amount of sucrose (granulated sugar) added to the medium. Furthermore, as in Examples 3 and 4, by using a fermentation medium containing a pH buffer and adjusted to a pH of 7.2 or less, for example, pH 7.0, the transition to an acidic range during cultivation was delayed, thereby ensuring sufficient cultivation time in the first stage, and further increases in production amounts were found.

[0069] Tables 3 to 6 show examples of formulations for health drinks, capsule-type health foods, lotions, and nutritional supplements for animals and plants, which were produced using the obtained health and nutritional food concentrates.

[0070] The health drink was prepared by blending 2 to 50% of the culture filtrate with other ingredients as shown in Table 3.

[0071] [Table 3]

[0072] The capsule-type health food was prepared by concentrating the culture filtrate 20 times and blending it with other ingredients as shown in Table 4.

[0073] [Table 4]

[0074] The lotion was prepared by blending 2% of the culture filtrate with other ingredients as shown in Table 5.

[0075] [Table 5]

[0076] The plant and animal nutrients were prepared by blending 20% ​​of the culture filtrate with the other ingredients shown in Table 6.

[0077] [Table 6] [Accession number]

[0078] 1) Name of recipient organization: National Institute of Technology and Evaluation, National Patent Microorganism Deposit Center (NPMD) 2) Date of receipt: December 24, 2021 3) Receipt number: NITE ABP-03577

Claims

1. A method for producing a health food material, comprising: preparing a fermentation medium by adding a water-soluble component of yeast as a nitrogen source to a medium base material containing a starch saccharified product, sucrose, or a mixture thereof; inoculating the medium with Paenibacillus polymyxa AK-T1-11 strain (accession number: NITE ABP-03577) and culturing the strain; and separating the liquid component containing the produced extracellular polymeric substance to prepare a food stock solution.

2. 2. The method for producing a health food material according to claim 1, wherein the starch saccharification product is a starch saccharification product obtained by hydrolyzing starch containing corn starch with amylase.

3. 3. The method for producing a health food material according to claim 1 or 2, wherein the culture is performed in a first stage at a pH of 5.5 to 7.2 and at 28 to 32°C for one week or more, and in a second stage at a pH of 4.0 to 6.0 and at a temperature of 8 to 13°C for one week or more.

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