JP7828634B2 - Health food ingredients - Google Patents

Description

NPMD NITE BP-03577

This invention relates to a health food ingredient used in functional foods and the like that are beneficial for promoting health, and a method for producing the same.

Enzamin (registered trademark) is a fermented metabolite extract that is believed to enhance human health by increasing metabolic activity to efficiently produce various components necessary for the body, and by rejuvenating T lymphocytes to strengthen immune function. Its manufacturing method is also disclosed in a patent publication (Patent Document 1).

Using the above manufacturing method, a saccharified product obtained by hydrolyzing starch containing cornstarch with amylase is used as a culture medium base. Yeast extract is added as a nitrogen source to prepare a fermentation medium. Paenibacillus polymyxa Akazawa strain, originally called Bacillus subtilis AK in the patent application mentioned above and now known as Akazawa (AK) strain, is inoculated into this medium. After long-term cultivation, the resulting liquid component is separated and used as a food stock solution. This can then be used to manufacture health foods, nutritional supplements, and cosmetics (Patent Document 2).

Patent No. 3902015 Publication Japanese Patent Publication No. 2012-143187

However, the manufacturing method for the health food described above did not identify any indicator components useful as health foods. Therefore, there was still room for further improvement in the bacterial strains and culture conditions to produce specific components more efficiently.

While many of the conventional natto bacteria, or Bacillus species, known as AK bacteria, have been selected through subculturing and long-term dry storage to produce high levels of physiologically active substances, the specific functional components produced at each stage of fermentation and in what quantities remained unknown.

Therefore, the culture process was merely based on past experience and trials. It was necessary to determine the temporal changes in the concentration of specific indicator components and to develop optimal culture conditions to produce health foods more efficiently.

Therefore, the objective of this invention is to solve the above-mentioned problems, identify a bacterial strain that more efficiently produces useful specific indicator components, and, by culturing it under predetermined culture conditions, create a health food that more fully exhibits the health-promoting effects required for functional foods, and to manufacture such excellent health foods more efficiently.

To solve the above problems, this invention improves conventional strains of Paenibacillus polymixa and further research, including on culture conditions, has resulted in a health food ingredient containing extracellular polymeric substances as an essential component, produced by Paenibacillus polymixa strain AK-T1-11 (receipt number: NITE ABP-03577), cultured in a fermentation medium consisting of starch saccharified sucrose or a mixture thereof, and containing water-soluble components of yeast as a nitrogen source.

The health food ingredients described above are obtained by culturing Paenibacillus polymixa AK-T1-11 strain in a specified culture medium, which efficiently produces extracellular polymers that are indicator components useful for human health. Therefore, health food ingredients using these strains as essential components can be obtained.

The aforementioned extracellular polymeric substance includes polysaccharides called extracellular polysaccharides (EPS), which include, for example, one or more polysaccharides selected from levan, levan oligosaccharides, and β-glucans. A typical example of β-glucan is curdlan.

By incorporating the required amounts of such health food ingredients into food products, it is possible to create health foods that more effectively exhibit the health-promoting effects required for functional foods and similar products.

To manufacture health foods using such health food ingredients, a method can be employed in which starch hydrolysates, sucrose, or mixtures thereof are used as a culture medium base, yeast extract is added as a nitrogen source to prepare a fermentation medium, Paenibacillus polymixa strain AK-T1-11 is inoculated into this medium and cultured, and the resulting liquid component containing extracellular polymers is separated to be used as a food stock.

According to the above manufacturing method, the specified bacterial strain efficiently utilizes sugars and nitrogen sources in the specified culture medium, producing fermentation products such as extracellular polysaccharides (EPS) with various activities, including levan and β-glucan, oligosaccharides, lipids, amino acids, cell wall components of the bacterial cells, and nucleic acids.

Incidentally, levan and its oligosaccharides are sugars beneficial for health promotion and can be used in functional foods, pharmaceuticals, and animal feed. Due to their high hydrophilicity, their use in cosmetics is also being researched.

Furthermore, β-glucan is a substance found in the cell walls of bacterial cells, and is a type of insoluble dietary fiber. It has the effect of lowering blood cholesterol levels. Additionally, curdlan, which contains only β-1,3-links, is used as a food additive as a thickening polysaccharide.

Furthermore, β-glucan also has the effect of activating immune cells and enhancing immunity by binding to foreign substance receptors on the cell membranes of innate immune cells such as macrophages and natural killer (NK) cells.

The above-mentioned starch syrup is preferably a low-molecular-weight starch syrup obtained by hydrolyzing corn starch with amylase. In addition to or in place of the starch syrup, sucrose can be used. By using sucrose as described above, the production of extracellular polysaccharides can be significantly increased.

Furthermore, it is preferable that the fermentation medium be adjusted to a pH of 7.0 or lower. By adding an appropriate amount of 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.

The above culture process is preferably carried out as follows: First, the culture is performed under conditions of pH 5.5–7.2 and 28–32°C for at least one week; followed by a second stage of culture under conditions of pH 4.0–6.0 and 8°C to less than 13°C for at least one week.

The two-stage culture method described above can increase the production of specific types of active ingredients, particularly EPS. The EPS obtained in this way further enhances the amount and function of the active ingredients produced by conventional culture methods.

This invention's health food ingredient contains a specific extracellular polymer substance produced when a particular strain of Paenibacillus polymixa is cultured in a predetermined culture medium. Therefore, it has the advantage of creating health foods that more effectively exhibit the health-promoting effects required for functional foods.

Furthermore, this invention's method for producing health food ingredients has the advantage of efficiently producing health food ingredients that more fully exhibit the health-promoting effects required for functional foods, by selecting strains of fermenting microorganisms that produce useful indicator components as efficiently as possible and adjusting them to predetermined culture conditions.

Chromatogram showing the relationship between retention time and detection intensity of polysaccharides with known molecular weights as measured by HPLC. This chart shows the relationship between the retention time of polysaccharides with known molecular weights measured by HPLC and their log molecular weight (MW) as a molecular weight calibration curve. This chromatogram shows the molecular weight distribution of the liquid component after culturing in Example 1, and the relationship between retention time and log molecular weight (MW) as measured by HPLC. This chromatogram shows the molecular weight distribution of the liquid component after culturing in Example 2, and the relationship between retention time and log molecular weight (MW) as measured by HPLC. This chromatogram shows the molecular weight distribution of the liquid component after culturing in Example 3, and the relationship between retention time and log molecular weight (MW) as measured by HPLC. This chromatogram shows the molecular weight distribution of the liquid component after culturing in Example 4, and the relationship between retention time and log molecular weight (MW) as measured by HPLC.

The health food material in this embodiment of the invention contains, as an essential component, an extracellular polymer substance produced by a predetermined strain of Paenibacillus polymixa cultured in a predetermined culture medium. It can be produced by preparing a fermentation medium by using starch hydrolysate, sucrose, or a mixture thereof as a culture medium base, adding a water-soluble component of yeast as a nitrogen source, inoculating and culturing Paenibacillus polymixa strain AK-T1-11, and then separating the liquid component containing the produced extracellular polymer substance to obtain a food stock solution.

The starch saccharified product used as a culture medium base is obtained by hydrolyzing plant-derived starches such as cereals, potatoes, plant roots and stems, and legumes with the digestive enzyme amylase in the final step of the decomposition process. It is particularly preferable that the starch saccharified product is a low molecular weight product obtained by hydrolyzing starches containing cornstarch with amylase.

Sucrose, as a carbon source, is a disaccharide obtained from many plants, and it is preferable to use granulated sugar, which has sucrose as its main component. In this invention, the carbon source can be supplied solely by sucrose.

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

Furthermore, it is preferable to include a pH buffer in the fermentation medium and adjust the pH to 7.0 or lower. For example, if easily metabolizable components such as glucose are used as the carbon source, acid tends to accumulate in the medium, and a decrease in pH can damage the microorganisms involved in fermentation.

Examples of pH buffering agents include dipotassium hydrogen phosphate and disodium hydrogen phosphate. Additionally, to properly adjust the osmotic pressure inside and outside the bacterial cells, appropriate amounts of sodium chloride, calcium chloride, etc., may be added as needed.

The fermenting bacteria inoculated into the fermentation medium containing the essential components mentioned above is Paenibacillus polymixa AK-T1-11 strain. Furthermore, one or more mutant strains with slightly different characteristics, such as preferred growth conditions and the ability to produce extracellular macromolecules, may be included, to the extent that they do not hinder the objective of this invention.

Based on the latest API50CH biochemical classification and the results of the complete bacterial 16S rRNA sequencing, strain Paenibacillus polymyxa AK-T1-11 has been identified as Paenibacillus polymyxa, a species of the Paenibacillus genus that was moved from the Bacillus genus in the 1993 bacterial species reclassification.

This Paenibacillus polymyxa AK-T1-11 strain efficiently grows in a culture medium based on a saccharified product or sucrose obtained by hydrolyzing corn starch with amylase, or a mixture thereof, to which a small amount of water-soluble yeast components such as yeast extract are added as a nitrogen source. Unlike ordinary bacteria, it can fix nitrogen from the atmosphere, and therefore easily grows even in culture media containing only a very small amount of nitrogen source.

This is effective in preventing the generation of putrid odors and the production of allergens. Furthermore, because it forms spores resistant to high temperatures and ultraviolet light, it can survive even in harsh environments and conditions such as high and low temperatures, making preservation easy. This property can be used to suppress the decline in 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.

The mycological characteristics of the above strain are as follows:
<Paenibacillus polymixa AK-T1-11 strain>
(a) Morphological properties
(1) Cell shape and size: Bacilli 0.6–0.8 × 3.0–50 μm, chain-forming, simple to short chains
(2) Presence or absence of cell pleomorphism: None
(3) Motility: Yes (Peritrichous flagella)
(4) Presence or absence of spores: Present, oval shape, located near the center of the bacterial body, and swollen.
(b) Culture properties
(1) Culture on meat juice agar plate: circular viscous colonies
(2) Meat juice liquid culture: bacterial cells aggregate at the top
(c) Biochemical properties
(1) Gram reaction: Indeterminate
(2) Reduction of nitrates: Positive
(3) VP test: Positive
(4) Urease: Negative
(5) Indole production: Negative
(6) Production of hydrogen sulfide: Negative
(7) Use of citric acid: Positive
(8) Catalase: Positive
(9) Oxidase: Negative
(10) Growing range: pH 5.5–7.6, temperature 10°C–45°C

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

The preferred first-stage culture conditions used in this invention vary in detail depending on the bacterial strain, but are generally a pH of 5.5 to 7.2 and a temperature of 28 to 32°C for more than one week but less than two months. Under conditions with a stronger acidity than those described above and a temperature below the specified temperature, even after culturing for more than two months, it is estimated that the specified bacterial strain used in this invention will not efficiently utilize sugars and nitrogen sources, and the expected effects of the resulting food product will not be fully obtained. Furthermore, under alkaline conditions exceeding the above pH range and high temperatures exceeding the specified temperature, cultivation will be insufficient, and the specified bacteria will not efficiently utilize sugars and nitrogen sources, resulting in a functional food product that will not fully achieve the expected effects, similar to the above.

The preferred second-stage low-temperature culture conditions used in this invention are a pH of 4.0 to 6.0, a culture temperature of 13 to 17°C, preferably 8°C or higher and less than 13°C, and a culture period of one week or more and less than four months.

Under conditions of a stronger acidity range and below the specified temperature, even after culturing for more than four months, it is estimated that various active amino acids, lipopolysaccharides, lipids, etc., will not be sufficiently reduced in molecular weight as fermentation products, and the desired effects will not be fully obtained in the resulting food product.

Furthermore, if cultured at temperatures exceeding the specified temperature under neutral or alkaline conditions beyond the above-mentioned acidic range, the activity is presumed to decrease, and the resulting functional food product will not fully achieve the intended effect, similar to the above.

Thus, the preferred culture method used in this invention involves, as a first step, culturing at a pH of 5.5 to 7.2 and a temperature of 28 to 32°C for at least one week, preferably less than two months, followed by a second step of culturing at a pH of 4.0 to 6.0 and a temperature of 8°C to less than 13°C for at least one week, preferably less than four months.

To separate the generated liquid components, a continuous centrifuge is used. The resulting food-grade stock solution can be used as is, but it can also be concentrated or diluted as needed.

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

Furthermore, in addition to this qualitative analysis, quantitative analysis can be performed by treating the sample with ethanol precipitation, then hydrolyzing it with dilute sulfuric acid to decompose it into its constituent sugars, and finally quantifying the constituent sugars using HPLC.

<Preparation of culture medium>
Fermentation media were prepared according to the following procedure using the proportions shown in Table 1 (all "parts" below refer to parts by mass).

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

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

After dissolving the rice syrup, the pH was adjusted by adding purified water up to 100 parts while simultaneously adding 0.2 parts of 10% sodium hydroxide.

pH and Brix were measured and confirmed to be within the specified limits. If within the limits (pH 7.2–7.6, Brix 4.5–4.8), the sample was dispensed into culture vessels, sterilized by autoclaving (121°C, 15 minutes), and then rapidly cooled by water cooling. The pH limit was set assuming that the culture medium pH would quickly reach 5.5–7.2 after incubation.

[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 stirred until dissolved.
Next, the pH of the culture medium was adjusted by adding purified water up to 100 parts while simultaneously adding 0.2 parts of 10% sodium hydroxide.

pH and Brix were measured and confirmed to be within the specified limits. If within the limits (pH 7 or less, Brix 4.5–4.8), the solution was dispensed into culture vessels, sterilized by autoclaving (121°C, 15 minutes), and then rapidly cooled by water cooling.

<Inoculation and cultivation of AK bacteria>
The AK-T1-11 strain, which had been stored at -80°C in spore form adsorbed onto strain preservation beads, was subjected to a heat treatment at 85°C for 10 minutes (a treatment to promote spore germination) immediately before inoculation into the culture medium. Then, one bead per 500 mL was added to 200 mL of enrichment medium placed in a 500 mL container of corvette, and incubated overnight in a rotary shaking incubator at 30°C and 150 rpm.

Next, after the shaking culture is complete, the culture medium is immediately aseptically inoculated into 100 mL each of stainless steel culture containers containing 30 L of fresh liquid medium, and then left to stand in a 30°C constant temperature room for 1 to 2 months.
Stage-by-stage culturing was performed.

After the prescribed incubation period, the culture vessels were moved to a constant temperature room at 8-10°C, where a second stage of low-temperature incubation was performed for 2-4 months.

<Measurement of molecular weight distribution>
HPLC was performed on the culture medium after the low-temperature incubation described above, using Shimadzu products including a liquid transfer pump (LC-20AD), system controller (CBM-20ALite), degassing unit (DGU-20A3R), column thermostat (CTO-20A), UV-VIS detector (SPD-20A), differential refractometer detector (RID-20A), and LC workstation (Lab solution single).

Size exclusion chromatography was performed using a linked Shodex Sugar KS-803 (φ8 mm × 300 mm) and KS-804 (φ8 mm × 300 mm) column to measure the molecular weight distribution of sugars. The procedure involved injecting 20 μL of solution into a column at an 80°C constant temperature bath, using water as the mobile phase, a flow rate of 1.25 mL/min, and a differential refractometer detector.

The molecular weights of unknown components were determined by first measuring the molecular weights of components with known molecular weights and creating a molecular weight calibration curve equation based on the relationship between retention time (elution time) and molecular weight. The components with known molecular weights were glucose (molecular weight: MW180), sucrose (MW342), and pullulan (MW6,100, MW9,600, MW22,000, MW47,100, MW107,000, MW194,000, MW337,000), a type of polysaccharide composed solely of glucose. The chromatograms obtained at a concentration of 1,000 mg/L are shown in Figure 1.

The molecular weight calibration curve equation was calculated using the least squares method, with the retention times of the nine components as explanatory variables and the logarithm of the molecular weight as the dependent variable. The molecular weight calibration curve is shown in Figure 2, and the equation, calculated in the same manner as described above, is as follows:

Molecular weight calibration curve equation: y = -0.4636x + 9.2797
Contribution rate: 0.9964

The molecular weight distribution of the culture media from Examples 1-4, collected after the start of cultivation, was measured using the HPLC method described above, and the results are shown in Figure 3-6.
Furthermore, polysaccharides contained in the culture medium after the low-temperature culture were separated by ethanol precipitation treatment, decomposed into monosaccharides by hydrolysis, and then measured by HPLC, which detected fructose. This determined that the polysaccharides contained in the Enzamin stock solution are levan, which forms a polymer in which fructose is linked by glycosidic bonds.
Furthermore, when the water-insoluble polysaccharides contained in the culture medium sediment after the low-temperature culture were separated and decomposed into monosaccharides by hydrolysis, and then measured by HPLC, they were determined to be curdlan, which is a β-1,3-glucan in which glucose is linked by glycosidic bonds.

Thus, two types of polysaccharides were identified, and as is clear from the results shown in Figures 3-6, it was confirmed that the disaccharide (sucrose) was broken down into monosaccharides after culturing, and that oligosaccharides and two types of polysaccharides (levan and curdlan) were newly produced.

Comparative Example

Then, sodium hydroxide was added to adjust the pH to within the range of 7.3 to 7.8, and this was placed in a culture vessel and autoclaved at 120°C for 20 minutes. After cooling, Paenibacillus polymixa (the conventionally known Bacillus subtilis AK strain: FERM P-18291) was inoculated, and fermentation was carried out for 60 days at a constant temperature of 30±2°C with a pH of 4.5 to 6.5. Subsequently, the culture solution was matured for 180 days at a constant temperature of 15±2°C with a pH of 4.0 to 6.0 to make it clear. This was filtered to obtain 54.3 parts of a liquid health food stock solution (hereinafter referred to as the culture filtrate).

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

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

In contrast, in Examples 1-4 described above, since the Paenibacillus polymixa AK-T1-11 strain was used, significant polysaccharide production was observed, with levan production at approximately log(MW) 5.5 and curdlan production at approximately log(MW) 6.0.

The production of levan and curdlan increased significantly with increasing amounts of sucrose (granulated sugar) added to the culture medium. Furthermore, as in Examples 3 and 4, using a fermentation medium containing a pH buffer and adjusted to a pH of 7.2 or lower, for example, pH 7.0, delayed the transition to an acidic range during cultivation. This allowed for sufficient cultivation time in the first stage, resulting in a further increase in production.

Tables 3-6 show examples of formulations for health drinks, capsule-type health foods, lotions, and animal and plant nutrients manufactured using the obtained health and nutritional food concentrate.

The health beverages were prepared by blending 2-50% of the culture filtrate with other ingredients as shown in Table 3.

The capsule-type health food was prepared by concentrating the culture filtrate 20 times and adding other ingredients as shown in Table 4.

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

The plant and animal nutrient solution was prepared by mixing 20% of the culture filtrate with the other ingredients shown in Table 6.

1) Receiving organization: National Institute of Technology and Evaluation, Patent Microbial Depository Center (NPMD)
2) Date of receipt: December 24, 2021 3) Receipt number: NITE ABP-03577

Claims (3)

A method for producing a health food ingredient, comprising: preparing a fermentation medium by using starch hydrolysate, sucrose, or a mixture thereof as a culture medium base, adding water-soluble components of yeast as a nitrogen source; inoculating this medium with Paenibacillus polymixa strain AK-T1-11 (receipt number: NITE ABP-03577) and culturing it; and separating the resulting liquid component containing extracellular polymer substances to obtain a food stock solution. The method for producing a health food ingredient according to claim 1 , wherein the starch saccharified product is a starch saccharified product obtained by hydrolyzing starch containing corn starch with amylase. A method for producing a health food material according to claim 1 or 2 , wherein the culture is performed in a first stage of culturing at a pH of 5.5 to 7.2 and a temperature of 28 to 32°C for one week or more, and in a second stage of culturing at a pH of 4.0 to 6.0 and a temperature of 8°C or higher and less than 13°C for one week or more.