JP2008005702A - Bacillus macroides bacterium and food using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find a component most contributing to the prevention and treatment of diseases, the prevention of aging, the enhancement in the immunizing power and so on, and to provide a novel health food or beverage using this component. <P>SOLUTION: The present invention provides Bacillus macroides Excel 001 (NITE BP-29) strain which forms whitish brown colonies with irregularly shaped edges on the LB (Luria-Bertani) agar medium and spores of this strain formed in the conditions with insufficient nourishment. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to Bacillus macroides bacteria and foods using the same. More specifically, it belongs to the technical field of Bacillus macroides, which can be used as a health food for the purpose of disease prevention, aging prevention, immunity improvement and the like.

Conventionally, various health foods and beverages have been proposed for the purpose of disease prevention, aging prevention, immunity improvement and the like.
For example, (Patent Document 1) discloses a method for producing an agaricus blazei murrill mycelium extract. Specifically, as shown in the example of claim 1, the specific production procedure is as follows. (A) Using a culture medium based on water, the mycelium of Agaricus blazei muryl can be cultured. Culturing 30 to 150 g of Agaricus mycelium per liter of culture medium on a medium at 0 to 25 ° C. for at least 1 week, (b) at least 30 ° C. over 1 to 4 days at a pressure of 1 to 10 atm. Separating the mycelium of Agaricus blazei murrill from the above medium, and (c) raising the liquid temperature to 50 ° C. or more at a stretch while continuing aeration under the same conditions as in step (b) for 1 to 4 hours After being kept, it is allowed to cool to room temperature, kept at this temperature for 1 to 15 hours, and then subjected to the first filtration, (d) left at a temperature of 15 to 30 ° C. for 1 to 4 hours to form a culture system After breeding existing bacteria, Incubate for 1 to 5 days at room temperature while continuing aeration under the same conditions as in (b) to propagate the mycelium of Agaricus blazei and Muryl, stop aeration and hold for 12 to 36 hours, and The step of performing the second filtration while aeration is performed under the same conditions as in step (b), (e) after incubating at room temperature for 2 to 7 hours while continuing the aeration under the same conditions as in step (b), The temperature of the culture system is raised to at least 60 ° C. and maintained at this temperature for 30 minutes to 4 hours to kill the propagated germs, allowed to cool to room temperature, incubated at this temperature for 1 to 6 hours, and final filtration (F) boiling the culture system for at least one day, allowing it to cool to room temperature, and incubating at this temperature for 1 to 6 hours, (g) room temperature while performing aeration under the above conditions Incubate with agaricus, brazei and murrill mycelium The target agaricus brazei muryl hyphae through a step of adjusting the concentration, (h) a stage of stopping aeration and incubating, and (i) a stage of lowering the aeration pressure as much as possible and incubating at room temperature It is said that body extract can be obtained.

The above-mentioned extract is currently manufactured and sold under the trade name “Yamato Shinto” by the agricultural union corporation Yamato Shingo Production Association, and actually has preventive and therapeutic effects on a number of diseases such as cancer, liver disease and diabetes. It has been reported and has been well received.
However, what ingredients in “Yamato Shrine” are most effective in preventing and treating diseases, preventing aging, improving immunity, etc., and the ingredients and disease prevention and treatment effects The cause-and-effect relationship with the others has not been clarified yet.

Japanese Patent No. 2887756

  Therefore, in view of the above-described conventional situation, the present invention finds a component that contributes most to prevention / treatment of diseases, prevention of aging, improvement of immunity, etc. by analyzing “Yamato Shrine” and its components. The purpose is to provide new health foods and beverages that use food.

  In order to solve the above problems, the present inventor conducted intensive studies, and as a result, the same white-brown colony was found in all batches in the culture of “Yamato Shrine”. This colony was identified as Bacillus macroides from the base sequence of the rRNA gene, thereby completing the present invention. That is, this invention is comprised from the following (1)-(4).

(1) A Bacillus macroides Excel001 (NITE P-29) strain that forms white-brown colonies with irregular edges on an LB (Luria-Bertani) agar medium.
(2) A Bacillus macroides Excel001 (NITE P-29) strain in which the 16S rRNA gene has the base sequence shown in SEQ ID NO: 1.
(3) The Bacillus macroides Excell001 (NITE P-29) strain described in (1) or (2) above, which is further spored by being in a nutrient-deficient state.
(4) A food comprising a culture of the Bacillus macroides strain according to any one of (1) to (3) above.

By using the Bacillus macroeides according to the present invention, it is possible to obtain a health food / beverage having features such as disease prevention / treatment action, anti-aging action, or immunity improvement action.
Furthermore, the effect of the Bacillus macroeidobacterium can be further enhanced by allowing the Bacillus macroeides to sprout in a nutrient-deficient state. The reason for this is not clear, but it is presumed that spore formation may affect the intestinal immunity by reaching the intestinal tract without being affected by digestive enzymes and sprouting there.

Hereinafter, embodiments of the present invention will be described in detail.
The strain according to the present invention has been found on the occasion of analysis of “Yamato Shrine” manufactured and sold as an agaricus mycelium extract.

First, “Yamato Shrine” is placed in an appropriately sized Erlenmeyer flask, sealed, and sterilized by autoclaving at 95 ° C. for about 5 minutes. Separately, a sterile petri dish containing LB agar medium is prepared. On this LB agar medium, Yamato Shrine cooled after autoclaving was placed and cultured for 16 hours in a 37 ° C. incubator. As a result, a white-brown colony with irregular edges was formed in common with all the batches, and this colony was isolated, and the bacteria were identified as follows.

  That is, genomic DNA was extracted from the isolated colony, and the base sequence was determined after amplification of about 500 bp of the 16S rRNA gene by PCR using the extracted genomic DNA as a template. The base sequence is as shown in SEQ ID NO: 1. Since this rRNA gene sequence showed 99% or more homology with the Bacillus macroides rRNA gene, it was named Bacillus macroides Excel001 strain from the name of the applicant. A deposit application was filed with the Microorganism Deposit Center on October 13, 2004, and the deposit number (NITE P-29) was notified on November 1, 2004.

  The above-mentioned Bacillus macroides (NITE P-29) strain forms white-brown colonies with irregular edges on the LB agar medium as described above. In addition, the central part of the colony becomes dark brown after long-term storage at room temperature, and the color of the medium around the colony becomes dark brown.

  Cultures of this strain, for example, are suspended in a liquid to prepare a health drink, and by taking an appropriate amount, various excellent effects such as disease prevention / treatment action, anti-aging action, and immunity improvement are obtained. be able to. The target diseases are wide-ranging, and include, for example, cancer, liver disease, diabetes, malignant lymphoma, skin disease such as Basedor's disease, atopic dermatitis, impotence, hypertension, etc. is not. Although its mechanism of action is unknown, it is presumed to have some effect on the patient's autoimmune function. Moreover, it may be taken by healthy people and is effective for maintaining health.

When preparing a beverage containing a culture of the Bacillus macroides (NITE P-29) strain, the concentration of the bacterium can be appropriately set according to the symptoms of the patient. For example, 1 × 10 ^{8 to} 5 × 10 ⁸ / ml per day is appropriate. Moreover, the form of food may be added to yogurt or the like as well as beverages, or powdered dry cells may be used as a health food as they are. Alternatively, it may be tableted for convenient consumption. In tableting, excipients such as lactose can be used as appropriate.

  Furthermore, it has been found that the Bacillus macroides (NITE P-29) strain of the present invention has a spore-forming effect such as an improvement in immunity when ingested as a food / beverage. Spore means a state in which a spore composed of a layer structure such as core, cortex, spore coat, etc. is formed in a microbial cell due to lack of nutrient sources, and is resistant to heat, drying, radiation, acid, alkali, etc. Has resistance. Moreover, although the strain which concerns on this invention maintains a strong dormant state when it makes it spore, it confirmed that it germinated immediately by adjusting culture conditions, and restarted proliferation division.

  As mentioned above, the reason why the effect is increased by spore formation is not clear, but in one case, when it is ingested by the human body, it reaches the intestinal tract without being affected by digestive enzymes and germinates there. Therefore, it is presumed that this is likely to affect intestinal immunity.

  The method for spore formation is not particularly limited, and the target spore can be collected by culturing a Bacillus macroides (NITE P-29) strain in an appropriate medium lacking nutrient sources. Specifically, for example, a neutral or alkaline medium (such as a nutrient medium, yeast extract / albumin medium, or peptone medium) containing Mg ions, copper ions, and the like is aerobic at a temperature of about 20 to 30 ° C. for several days. And a method of culturing the strain until the carbon source is exhausted in a medium containing 1 to 5% by weight of corn steep liquor and a carbon source, and then aeration for several tens of hours. . The aerobic culture is performed by shaking culture using a shaker or by stirring while feeding air when using a jar fermenter or the like.

  The sporulated Bacillus macroides (NITE P-29) strain can be used in combination with a strain that does not sporulate.

(Example 1) Preparation of beverage containing spores of Bacillus macroides (NITE P-29) (Purpose) A low-nutrient medium, MCM starvation buffer, was used to produce Bacillus macroides. The (NITE P-29) strain is sporulated to produce a beverage level spore water.

(Method) MCM starvation buffer (10 mM MOPS, 2 mM CaCl ₂ , 4 mM MgSO ₄ pH 7.5 (adjusted with NaOH)) was prepared using a commercially available Volvic water (mineral water), and filtered with a 0.25 μm filter. did.

  Subsequently, the Bacillus macroides (NITE P-29) strain was cultured overnight in 50 ml of LB medium (Gibco-BRL), and centrifuged at 3000 rpm for 10 min to prepare a pellet. Then, it was washed twice with 50 ml of MCM starvation buffer and replaced with 50 ml of MCM starvation buffer. It was shaken at 30 ° C. for 2 days, centrifuged at 3000 rpm for 10 min, the pellet was washed 3 times with 50 ml of Volvic water, and the pellet was replaced with 50 ml of Volvic water.

  All steps were performed aseptically, and all containers such as Erlenmeyer flasks were sterilized commercially available. Moreover, a part of sample was collect | recovered at each stage, such as washing | cleaning, and it seed | inoculated to LB culture medium and confirmed loss etc.

(Results and Discussion) Spore water of Bacillus macroides (NITE P-29) strain was prepared at the beverage level. As a result, we succeeded in producing spore water containing 1.14 × 10 ⁴ / μl in total of viable bacteria and spores. Spores were confirmed by culturing on LB plates after heat treatment at 95 ° C. for 5 minutes. In addition, it became clear that some bacteria (spores) were lost during the washing | cleaning by Volvic water. As a result of inoculating the supernatant stored at each centrifugation stage on an LB plate, it was found that viable bacteria and spores could not be completely pelleted under centrifugation conditions of 3000 rpm × 10 min. There is still room for improvement in the number of revolutions and time of centrifugation.

When the prepared spore water was sampled by 5 persons (corresponding to 5.7 × 10 ⁷ viable bacteria and spores) by 2 persons including the present inventor, no abdominal pain was observed, and atopic dermatitis tended to improve. Indicated.

  Next, in consideration of diluting the spore water and drinking it for a long time, the long-term storage stability of the spore in Volvic water was examined. Specifically, the number of viable bacteria and spores on the 4th, 12th, and 31st days of spore water was examined. As a result, 670 / μl and 154 / μl of spores were detected on the 12th and 31st days of spores, respectively. Although a decrease in the number of spores was observed, it was revealed that a sufficient amount of spores were maintained over a long period of time. Therefore, it is very advantageous when making a product.

(Example 2) Determination of optimum conditions for producing spore water of Bacillus macroides (NITE P-29) strain (Purpose) Most efficient in producing spore water of Bacillus macroides (NITE P-29) strain Of good sporulation conditions and spore recovery method.

(Method) Basically, a beverage level spore water was prepared in the same manner as in Example 1. The changes are as follows. The change in the number of spores was examined by shaking in MCM starvation buffer for 10 days. In addition, the number of rotations at the time of washing with Volvic water was 10,000 rpm and 15000 rpm, and the amount of spores remaining in the supernatant was examined.

  After shaking with MCM starvation buffer for 10 days, the supernatant was collected by centrifugation at 2000 rpm × 10 min. 1 ml of this supernatant was centrifuged at 10,000 rpm × 10 min, and 100 μl of the supernatant was recovered. The remaining supernatant and pellet were centrifuged again at 15000 rpm × 10 min, and 100 μl of the supernatant was recovered. All remaining supernatant was removed and the pellet was resuspended with 1 ml of Volvic water. After centrifugation at 15000 rpm × 10 min, the operation of removing the supernatant and resuspending again with 1 ml of Volvic water was repeated twice. Finally, it was suspended in 1 ml of Volvic water and seeded on an LB plate.

(Results and Discussion) First, when the number of spores immediately after MCM starvation buffer replacement was examined (negative control), no spore formation was observed, and it was found that no spore formation occurred in a high nutrient LB medium. .
Next, the number of spores on the second, fourth, sixth, eighth and tenth days after MCM starvation buffer replacement was measured. As a result, the number of spores on day 6 was the largest, and almost no spores could be detected after day 8. From this result, it was found that long-term shaking with MCM starvation buffer resulted in a decrease in the number of spores.

  In addition, since it was revealed in Example 1 that spores were lost during washing or the like, optimal centrifugation conditions for producing spore pellets were determined. As a result, it was revealed that most of the spores remained in the supernatant at 2000 rpm. Using this, it is possible to remove (reduce) pellets of live and dead bacteria in the MCM starvation buffer by centrifuging at 2000 rpm for 10 minutes.

Claims (4)

  A Bacillus macroides Excel001 (NITE P-29) strain that forms white-brown colonies with irregular edges on LB agar.   The Bacillus macroides Excel001 (NITE P-29) strain, wherein the 16S rRNA gene has the base sequence shown in SEQ ID NO: 1.   The Bacillus macroides Excell 001 (NITE P-29) strain according to claim 1 or 2, which is further sporulated by being in a nutrient-deficient state. The foodstuff characterized by including the culture | cultivation of the Bacillus macroeides strain in any one of Claims 1-3.