JPH03297358A - Preparation of fermented soybean having low vitamin k content - Google Patents

Abstract

PURPOSE:To prepare the subject fermented soybean useful for patients required to reduce the ingestion of vitamin K, such as thrombotic patients, by employing a Bacillus natto variant A-1 having low vitamin K productivity. CONSTITUTION:When fermented soybean is prepared, a Bacillus.natto variant A-1 (Bacillus sp.A-1) (FERM p-11344) having low vitamin K productivity is employed.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing natto with a low vitamin K content using a mutant strain of Bacillus natto with a low vitamin-producing ability.

[Conventional technology]

Bacillus natto is a type of Bacillus subtilis and has a very strong vitamin-producing effect, reaching 12 to 13 times that of E. coli, which is generally said to have a strong vitamin-producing effect. Therefore, there is a large amount of vitamin K in natto, 6000-8000n.
g/g natto), which is much higher than green vegetables and seaweed, which usually contain a lot of vitamin K. Therefore, eating natto is generally recommended from a nutritional standpoint.

[Problem to be solved by the invention]

However, since vitamins are also blood clotting factors,
For example, it is generally recommended that patients taking anticoagulant therapy to prevent thrombosis after surgery or people at risk of thrombosis refrain from eating natto.

Therefore, if natto with low vitamin content can be provided, general consumers and people suffering from such symptoms can eat it with peace of mind, and this will bring great industrial benefits. .

Therefore, an object of the present invention is to provide a new method for producing natto with a low vitamin content.

[Means to solve the problem]

The present inventors believe that since soybeans, which are the raw material for natto, have very low vitamin content, if we develop natto bacteria with low vitamin production ability, we will be able to produce natto with low vitamin content. I focused on the idea that it might be possible to do something,
After extensive research, we discovered that a mutant strain obtained by mutating conventional commercially available Bacillus natto bacteria is a bacterium that can achieve the desired purpose, leading to the completion of the present invention.

The present invention uses Bacillus natto mutant strain A-1 (Bacillus sp.), which has low vitamin production, when producing natto.
A-1) A method for producing natto with a low vitamin content is provided.

The present invention will be explained in detail below.

I. Means for creating mutations Mutations are induced by treating conventional MII fluid of commercially available vegetative cells of Bacillus natto from which spores have been removed with N-methyl-N'-nitro-N-nitrosoguanidine. This suspension is appropriately diluted, plated on a Triptose Blood agar medium containing 20 Hg/m+ beromomycin and kanamycin, and cultured overnight at 37°C. The resulting colonies were treated with 5.0 Hg/ml Menadi.
on Sodium B15ul-"Transfer to a Triptose Blood agar medium containing iLe (Md) and the same medium without it, and after culturing overnight at 37°C, colonies with increased growth due to Md are isolated as mutant strains. In this way, Using the obtained mutant strain, natto is produced by a conventional method, and the one that produces normal natto is selected, and the vitamin content of this natto is adjusted.

(b) Identification of mutants As shown in the Examples below, mutant strain A can produce high-quality natto by conventional methods and produce natto with aML of vitamins less than 1/3 of normal natto. -1 was obtained by the method described above. At that time, the content of vitamins is measured by
This was done by culturing the bacteria in a liquid solution using 2% soybean broth or solid culture using boiled beans, and then determining the amount of vitamins produced in or on the culture medium. This mutant strain A-
It was found that the vitamin content of Bacillus 1 was less than 2,000 ng/g of natto, which is less than 1/3 of that of conventional natto bacteria.

Other mycological properties of this strain are those of commercially available Bacillus natto [
Food Research Institute Report (Rep, Natl, Food Res,
In5t.

No, 50. '1g-21, 1987) and Soybean Monthly Report, December issue, 21-29 (1985).

That is, this mutant strain A-1 is an aerobic, Durham stain-positive spore-forming rod, and the size of the bacterium (1 x 2 to 3 μ),
Bergey's characteristics such as colony shape, suitable growth temperature (35-45℃), fermentability of various sugars, and GC content of DNA
Manual 8th edition Bacillus subtilis (Bacillus 5
It belongs to the so-called Bacillus natto because it has properties consistent with those of Bacillus natto (ubt+Ius), produces mucilage, and requires biotin. This mutant strain A-1 (Bacillus sp.
, A-1) was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on March 6, 1990, as Microtechnology Research Institute Deposit No. 11344.

■ Natto production and effect test Results of vitamin K in natto prepared using commercially available Bacillus natto and mutant strain A-1 of the present invention according to conventional methods, extracted with hexane, and determined by high performance liquid chromatography. In contrast, natto prepared using commercially available Bacillus natto contained 6540 ng/g of vitamin K, whereas the vitamin content of natto prepared using mutant strain A-1 was 1940 ng/f. No difference was observed between the two in terms of natto properties such as stringiness, stiffness, and taste.

〔Effect of the invention〕

As described above, the natto obtained by the method of the present invention has a vitamin content of 1/3 or less than that of normal natto, which is lower than that of seaweed,
It has almost the same level of 8H as the vitamin contained in seaweed such as wakame. Therefore, general consumers, such as those with thrombosis, who need to limit their intake of radiotherapy and vitamins, can safely consume it.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example A strain that produces high-quality natto was isolated from commercially available Bacillus natto (manufactured by Miyagino Natto Seisakusho) and used as a parent strain. This parent strain was inoculated into a 2% soybean broth medium (PH6, 8) and cultured with shaking at 37°C for 90 minutes. Collect bacterial cells by centrifugation, 300μg
/ml of N-methyl N'-nitro-N-nitrosoguanidine in a trismaleate buffer solution containing 3
Mutation treatment was performed by allowing the mixture to stand at 7°C for 20 minutes.

Next, this kake 7Ij! After collecting and washing the bacterial cells from the J solution on a membrane filter using a filter filtration device, Tr
The cells were suspended in ipLose Blood medium and cultured with shaking at 37°C for 5 hours to express the mutant trait. This culture solution was appropriately diluted, plated on TripLose Blood agar medium containing 2.0 μg/ml of paromomycin and kanamycin, and cultured overnight at 37°C. The obtained colonies were treated with 5.0 μg/ml Menadion Sod.
Transferred to Triptose Blood agar medium containing ium B15ulf'ite (+'vld) and the same medium without it, and cultured overnight at 37°C. Strains with increased colony growth were isolated as mutant strains using Md. and stored in a test tube slant (296 soybean broth agar medium).

Take 1 platinum loop of spores from a test tube slant and add 200ml
The suspension was suspended in 1% sterilized saline and sprayed onto hot steamed soybeans at about 80°C. The steamed soybeans are placed in a small Styrofoam container and the soybeans are covered with a thin polyethylene film.
The mixture was covered with a lid and fermented at 40°C for 16 hours. After that, it was refrigerated in a refrigerator at 5°C for one day, and then vitamin K was extracted from the natto using hexane, and SEP-PAKSi I
It was purified using an ica cartridge, and its content in vitamins was measured by absorbance at 248 nm using high performance liquid chromatography. The vitamin content of natto made from the parent strain, commercially available Bacillus natto, was 6540 ng/g, while the natto that showed the lowest vitamin content was 1940 ng/g, which was less than 30%. The quality of this natto, such as stringiness, aroma, and taste, was completely comparable to that of the parent strain. The natto bacteria that produces No. 2 natto was named mutant strain A-1.

Claims (1)

[Claims] 1. When producing natto, Bacillus natto mutant strain A-1 (Bacillus sp.
) A method for producing natto with a low vitamin K content. 2. Bacillus natto mutant strain A-1 (B) with low vitamin K production
acillus sp. A-1) Microtechnical Research Institute No. 11344
issue.