JPH0265749A - Fermented food - Google Patents

Abstract

PURPOSE:To obtain a fermented food having flavor, excellent digestion and shelf stability and high nutritive value free from stringiness by fermenting a protein-containing solid food with specific Bacillus natto HOS80. CONSTITUTION:A protein-containing solid food (preferably beans, cereals, grains, potatoes, fruits, mushrooms and algae) is fermented with Bacillus natto HOS80 which belongs to the genus Bacillus subtilis, has >=100 units of gamma-GTPase activity and provides aged protein having substantially no stringiness to give the aimed food.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to fermented foods, and particularly to a novel fermented food that is fermented by a specific microorganism and has substantially no stringiness.

[Conventional technology and issues to be solved]

Aged natto is an excellent product that has the advantages of being not only highly digestible and nutritious due to the moderate decomposition of high-quality vegetable protein, but also delicious, inexpensive, and long-lasting. It is a balanced food. Moreover, recently it has been discovered that natto contains large amounts of fibrinolytic enzymes, which can be used to prevent and treat cerebral thrombosis, and the value of natto has been reconsidered.

However, since natto has a unique stringy property, when eating natto, the sticky substance may stick to the lips or tableware and cannot be easily removed, causing discomfort or disgust to people. In addition, after ripening natto, it is difficult to handle, post-processing is not possible, and its stringy properties limit the spread of natto or limit its use as a side food.

Therefore, research was conducted on the stringiness of natto, and a strain of natto bacteria that does not have stringiness was developed. In other words, Professor Hara of Bonyo University extracted a plasmid from Bacillus natto and discovered that natto produced using Bacillus natto without the plasmid did not become stringy.

Specifically, by extracting a plasmid from Bacillus natto, the 1-GTPase (glutamyl transpeptidase gluLamyl transp) of Bacillus natto was extracted.
eptidase) activity is approximately 0, Bacillus natto (hereinafter referred to as HO5-0 strain) with a γ-CTPase activity of approximately 0 is obtained from a Bacillus natto mutant strain obtained by mutating the known nattotsuki. was selected and cultured, and the natto produced using the HOS-0 strain had almost no stringiness.

However, natto produced using the HOS-0 strain had a problem in that almost no hydrophobic amino acid (tyrosine) was precipitated and a strong bitter taste remained. Therefore, it is necessary to add salt to the produced natto to remove its bitter taste, which limits its use.

The present inventor has been conducting research on the stringiness of Bacillus natto for many years, and as a result, the r GTPase activity was 10
Despite having 0 or more units, they discovered a natto bacterium that has virtually no stringiness in aged natto and has very little bitterness, leading to the present invention.

[Means to solve the problem]

The gist of the fermented food according to the present invention is that the solid food containing protein is prepared from Bacillus subtilis (8uci
llus 5ubLilis), 7-GTPa
The reason for this is that it is fermented using Bacillus natto HOS 80, which has a se activity of 100 units or more and the resulting protein is substantially non-stringy.

[For production]

Here, "having substantially no stringiness" means, for example, drying, 21! This means that Jukusei protein in its unprocessed state, which has not been subjected to any means such as drying, drying, or powder mixing, does not have the stringiness characteristic of Seiyu protein. Also, ``aging'' means ■Protein is broken down and flavor comes out. An increase in peptides is seen. ■In the later stage, crystals of hydrophobic amino acid (tyrosine), which is an amino acid produced by decomposition, are formed. ■Describes the condition of becoming soft.

Bacillus natto HO580 used to produce the fermented food according to the present invention is produced from a Bacillus natto mutant strain obtained by mutating a known Bacillus natto strain.

Examples of Bacillus natto used to obtain mutant strains include Bacillus subtilis.
Biotin belonging to Bacillus subtilis
Any known auxotrophic Bacillus natto can be used as a parent strain. Specific examples include Miyagino natto bacteria,
Examples include Takahashi bacteria, Asahi bacteria, Matsumura bacteria, and Naruse bacteria.

Methods of mutation include, for example, a method of contacting with a mutagen, a method of genetic manipulation, and an X-ray method.

Any known method can be employed, such as a method of irradiating with ultraviolet rays, light, etc.

According to a method of contacting a suitable mutagen as a mutation method, a known parent strain, Bacillus natto, is cultured in a nutrient medium containing a mutagen, and the resulting mutant strain produces y-GTP.
Strains with ase activity of 100 units or more are screened. Fermented foods, such as natto, are produced using the mutant strain with the obtained γ-GTPase activity of 10 (position 1 or higher).
Among them, strains are selected that can produce natto that has no flavor and is substantially non-stringy.

Here, in order to precipitate tyrosine in the produced fermented food and obtain a fermented food with less bitterness, it is necessary to use one of the mutant strains.
- The parent strain is selected so that the GTPase activity is 100 units or more, preferably 20 (1st place or more).

In addition, any known mutagen can be used, such as acridine orange, N-methyl-N'~
Mention may be made of agents such as nitro-N-nitrosoguanidine and dimethyl sulfate.

Furthermore, the contact concentration of the mutant na varies depending on the mutagen used and is not particularly limited, but may be the same as that normally used when such operations are performed. For example, acridine orange usually contains 1 to 200 mcg (microdulum)/l.
It may be about a1.

Any known nutrient medium can be used, such as meat extract, peptone, calf plasma, agar, gelatin,
Examples include a medium to which salt is added, a meat juice medium, a meat juice agar medium, a meat juice gelatin medium, lidmus milk, a MEM medium, and the like.

Culture is always carried out using known methods such as static culture and shaking culture.
The culture time may be approximately 1 to 5 days at a temperature of ~45°C.

Specific examples of mutant strains obtained in this way include:
Bucillus 5ubLilis HOS 80
(The 10th microbiological research institute at the Institute of Microbial Technology, Agency of Industrial Science and Technology)
It has been deposited under the accession number No. 195 (FERM P-10195). Hereinafter referred to as "rHO580 strain". ) can be mentioned. The HOSIIO strain is a mutant strain obtained by mutating Miyagino natto bacteria using Miyagino natto bacteria as a parent strain.

The mycological properties of strain 80380 are shown.

(a) Shape, deaf shape; rod-like size; 2.3 to 3.5 X O, 7 to 0.9 μm Presence or absence of spores; size of spores; 0.8
Shape of spores; presence or absence of ellipsoidal sporangia expansion; no spores; center Durham staining; positive (b) Growth status on ordinary agar medium (cultured at 25'C for 25 hours) Shape; annular surface Rough, wrinkled periphery; Wavy hue; Opaque, cream color (c) Condition of gelatin puncture culture growth; + Liquefaction; Laminar (d) Growth on anaerobic agar medium; (e) Sabouraud F ! Presence or absence of growth in Tong culture medium: + (f) Presence or absence of growth in lidmus milk: Assimilated.

Decomposes casein without coagulation (g) Physiological properties Catalase;+ Oxidase;+ Hydrolysis of starch;+ Hydrolysis of gelatin;+ Lysine decarboxylase; Arginine dihydrase; Ornithine decarpoquinlase; Generation of indole Nitrate reduction;+ Nisculin;+ Urease;- Use of citric acid;+ Phenylalanine deaminase; Yolk reaction; Growth range: 55°C 50°C; +.

20°C: +.

7°C 5°C; Production of gas from glucose; Production of acetoin: + Presence or absence of acid production from the following sugars Glucose; + Xylose; + Fructose; + Mannitol; + Maltose; + Nucrose; + Galactose; - Lactose ;± β-galactosidase; + biotin requirement; + growth in the presence of 5% sodium chloride; + growth in the presence of 7% sodium chloride; + lysozyme o, oot
Growth in the presence of χ;+Growth in the presence of azide 0°02χ;-The orchidistic properties of the above-mentioned mutant strain are similar to those of the parent strain Miyagino Natto Bacillus, except that the cell and spore sizes are slightly different. Although it is a failure, strain 80380 can be clearly distinguished from the parent strain Miyagino Natto in that it can produce natto that is substantially free of stringiness.

Fermented foods were produced using the obtained Bacillus natto HOS 80 according to a known method. First, a solid food containing protein is washed and then immersed in water to swell the solid food to about 1.5 to 3.0 times, preferably 2.0 to 2.5 times. Next, the solid food is heated with pressurized steam at a pressure of 1
．． 5-2.0g/c+g" for about 10-30 minutes, or boil it by direct heating, then boil it at 8.
Cool to below 0'C.

After that, inoculate this with HOS 80 and
Fermented foods are produced by culturing at a temperature of C for about 12 to 80 hours. The ripening time by HOS80 is P)1 of the parent strain.
．． It required about twice as much.

The fermented food obtained in this way does not have the stringiness seen in the parent strain, but may have a slimy feel in some cases. Furthermore, as the fermented food matured, the proteins were decomposed and the food became more flavorful and softer, and an increase in peptide content was observed. And 1. to Goho. When the hydrophobic amino acid (tyrosine) was decomposed, crystals were formed. Although the theoretical explanation for the precipitation of 1,000 rosin has not been fully elucidated,
This is thought to be because 0 has γ-GTPase activity of 100 units or more. Therefore, a fermented food with extremely low bitterness can be obtained, and the flavor is not at all dull.

Fermented foods according to the present invention include soybeans such as natto, peas such as green peas, legumes such as azuki beans, kidney beans, and fava beans, grains such as corn, wheat, barley, oat millet, and pearl barley, sesame, peanuts, Seeds such as ginkgo,
It is also manufactured using other solid foods that contain protein, such as potatoes, fruits, mushroom necks, and algae. The produced fermented food can be eaten as is, but it can also be made into various processed foods, such as those listed below, by taking advantage of the fact that it is substantially non-stringy and easy to handle.

(a) Processed foods mixed with powdered foods; such processed foods are served, for example, as side foods, sweets, snacks, and the like. Any known powdered food can be used, such as salt, starch, seaweed, chili pepper, sugar, maltose,
Examples include flour, various seasonings, and various spices. The amount of these powdered foods added is not particularly limited, and is appropriately selected depending on the properties of the powdered foods and the intended use of the resulting food.

(b1 After drying the fermented food according to the present invention, dextrin or the like may be sprayed onto the surface of the dried fermented food, and green seaweed, chili peppers, etc. may be attached on top of it to serve as a snack, or sugar may be added after drying. An aqueous solution such as water may be sprayed onto the surface of the cake to serve as a confectionery. Any known drying method can be used, such as freeze drying, vacuum drying, hot air drying, etc. The fermented food according to the present invention has substantially no stringiness and does not cause the foaming phenomenon seen in conventional natto when freeze-drying.
Freeze-drying can be performed without pre-freezing.

(C) The fermented food according to the present invention can be easily crushed compared to conventional natto, which has stringy properties, so it can be crushed according to a known method and used as raw materials for various foods, additives, etc. May be used. The fermented food powder according to the present invention can be used for virtually all processed foods, and specific examples include pastry products such as sausages, salami, and kamaboko, sweets, and steamed buns.

Example C] Next, the present invention will be explained in more detail using reference examples and examples.

Thoughts: I HOS80'') Meat extract 1%,
Miyagino natto bacteria cultured in a liquid nutrient medium (hereinafter referred to as "liquid medium") containing 1% peptone and 0.5% salt were collected by centrifugation and collected at 0.5 tag/aj! N-methyl-N
The suspension was suspended in 1/I OM phosphoric acid buffer (Pt+ 6.8) containing '-nitro N-nitrosoguanidine (NTG) and allowed to stand for 30 minutes while cooling with water. Thereafter, the soybeans were collected by centrifugation, washed three times with the same Bufler to remove NTG, and then spread on a plate medium containing 1% meat extract, 1% peptone, 0.5% salt, and 2% agar.
After 24 hours of incubation at °C, 2 of the grown colonies
00 indiscriminately, produced natto using each strain, selected a strain that could produce natto with virtually no stringiness even after repeated passage I8 cultivation, and
Measure TPase activity to determine T G T P asC
Bacteria with activity of 100 units or more were selected. In this way, a stable mutant strain, HO380 strain, was obtained.

Reference example 2 (measurement of rGTPase activity) γ-GT
Measurement of Pase activity is carried out using Biochikae High-Physical Acta (Biochi Steelica and Bioph).
ysica AcLa, 73 (1963) 679-6
According to the method described in 81), it was performed as follows.

(Preparation of al test 4 The H O380 strain obtained in Reference Example 1 was inoculated into 11f1 culture medium consisting of the following components, 165r, p
Shaking culture was carried out by culturing at 37°C for 7 days and 14 days, respectively, while shaking at . Each of the obtained culture solutions was centrifuged at 1 OO00r, p, +w for 5 minutes, and the supernatant was used as an enzyme solution.

Preculture medium (PI (adjusted to 6,8) Peptone 1.2% Citric acid 0.2% Glycerol 2.0% NH, CI 0.7% to 211PO40,05% gsOaIHto 0.05% FeCl57
HJ 0.004% biodin 0.1
1tg/d (b) Enzyme activity measurement method Each of the following components was added to 50mM) Squirrel buffer 2 with pH 9.0.
A substrate solution was prepared by dissolving in 0Id.

Base solution + r-glutamyl-ρ-nitroanilide monohydrate (
Base 'ff); 29 ■ Explosion C1 □
; 41■Grinlglycine; 166m
g For each of the enzyme solution from the 7-day culture and the enzyme solution from the 14-day culture, 0.1 mN of the enzyme solution and the obtained aqueous solution 3
ml and heated at 37°C for a specified time (0 minutes 54 minutes,
After reacting for 6 minutes, 8 minutes, and 10 minutes, the absorbance at a wavelength of 410n+m was measured using an absorption photometer. The results are shown in Figure 1.

The 11th position of γ-GTPage activity is the amount of enzyme that releases 1 μmol of P-nitroanilide under the above reaction conditions (37°C), and γ-GTPase activity is determined by multiplying the rate of change in absorbance over time by a constant value. Thank you for the value. 803
The 1-GTPase activity of the 80 strains was 291 units after 7 days of culture, and about 271 units after 14 days of culture.

The 1-GTPase activity of the parent strain Miyagino Nattokan was examined under the same conditions as in Reference Example 2.

That is, the parent strain was cultured with shaking for 7 days and 14 days under the same conditions as in Example 2, and then an enzyme solution was prepared in the same manner. Regarding the obtained enzyme solution, the substrate?
The absorbance was measured using an absorption photometer.

The results are shown in Figure 1.

The 7-GTPase activity of the parent strain was 455 units when cultured for 7 days and 388 units when cultured for 14 days. -0 strain, γ-G T
Pase activity was examined. That is, the HOS-0 strain was cultured with shaking for 7 days and 14 days under the same conditions as in Reference Example 2, and then an enzyme solution was prepared in the same manner. The obtained enzyme solution was mixed with a substrate solution in the same manner as in Reference Example 2, and the absorbance was measured using an absorptiometer. The results are shown in Figure 1.

The 1-GTPage activity of the HOS-0 strain was 6 units after 7 days of culture, and 17 units after 14 days of culture.

Selected soybeans were washed and soaked in water for 1-2 days until swelling to 2.0-2.5 times the original volume. The soybeans were steamed for 20 minutes under a pressure of 2.0 kg/cm'' using pressurized steam, cooled to below 80°C, and the HO580 strain obtained in Reference Example 1 was inoculated into the soybeans.

Soybeans inoculated with the 80380 strain were kept at 40°C for 24 hours to produce Kisei natto. The obtained natto had a good flavor peculiar to natto and also had excellent storage stability. Also,
Bitterness was extremely low.

In addition, the stringiness of the obtained adhesive was examined in the following manner.

(2) The two obtained natto were slowly pulled apart from each other, and the distance until the thread formed between the natto broke was measured.

As a result, just by pulling the natto apart m + w, the thread broke,
It had substantially no stringiness.

(2) The obtained long natto was dissolved in 200 d of water, and ethyl alcohol was added thereto so that the final consistency was 85%.

As a result, the solution became almost uniformly cloudy, and even when stirred with a glass rod (T), nothing adhered to the glass rod.

几51[-[ Natto was produced using the parent strain Miyagino natto bacteria under the same conditions as in Example 4N1. The resulting natto was examined for stringiness in the same manner as in Example 1.

■As a result, even if the stuck natto was separated by more than 101 cm, the threads did not break.

■As a result, filaments precipitate out, and when stirred with a glass rod,
The filament adhered to the glass rod and was able to be wound up.

1. The Nigraph pieces were washed and soaked in water for 4-8 hours until they swelled to 2-2.5 times their original volume. As in Example 1, this was heated for 7M for 20 minutes under a pressure of 2°0 Kg/cm'' using pressurized steam, and then cooled to below 80°C.
This graph piece was inoculated with the 80580 strain 1q obtained in Reference Example 1. Graph piece inoculated with 80380 strains,
A fermented graph piece was produced by holding at 40°C for 24 hours. The obtained fermented graph peas were non-stringy, soft and delicious, and had the unique flavor of graph peas.

The corn was washed and soaked in water for about 4 days until it swelled to 2-2.5 times its original volume. Example 1
In the same manner as above, aged fermented corn was produced.

The obtained fermented corn was not stringy, soft and delicious, and had the unique flavor of corn.

(Effects of the Invention) According to the fermented food of the present invention, it has the advantages of conventional natto as an excellent food, that is, it has good taste, excellent digestibility, high nutritional value, and has a long shelf life ( 3
It has now become possible to provide a food that is substantially free from stringiness, which has never been seen before, while retaining its advantages, such as the fact that it is

Therefore, since the fermented food according to the present invention is substantially free of stringiness, it does not cause discomfort to any person, and can be used not only as a so-called side food, but also for confectionery, etc. Furthermore, it has better effects than the second invention, such as being able to freely perform post-processing as a food ingredient.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between absorbance and time for calculating the γ-GTPase activity of aged proteins. However, ○ is a 7-day culture of HOS80 strain, Δ is a 14-day culture of the 80580 strain. Each day culture is shown. Patent applicant: Masami Hoshino

Claims (2)

[Claims] (1) A solid food containing protein belonging to Bacillus subtilis,
Bacillus natto HOS which has a γ-GTPase activity of 100 units or more and whose aged protein has substantially no stringiness
A fermented food product characterized by being fermented by a fermentation method of 80%. (2) The solid food containing the protein is beans, grains,
2. The fermented food according to claim 1, wherein the fermented food is seeds, potatoes, fruits, mushrooms, or algae.