JPS63148985A - Production of enzyme for hydrolyzing protein of fishes - Google Patents

Abstract

PURPOSE:To efficiently obtain protease capable of exhibiting high treating effects in hydrolytically treating proteins of fishes, by cultivating a novel microorganism belonging to the genus Aspergillus or variant strain thereof in a nutrient culture medium. CONSTITUTION:Aspergillus sojae S297 (FERM-P No.9073) separated from a commercially available mold starter for soy sauce or a natural or artificial variant strain thereof is inoculated into a solid culture medium, e.g. wheat bran, etc., then aerobically cultivated for 40-55hr to secrete protease in the culture medium (bran). After the cultivation, the bran is suspended in water to readily migrate the protease into an aqueous layer and remove insoluble substances. Thereby a crude enzymic solution is obtained. Purified protease is prepared from the crude enzymic solution by a normal purification means and utilized for hydrolytically treating proteins of fishes.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Industrial application field
The present invention relates to a method for producing a fish proteolytic enzyme using 97').

Approximately 100,000 tons of broth is produced each year when canning bonito, tuna, etc. This broth contains a large amount of protein, so it is treated with proteases and processed in various other ways, and used as a component of microbial culture medium and seasonings. has been done.

However, it is very difficult to eliminate the peculiar odor of fish and produce processed products with rich taste and flavor in an industrially advantageous manner, so only about 60% of the broth is actually used, with the remaining 40% being used. % are currently being discarded.

The present invention relates to a method for producing an enzyme suitable for treating these fish proteins, and the enzyme of the present invention exhibits a much higher treatment effect than any conventional commercially available enzyme. be.

(2) Conventional technology Examples of obtaining materials for beverages, seasonings, therapeutic foods, etc. by hydrolyzing fish protein with enzymes as a protein source have been seen for a long time (Tokai Suikenho Vol. 43, pp. 87-90, 1965) year; Tokai Water Research Report Vol. 73, pp. 103-112, 1973).

The new method uses commercially available alkaline proteases to act on fish proteins at their main active pH range of 7.5-9.0, stopping the degree of hydrolysis within the range of 15-25%. A hydrolyzate with less taste and fishy odor and its manufacturing method have been announced (Japanese Patent Application Laid-Open No. 1983-1992).
1-28370).

Furthermore, Masao Fujimaki and others have reported that actinase, an enzyme produced by Streptomyces griseus, decomposes fish proteins. (App Culture and Biological Chemistry Vol. 37, 2891
(Page 2898, 1973) However, these conventional methods are not always satisfactory.

(3) Problems to be Solved by the Invention The present invention processes fish protein contained in broth, etc., which is produced in large quantities during canning of bonito, tuna, etc. or processing of mackerel and sardines, and eliminates the peculiar odor of fish. Since the commercially available enzymes used to produce processed foods with excellent taste and flavor are not always satisfactory, the purpose of this project is to create novel and excellent enzymes that meet this purpose.

The present inventors have investigated enzymes obtained by culturing various protease-producing bacteria independently isolated and commercially available proteases.
We compared the proteolytic effects of fish and found that the protease produced by the bacterial strain Aspergillus sonii 5297 isolated by the present inventors was the most excellent, and completed the present invention.

(4) Structure of the Invention The present invention is directed to "Aspergillus 5ojae
(A1 Research Institute No. 9073) or a mutant strain thereof is cultured to produce and accumulate protease in a medium, and the protease is collected. ”
It is related to.

[Microbiological characteristics]

Microorganism according to the present invention, Aspergillus sonii 5297
is a new strain that the present inventors isolated from commercially available seed koji for mustard seeds.As described below, it produces an extremely excellent protease as a fish proteolytic enzyme, has an extremely short conidiophore, and has an abundant amount of microorganisms. It is distinguished from other Aspergillus sonii in that it quickly forms a spore head.

Hereinafter, the mycological properties of Aspergillus sonii 5297 will be explained based on the results of observation after growing it on a Czapek agar plate at 25°C.

(Growth) On the Czapek agar plate, the colony spreads quickly and the colony diameter is 7
The basal hyphal layer, which develops around cm, is white and dense, the conidiophores are extremely short, the center is hardly woolly raised, and the surface is flat. The formation of conidial heads and conidia is particularly good, and the colonies are bright yellow to yellow when young.
Later on, it becomes yellowish-green to green, and when it matures it becomes yellowish-brown, and as it gets older it turns brown with some yellowish-green. The surface of the village is cream-colored to light brown.

(Morphology) Conidia: 350-400 μm in diameter, radial when young, loosely radial when mature, yellow-green to brown.

Conidiophore: 200-500 μm in length, 10-13 μm in diameter at the lower part of the apical sac, the basal hyphal layer (4-7 μm), the wall surface is smooth, and only the lower part of the apical sac is rough.

Apical capsule: diameter 20-22 μm, slender spherical shape.

Formed from almost the front surface of the phialide bimodal sac, length 7-9 μm) x width (3-4 μm) Conidia: Diameter 5-6
．． 3 μm, spherical to fine spherical, Table and others: No ability to form pink pigment on Czapek agar plate containing p-anisaldehyde. Produces kojic acid. Aflatoxin B+, B2
, G+.

Does not produce G2.

The above mycological properties were analyzed by K. B. Raper & D.I.
, 1. The Genus Aspergillus, by John Fennell
s), pp. 375-403, 1965; Ushijima S, et al.
and Biological Chemistry (Agricu)
ultural and biological Che
mistry) Volume 46, pages 2365-2367, 19
1982; H, Murakami et al., Journal of Gen
Eral and Applied Microbio
28, pp. 55-60, 1982, it was confirmed that strain 3297 of the present invention belongs to Aspergillus sonii (Aspergillus 5ojae).

Microorganisms that can be used in the present invention are not limited to the Aspergillus sonii strain 5297, which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as Deposit No. 9073, but also natural or artificial mutant strains thereof. Naturally included.

Although the culture in the present invention may be a liquid culture, aerobic solid culture is generally preferred.

Usually, the strain 5297 of the present invention is inoculated into a solid medium of wheat bran, etc., and when cultured aerobically for 40 to 55 hours, it secretes protease into the medium (koji). After culturing, the protease is easily transferred into suiseki by QJing the koji in water, and a crude enzyme solution can be obtained by removing insoluble matter.

Purified protease can be obtained from this crude enzyme solution by a known enzyme purification method.

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

Example powder 4.8 kir, KHzPOa320 g 1L-
After sterilizing a solid medium consisting of 160 g of sodium glutamate and 81 g of water at 121°C under 1 atm for 20 minutes, conidia of Aspergillus sonii 5297 were inoculated, cultured with ventilation at 30°C for 2 days, and 6.9 kg of koji was inoculated. Obtained. Add this to 6 liters of water
01, stirred thoroughly, and centrifuged (9000 rpm).
) L, 55 Il of supernatant liquid (enzyme titer 220 U/mj was obtained. Next, this liquid was passed through a separation membrane PM100O using a Holofiber system (manufactured by Komatsu Yokakoki Co., Ltd.), and then concentrated using a separation membrane PM5. , purified and recovered.

The concentration obtained was 41, and the absorbance at 280 nm was 26.
Met.

After adding 420 g of dextrin (N501318, manufactured by Nippon Kizuka Kogyo Co., Ltd.) as a stabilizer to this liquid, freeze-drying was performed to obtain 422 g of enzyme powder.

The enzyme titer was 24000 U/g and the recovery rate was 83.
It was 7%.

This enzyme powder was used in subsequent tests as a standard of the enzyme of the present invention.

[Enzymological characteristics]

This standard enzyme does not consist of a single protease, but a mixture of many types of proteases. In order to clarify this, Table 1 shows the results of measuring the enzyme activity of six types of proteases that showed relatively strong activity.

*Literature■: Agricultural and Biolo
logical Chea+1stry37, 3685(1
973).

■：Method of Enzywology, G
, E., Perlmannand L., Loran
d, Academic Press, 18+ 397
(1970).

■ :Biochemica et Biophy
sica Acta 39ヱ.

443 (1975).

■: LAP-C-Te5t Wak manufactured by Wako Pure Pharmaceutical Company ■
Activity measurement method used.

The enzyme of the present invention contains many types of enzymes as shown in Table 1, and although these individual enzymes can be used separately, it is usually best to use them as a mixture to obtain suitable results. I can do it.

When fish proteins are degraded using the enzyme of the present invention, the enzyme can also be used after being immobilized on an inert carrier.

This decomposition treatment is often carried out at 50 to 60°C, but the enzyme of the present invention has better heat resistance than commercially available enzymes, so it can obtain suitable treatment effects in actual use situations.

Next, Table 2 shows the results of heat resistance tests of commercially available representative proteases and the enzyme of the present invention.

*The residual activity rate was determined by measuring the three types of enzyme activities contained in each test enzyme using the following method.

(b) The activity of neutral and alkaline brotinase is 20
The enzyme was incubated at 60°C in mM phosphate buffer (pH 7,0).
, and the remaining activity was measured by a modified Anson-Hagiwara method.

(L) The activity of carboxypeptidase was determined by the method of China et al.
ultural and biological Che
Mistry.

36, 1343 (1972)) using the substrate carbobenzoxy-glutamine-tyrosine.

(c) The activity of aminopeptidase was determined by treating the enzyme in 201 phosphate buffer (pH 7,0) at 60°C for 10 minutes and measuring the remaining activity with LAP C-Te5tWako (
(manufactured by Wako Hyakuyaku).

The thermostability of alkaline proteinase and carboxypeptidase was significantly superior to that of commercially available enzymes.

5 Effects In order to explain the effects of the enzyme of the present invention, test examples are shown below.

Test Example 1 Sensory test regarding the taste of fish protein degradation products obtained by treatment with the enzyme of the present invention and commercially available enzymes.

Test method Cultivator 350 (trade name of broth extract produced as a by-product during canned tuna production, manufactured by Yaizu Suisan Kagaku Kogyo Co., Ltd., moisture 40%) was used as a substrate, and 20 units of the test protease was used as a substrate.
ml and hydrolyzed at 50°C for 3 hours. After boiling the treated solution for 5 minutes, it was diluted 2 times with water and evaluated by 8 experts at 60℃ for strong flavor and low bitterness. A sensory test was conducted according to the Sensory Test Handbook (edited by the Society).

Test enzyme Test results The results of this test are shown in Table 3.

Table 3: This significant difference test was carried out using Kramer's Rapid Significant Difference Test Table (Sensory Test Handbook, p. 845).

The commercially available enzymes in the Motoichi test enzymes are commercially available enzymes 2 shown in Table 4.
A sensory test similar to that in Test Example 1 was conducted on five items, and the top three items were selected.

As is clear from the test results in Table 3, the broth extract treated with the enzyme of the present invention received the highest score and was sensually superior to the broth extract treated with any commercially available enzyme. It has been found.

Table 4 Test Example 2 Sensory test regarding the taste of fish protein degradation products obtained by treatment with the enzyme of the present invention and a known protease produced by Aspergillus sonii.

Test method Fish protein degradation products were prepared in the same manner as in Test Example 1, and a sensory test was conducted by nine experts to determine the test enzyme and protease P (in Test Example 1, the most sensually preferable decomposition product among the commercially available enzymes). A two-point preference test method (see the sensory test handbook) was used to compare the taste between the two products (see the sensory test handbook).

Test enzyme: The enzyme of the present invention and 8 strains belonging to Aspergillus sonii preserved at the Food Research Institute, which are known to produce proteases with relatively good heat resistance (preservation number: R192)
111, R19211[[, R6110I[, R131
2V,.

R6423n, S-12-3, 0-14-9■, 0
-2O-2) was selected and the respective bacteria were cultured in the same manner as in the previous example to prepare test enzymes.

Test results The results of this test are shown in Table 5.

Table 5 As is clear from the results in Table 5, many of the proteases produced by strains belonging to Aspergillus sonii were comparable to Protease P, which obtained the most favorable results among commercially available enzymes. Among them, the enzyme of the present invention obtained outstanding results.

Test Example 3 Fish proteolytic activity test of the enzyme of the present invention and a commercially available enzyme Test method Cultivator 350 (above-mentioned tuna broth extract) was passed through an Amberlite IR-1208 (H+ form) column to determine the amount of water contained therein. Adsorbs and removes free amino acids,
Fractions with an A260nm/A280nm ratio of 1.5 or less were collected, adjusted to pH 6.0, and then lyophilized to prepare a fish protein to be used as a substrate.

Add 1 part of the test enzyme to this 5.6% (w/v) fish protein aqueous solution.
00U/ml (titer determined by Anson-Hagiwara modified method)
was added at a ratio of
59, 1973), and the fish protein decomposition rate was calculated from the following formula.

Fish protein decomposition rate= (However, in the formula, [ ] indicates the analysis value of the α-amino group of the substance in [ ].) Test Results The results of this test are shown in Table 6.

Table 6 As shown in Table 6, the enzyme of the present invention showed a much higher rate of fish proteolysis than commercially available enzymes suitable for non-proteolysis and typical proteases of plant and animal origin. .

In addition, in this specification, the enzyme unit means, unless otherwise specified,
App Culture and Biological Chemistry Vol. 37, pp. 2685-2694, 1 by Nakadai et al.
According to the proteinase activity measurement method (modified Anson-Hagiwara method) reported in 1997, 1 μmole of tyrosine was absorbed per minute under conditions in which milk casein (manufactured by Merck & Co.) was decomposed with a test enzyme at 30°C and pH 7.0. It is expressed as the amount of enzyme released.

Claims (1)

[Claims] Aspergillus s.
A method for producing a fish proteolytic enzyme, which comprises culturing A. ojae) S297 (Feikoken Bacteria No. 9073) or a mutant strain thereof, producing and accumulating protease in a medium, and collecting the protease.