JP5637431B2 - Method for producing krill-derived proteolytic enzyme and proteolytic method using the enzyme - Google Patents

Description

The present invention relates to the proteolytic method using the production method and the enzymatic proteolytic enzymes from krill.

  Krills (nankoku krill, horned krill) are a kind of zooplankton, and the catch in Japan is about 60,000 tons per year. Krills are currently used mainly for fishing bait and colored fish feed, but because cheaper feeds are available from China and South Korea and the demand for fishing baits is lower, Other utilization methods with high added value are desired. In particular, one of the utilization methods for increasing the added value of krills is the development of food processing aids, quality improvers and functional foods by proteolytic enzymes (proteases) contained in krills.

  However, the water extract of crustaceans containing krills is a significant hindrance to the above-mentioned development of use because the blackening proceeds remarkably even if left alone for 1 day under refrigeration. This blackening occurs when phenols containing tyrosine are oxidized by endogenous tyrosinase, and further auto-oxidation proceeds to produce black melanin.

  Here, as a method of preventing blackening of crustaceans, in addition to a method of using a proteolytic enzyme inhibitor and sodium sulfite, kojic acid, catechins, cysteine, glutathione, or a mixture of two or more of vitamin C in combination. (Refer patent document 1), The method of heat-processing at 80-90 [degreeC] is proposed (refer nonpatent literature 1).

Japanese Patent Laid-Open No. 06-245690

"Studies on the use of horned krill", Iwate Prefectural Fisheries Technology Center Annual Report Vol.1997 Page.111-115 (1998)

  The proteolytic enzyme preparation obtained by water extraction of krills shows stable degradation activity for a long time at a reaction temperature of 37 [° C.], but the enzyme is gradually deactivated at a reaction temperature of 50 [° C.]. In order to use a proteolytic enzyme for the above-mentioned purpose, it is necessary to be able to react stably for a long time in a high temperature region where the degradation activity is high.

  All the blackening prevention methods so far have significantly reduced the function of proteolytic enzymes, and were not intended to utilize proteolytic enzymes. The development of a technique for preventing blackening while maintaining the activity of proteolytic enzymes has been awaited.

The present invention, while suppressing darkening due melanogenesis, stable reaction using the methods and the enzyme to produce a krill-derived proteolytic enzymes capable of performing proteolysis was cracking activity is high hot It aims to provide a way to do .

A method for producing a krill-derived proteolytic enzyme for solving the above-mentioned problem is a step of producing or preparing a supernatant obtained by homogenizing krill with water and then heat-treating the supernatant in the presence of calcium ions. It is characterized by having.

  The step is preferably a step of heat-treating the supernatant for 10 to 20 [min] in the temperature range of 50 to 55 [° C.] in the presence of 0.5 to 1 [mM] calcium ions.

  According to the method of the present invention, as will be described in detail later, the specific activity of the proteolytic enzyme can be increased by thermal stabilization with calcium ions while suppressing blackening. Moreover, when the supernatant solution after the heat treatment is used as an enzyme sample, calcium ions are added to the reaction system so that the concentration is 0.5 to 1 [mM], and protein decomposition is performed at a reaction temperature of 50 [° C.] It is possible to maintain a high degradation activity for a longer time.

The azocasein-degrading activity (relative value of specific activity) of the supernatant solution obtained by heat-treating the crude enzyme extract at each temperature and 20 [min] in the presence or absence of 1 [mM] calcium ion, and the supernatant Explanatory drawing of the melanin production amount (relative value) when left at 4 [° C.] for 1 day. In the presence of 1 [mM] calcium ion, the crude enzyme extract was 55 [° C.], the azocasein-degrading activity (relative value of the specific activity) of the supernatant solution obtained by heat treatment at each time, and 4 [ [° C.] Explanatory drawing of the amount of melanin production (relative value) when left for 1 day. Explanatory drawing of the experimental result of the time-dependent change of the amount of azocasein decomposition | disassembly at each calcium ion concentration and reaction temperature 50 [degreeC].

Embodiments of a method for producing a krill-derived proteolytic enzyme of the present invention and a proteolytic method using the enzyme will be described.

Example 1
(Preparation of crude enzyme extract)
For example, krill 40 [g] stored frozen at −80 ° C. under ice-cooling with a double volume (80 [ml]) of cold water using a HSIANGTAI homogenizer (main body: HG-200, shaft generator: K30). Homogenized. This homogenate was centrifuged (10,000 × [g], 10 [min], 4 [° C.]) to obtain a supernatant (crude enzyme extract).

(Heat treatment)
Add 10 [mM] calcium chloride 0.3 [ml] (+ Ca ion: final concentration 1 [mM]) or water 0.3 [ml] (-Ca ion) to the supernatant 2.7 [ml] and mix. The obtained samples were heat-treated at respective temperatures of 37, 45, 50, 55, 60, and 70 [° C.] for 20 [min]. After the heat treatment, centrifugation (10,000 × [g], 10 [min], 4 [° C.]) is carried out, and the supernatant liquid containing the proteolytic enzyme (protease) is subjected to melanin production, protein quantification, proteolysis Activity was measured.

(Measurement of melanin production)
A part (about 1.5 [ml]) of the supernatant liquid obtained under the above conditions was placed in a test tube and allowed to stand at 4 [° C.] for 24 [hr]. This solution was diluted 5-fold with distilled water, and the absorbance of 405 [nm] was measured for a solution obtained by solubilizing the supernatant after centrifugation with an equal amount of 1M sodium hydroxide. The amount of melanin production) was quantified. In addition, the melanin production amount was shown by the relative value which set 100% as the non-heat processing.

(Quantification of protein)
The protein concentration in each supernatant obtained by the above procedure was measured according to the microbiuret method (see Itzhaki, RF and Gill, DM (1964) Anal. Biochem. 9 401-410). The standard protein used was bovine serum albumin.

(Measurement of proteolytic activity)
Proteolytic activity was measured by a method in which azocasein was used as a substrate and the amount of free tyrosine produced by the degradation of the substrate was measured with an absorbance of 335 [nm].

  Specifically, 150 [μl] of water was added to 700 [μl] of 1 [%] (w / v) azocasein dissolved in 100 [mM] phosphate buffer solution (pH 7.0), and the protein concentration was adjusted to 0. 150 [μl] of the supernatant solution of each condition prepared so as to be 5 [mg / ml] was added, and a hydrolysis reaction was performed at 37 [° C.] for 1 [hr].

  10 [%] (v / v) trichloroacetic acid 2 [ml] was added to stop the reaction, and the absorbance at 335 [nm] of the supernatant after centrifugation was measured. In addition, about the supernatant liquid of each condition which carried out enzyme inactivation over 95 [degreeC] over 10 [min], what performed the same process as the above was used for the blank. The results were summarized as relative activities when the activity of the crude enzyme extract not subjected to the heat treatment was 100%.

  Fig. 1 shows the results of azocasein degradation activity (relative value) and melanin production (relative value) of the supernatant solution obtained by heat-treating the crude enzyme extract at each temperature for a predetermined time in the presence or absence of calcium ions. . As for blackening, that is, melanin production amount, strong blackening was observed up to 45 [° C], but from 50 [° C] onward, the melanin production amount became 50% or less before heat treatment, and there was a clear blackening suppression effect. It was seen. Regarding the degree of blackening, no difference was observed between the presence and absence of calcium ions.

On the other hand, with regard to proteolytic activity, an increase in activity was observed by heat treatment at 45 to 60 [° C.] regardless of the presence or absence of calcium ions. This is presumed to be due to the contribution of thermal stabilization by endogenous calcium ions contained in seawater and krill, but the addition of 1 [mM] calcium ions further increases the specific activity.
The temperature range of 50 to 60 [° C.] is a preferable condition in which the specific activity of proteolysis is increased by 10 [%] or more than before the heat treatment while suppressing the melanin production to 50 [%] or less before the heat treatment. In particular, the heat treatment at 55 [° C.] is the most preferable condition that can suppress the melanin production to 35 [%] or less before the heat treatment and increase the specific activity of the proteolytic enzyme by 20 [%] or more.
In addition, when the calcium ion concentration was changed, for example, at 0.5 [mM], the proteolytic activity had a level equivalent to 1 [mM]. Since the increase is limited to about 10 [%] before the heat treatment, it has been found that 0.5 to 1 [mM] calcium ion is the optimum concentration for carrying out the present invention.

(Example 2)
The optimum treatment time was examined for heat treatment at 55 [° C.] in the presence of 1 [mM] calcium ion, which was a preferable condition for increasing proteolytic activity while preventing blackening in Example 1.

(Heat treatment)
With respect to a sample obtained by adding 10 [mM] calcium chloride 0.3 [ml] (final concentration 1 [mM]) to 2.7 [ml] of the crude enzyme extract and mixing, 55 [° C.] 2, 5, 10, Heat treatment was performed under the conditions of 20, 40, and 60 [min]. After the heat treatment, centrifugation (10,000 × [g], 10 [min], 4 [° C.]) was carried out, and the supernatant containing the proteolytic enzyme (protease) was used in the same manner as in Example 1 to produce melanin, protein And proteolytic activity were measured.

  FIG. 2 shows the results of azocasein degradation activity (relative value) and melanin production (relative value) in the supernatant solution obtained by heat-treating the crude enzyme extract at 55 [° C.] in the presence of calcium ions for each predetermined time. Show. Regarding the blackening, the amount of melanin produced by heat treatment of 10 [min] or more became 50 [%] or less before the heat treatment, and a clear blackening prevention effect was recognized. Regarding the proteolytic activity, an increase of 20 [%] or more was observed for 5 to 20 [min]. Therefore, in the case of heat treatment at 55 [° C.], it has been found that the treatment time of 10 to 20 [min] is a condition for increasing the proteolytic activity while preventing blackening.

  As described above, the specific activity of the proteolytic enzyme can be increased, that is, the crude purification can be performed at the same time, while suppressing blackening, which is a major obstacle to enzyme preparation, by a simple and inexpensive method combining heat treatment and calcium ion addition. It becomes possible.

Example 3
The supernatant obtained by heat treatment over 20 [min] at 55 [° C.] in the presence of 1 [mM] calcium ion, which was the most preferable condition for preventing the blackening and increasing the proteolytic activity according to Example 2. Was used as a sample. In this example, the optimum amount of added calcium capable of suppressing deactivation at a high temperature (50 [° C.]) was examined.

(Measurement of proteolytic activity)
In the presence of calcium ions (final concentration: 0.5 to 1, 5 and 10 [mM]) or in the absence, the reaction was allowed to proceed for 0, 5, 10 and 25 [hr] at a temperature of 50 [° C.], respectively. The activity was measured in the same manner as in Example 1 except that.

  The results of this example are shown in FIG. At a reaction temperature of 50 [° C.], when calcium ions are not added, the rate of increase gradually decreases and deactivation proceeds. On the other hand, when 0.5 to 1 [mM] calcium ion is added to the reaction system, the heat resistance of the degrading enzyme increases and the progression of inactivation can be suppressed most. After a reaction time of 25 [hr], the reaction can proceed with an activity about 1.7 times that of calcium-free addition.

  On the other hand, when 5 [mM] calcium ion is added to the reaction system, the effect of heat resistance cannot be obtained as much as 0.5 to 1 [mM]. However, after the reaction time of 25 [hr], about 1. The reaction can proceed with 5 times the activity. Further, when 10 [mM] calcium ion was added to the reaction system, almost no heat-resistant effect was obtained, and the activity was the same as when calcium ion was not added.

  As described above, when 0.5 to 1 [mM] calcium ion is added to the reaction system, the heat resistance of the proteolytic enzyme contained in krill increases, and the protein is stably stabilized at a high temperature of 50 [° C] where activity is high. It becomes possible to perform decomposition.

Claims (3)

A method for producing a krill-derived proteolytic enzyme,
A method comprising preparing or preparing a supernatant obtained by homogenizing krill with water and then heat-treating the supernatant in the presence of calcium ions.   The step is a step of heat-treating the supernatant for 10 to 20 [min] in the temperature range of 50 to 55 [° C.] in the presence of 0.5 to 1 [mM] calcium ions. The method for producing a proteolytic enzyme according to claim 1. A protein degradation method using a krill-derived proteolytic enzyme,
Calcium ions to a concentration range of 0.5 to 1 in the supernatant heat treated according to the method of claim 2 wherein [mM] was added, degradation of the protein at a temperature of 50-60 [° C.] The method characterized by performing.

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