JPH03123469A - Method for enhancing angiotensin conversion enzyme inhibiting activity in krill - Google Patents

Abstract

PURPOSE:To obtain a krill liquefied material capable of more effectively providing blood pressure depressing action in small amount of intake and readily drinking and enhance angiotensin conversion enzyme inhibiting activity in krill by fermenting krill with lactic acid bacterium. CONSTITUTION:Angiotensin conversion enzyme inhibiting activity in krill is enhanced by fermenting krill with lactic acid bacterium. Furthermore, as the lactic acid bacterium, Lactobacillus bulgaricus, Lactobacillus acidophilus, Streptococcus thermophilus, etc., is preferably used and the fermentation is preferably carried out by fermenting krill for 1-10 day while retaining pH for 5-6 day and further previously decomposing krill with a proteolytic enzyme before fermentation.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for enhancing angiotensin-converting enzyme inhibitory activity in krill. It is possible to obtain a liquefied krill product that has a blood pressure lowering effect and is easy to eat and drink.

[Conventional technology]

Today, hypertension is one of the diseases that not only has a high mortality rate but also is a problem in a wide range of age groups, from the elderly to the young. Hypertension includes secondary hypertension and essential hypertension.The former refers to hypertensive hypertension when there is a disease that causes an increase in blood pressure, while the latter refers to high blood pressure when there is no disease that causes an increase in blood pressure. , hypertension itself is a disease, accounting for 80% of hypertension cases. The renin-angiotensin system, which is involved in blood pressure regulation, is deeply involved in all types of hypertension, and in patients with essential hypertension, there is great hope that the renin-angiotensin system will suppress blood pressure increases.This renin-angiotensin system is known as ACE. An enzyme exists, and this enzyme converts dipeptide (His-Leu) from the C-terminus of angiotensin I present in the blood.
is released and converted to the active form of angiotensin H. Angiotensin ■ has a strong vasoconstrictive effect and increases blood pressure.

Therefore, by inhibiting ACE activity, it is possible to suppress the increase in blood pressure and lower the blood pressure.

Currently, proline derivatives are commercially available as ACE inhibitors, and are being actively developed as drugs through drug design. On the other hand, AC from natural products
A search is underway for substances with E inhibitory activity, and it is known that inhibitors exist in the decomposition products of krill, casein, and gelatin.

[Problem to be solved by the invention]

As mentioned above, krill is known to have a blood pressure lowering effect, but it is difficult to ingest it in its raw form and difficult to eat in large quantities. In addition, it is not preferred as a food because it has the unique odor and taste of krill.

Therefore, an object of the present invention is to provide a liquefied krill product that can more effectively lower blood pressure when ingested in small amounts and is easy to eat and drink.

(Means for Solving the Problems) As a result of various studies, the present inventors found that the ACE inhibitory activity of krill can be enhanced by fermenting krill with lactic acid bacteria and liquefying it, and that the desired liquefied krill product (lactic acid It was found that a fermentation liquid) was obtained.

The present invention was made based on the above findings, and provides a method for enhancing angiotensin-converting enzyme inhibitory activity in krill by fermenting krill with lactic acid bacteria.

Hereinafter, the method for enhancing angiotensin-modifying enzyme inhibitory activity in krill according to the present invention will be described in detail.

The krill used in the present invention includes raw krill such as shelled krill, shelled krill, frozen products thereof, shelled krill exposed to water (surimi), frozen products thereof, and heat-treated krill. can be used in any condition.

The lactic acid bacteria used in the present invention are commonly used types of lactic acid bacteria, such as Lactobacillus bulgaricus (Lactobacillus +1
us bulgaricus), Lactobacillus acidophilus (
Lactobaciflus acidophilu
s), Streptococcus thermophilus (Strept.

coccus therv+ophillus), etc. can be used.

Fermentation is usually carried out for 1 to 10 days, preferably 2 to 5 days. Even after 10 days, the ACE inhibitory activity of the lactic acid fermentation liquid will not be enhanced, and if it is shorter than 1 day, the inhibitory activity will not be enhanced.

The pH during the fermentation period is preferably adjusted to 5 to 6, and if the pH is not adjusted, it gradually decreases immediately after the start of fermentation and decreases to around 13.0 on the second day during one fermentation period. By maintaining the pH within the range of 5 to 6, enhancement of the ACE inhibitory activity of the lactic acid fermentation liquid is further promoted.

Further, the fermentation temperature is preferably about 0°C to about 40°C.

In addition, if the raw material krill is previously decomposed with a protease before fermentation, the enhancement of the ACE inhibitory activity of the lactic acid fermentation liquid can be further promoted.

The proteolytic enzymes mentioned above include commercially available pronase,
Any proteolytic enzyme preparation such as Samoase (thermolysin) and Protease A can be used.

A preferred embodiment of the enhancement method of the present invention is listed below.

Approximately the same amount of water is added to the raw material krill and pulverized, followed by the addition of proteolytic enzymes to decompose the krill.

Add lactic acid or the like to this to adjust the pH to 5-6.

After that, add lactic acid bacteria, and maintain the pH at 5-6.
Ferment for ~10 days to obtain a krill lactic acid fermentation liquid.

〔Example〕

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

The protein concentration and ACE inhibitory activity of the krill lactic acid fermentation liquid obtained in the examples were measured as follows.

1) Method for Measuring Protein Concentration Protein concentration was determined by appropriately diluting the 0 fermentation liquid measured using the Folin-Lowry method and measuring it by a conventional method.

The amount of protein was converted into the amount of tyrosine.

2) Measuring method of ACE inhibitory activity Cu5hn+an modified method (D, W Cushman a
nd H, S, Cheung Biochem, P
harmacol, 20. p 19671971)
was used.

The fermentation liquid was diluted to a certain amount of protein (in terms of tyrosine) and used for the reaction.

ACE (manufactured by Sigma) 22gg/mil solution 20μ
P.i. and 50 μ2 of the diluted fermentation liquid were placed in a test tube, and 0.5 M phosphoric acid of pH 8.3 and 1.5
240μ2 of a solution of 5mM Hiprylu, Histidyl, Leucine (manufactured by Sigma) was added to a buffer containing MNaCl, and the mixture was reacted at 37°C for 60 minutes. After that, 1.5 mfl of 0.28 N Na OH was added to stop the reaction, and then 10 μl of 2% O phthalaldehyde in methanol solution was added. After exactly 10 minutes, 3NHC1 was added to stop the fluorescence reaction. The reaction solution was diluted 10 times, and the fluorescence intensity was measured at an excitation wavelength of 340 nm and a fluorescence wavelength of 455 nm.

The formula for calculating the inhibition rate is: [1-(S-B)/AI
trol) with 20μ of water instead of ASACE.
Let B be the fluorescence intensity of the reaction mixture.

Example 1 An equal amount of water was added to 200 g of each of shelled krill, shucked krill, frozen shucked krill, boiled shucked krill, water-bleached shucked krill (surimi), and frozen krill surimi, and pulverized them. p in the pore acid
Adjust H to 5.6 and autoclave to 121°Cl2.
After sterilization for O minutes, grapes $J! 10 g and 20 g of lactic acid bacteria culture (previously cultured in milk medium using commercially available yogurt (Kanin Nature)).
Ferment for 5 days at °C. The pH during this time is maintained at 5.6. After fermentation is complete, separate the supernatant and residue by centrifugation, filtration, etc.
The mixture was sterilized at 80°C for 130 minutes to obtain a krill lactic acid fermentation liquid. The ACE inhibitory activity of this krill lactic acid fermentation liquid was measured. As a control, water extracted from shelled krill is shown.

The results are shown in Table 1 below.

Table 1 From the results in Table 1, when looking at the inhibitory activity by form, it can be seen that all forms exhibit almost the same inhibitory activity.

Furthermore, when we measured the protein concentration of the above-mentioned krill lactic acid fermentation liquid, all of them showed the same value, except for slightly more shells.

This shows that in the present invention, there is no difference in ACE inhibitory activity no matter what form of krill is used as the raw material.

Example 2 Add an equal amount of water to 200 g of shucked krill, grind it, adjust the pH to 5.6 with porous acid, and boil it in an autoclave at 121°.
After sterilization with Cl2O, 10 g of glucose and lactic acid bacteria, B. bulgaricus (L,
bu1garicus+ JAM 1120), L. acidophilus, IAM
12475), Streptococcus thermophilus (Str, the
Add 20g of fermentation liquid of rn+ophillus, IFO3863) and ferment at 30°C for 5 days. During this period, the pH is maintained at 5.6. After fermentation is complete, the supernatant and the residue are separated by centrifugation, filtration, etc., and sterilized at 80°C for 130 minutes.
A krill lactic acid fermentation liquid was obtained. The ACE inhibitory activity of this krill lactic acid fermentation liquid was measured. As a control, water extracted from shelled krill is shown.

Table 2 From the results in Table 2, it can be seen that there is almost no difference when looking at the inhibitory activity on the side of the image, and the inhibitory activity is enhanced in all cases compared to the water-extracted product.

Furthermore, when the protein content of the above-mentioned krill lactic acid fermentation liquid was measured, there was no difference depending on the bacteria.

This shows that in the present invention, commonly used lactic acid bacteria can be used as the lactic acid bacteria used for fermentation.

Example 3 An equal amount of water was added to 200 g of shucked krill, and after grinding, the pH was adjusted to 5.6 with lactic acid.
After sterilization with Cl2O for a minute, 10 g of Grape Ii and lactic acid bacteria culture [prepared with commercially available yogurt (Snow Brand Nature)]
20g of cultured in milk medium] was added and fermented at 30'C for 1 to 10 days. The pH during this time is maintained at 5.6. After the fermentation was completed, the supernatant and the residue were separated by centrifugation, filtration, etc., and sterilized at 80'C for 130 minutes to obtain a krill lactic acid fermentation liquid. Regarding this krill lactic acid fermentation liquid, AC
E inhibitory activity was measured. As a control, water extracted from shelled krill is shown.

The results are shown in Table 3 below.

Table 3 From the results in Table 3, when we look at the inhibitory activity in terms of the number of days of fermentation, it peaks between days 2 and 5, and the inhibitory activity does not increase much compared to the water-extracted product when it is less than 2 days or more than 5 days. It turns out that it has not been done.

Furthermore, when the protein content of the above-mentioned krill lactic acid fermentation liquid was measured, it rapidly increased by the second day and remained almost unchanged thereafter.

From this, in the present invention, fermentation is carried out for 1 to 10 days.
It turns out that it is preferable to set the period of time to 2 to 5 days.

Example 4 An equal amount of water was added to 200 g of shucked krill, and after grinding, the pH was adjusted to 3 to 7 with lactic acid, and the other was sterilized in an autoclave at 121°C for 20 minutes, and then 1110 g of grapes were added. Add 20 g of lactic acid bacteria culture [previously cultured in milk medium using commercially available yogurt (Nachele Snow Brand)] and ferment at 30°C for 5 days. During this time, the pH is maintained at the adjusted pH. After fermentation is complete, separate the supernatant and residue by centrifugation, filtration, etc., and hold at 80°C.
The mixture was sterilized for 130 minutes to obtain a krill lactic acid fermentation liquid. The ACE inhibitory activity of this krill lactic acid fermentation liquid was measured. As a control, water extracted from shelled krill is shown.

The results are shown in Table 4 below.

Table 4 From the results in Table 4, looking at the inhibitory activity by pH, pH 5~
With a peak at pH 6, those at pH 3, 4, 7 and unadjusted do not have much enhancement compared to those extracted with water, and those at pH 5 and 6 clearly have inhibitory activity compared to those extracted with water. It turns out that it's good for you.

In addition, when the protein content of the above-mentioned krill lactic acid fermentation solution was measured, there was almost no difference in pH 3 or 4, although the unadjusted content was slightly less.

From this, in the present invention, the pH during the fermentation period is 5.
It can be seen that it is preferable to maintain the value at ~6.

Example 5 An equal amount of water was added to 200 g of shucked krill, and after grinding, proteolytic enzymes such as pronase (manufactured by Kaken Kagaku Co., Ltd.), samoase (manufactured by Daiwa Kasei Co., Ltd.), and protease A (manufactured by Sigma Co., Ltd.) were added at l OOU/g each. After digestion at 30°C for 3 hours,
Adjust the pH to 5.6 with lactic acid and autoclave to 121.
After sterilization with Cl2O for 1 minute, add 10 g of glucose and 20 g of lactic acid bacteria culture [previously cultured in milk medium using commercially available yogurt (Kanin Nature)].
Ferment at 0°C for 5 days. The pH between them was adjusted to p
Keep it at H. After the fermentation was completed, the supernatant and the residue were separated by centrifugation, filtration, etc., and sterilized at 80°C for 130 minutes to obtain a krill lactic acid fermentation liquid. The ACE inhibitory activity of this krill lactic acid fermentation liquid was measured. As a control, water extracted from shelled krill is shown.

The results are shown in Table 5 below.

Table 5 From the results in Table 5, when we look at the inhibitory activity for each enzyme, there is no difference depending on the enzyme, and it is clear that the inhibitory activity is higher than that of the unadded and water-extracted
It can be seen that the enzyme-digested product has enhanced inhibitory activity.

Furthermore, when we measured the protein content of the krill lactic acid fermentation liquid mentioned above, the amount of protein in the enzyme-digested product was slightly higher than that in the non-enzymatically digested product, but the protein content was almost the same.

This shows that digestion with a proteolytic enzyme before fermentation enhances the inhibitory activity, and that any commercially available enzyme preparation can be used.

〔Effect of the invention〕

According to the enhancement method of the present invention, the ACE [harmful activity] of krill can be enhanced, a blood pressure lowering effect can be more effectively obtained even with a small amount of intake, and a liquefied krill product that is easy to eat and drink can be obtained.

Claims (3)

[Claims] (1) A method of enhancing angiotensin converting enzyme (hereinafter referred to as ACE) inhibitory activity in krill by fermenting krill with lactic acid bacteria. (2) As lactic acid bacteria, Lactobacillus bulgaricus
Lactobacillus bulgaricus), Lactobacillus acidophilus
) or Streptococcus
, thermophilus), and fermentation is carried out for 1 to 10 days while maintaining the pH at 5 to 6. (3) The method according to claim (1), wherein the krill is degraded with a proteolytic enzyme before fermentation.