JPH04287693A - Production of epsilon-polylysine - Google Patents

Abstract

PURPOSE:To obtain in high yield the title compound to be used as a food additive, little in harsh taste, having cholesterol level-suppressing and reducing activity, antifungal activity and antiphage activity by digesting epsilon-polylysine of a specific polymerization degree with a neutral protease. CONSTITUTION:epsilon-Polylysine of the formula ((n) is >=20) with a polymerization degree of >=20 is dissolved in water and the pH value of the resulting aqueous solution is adjusted to 6-8 with 6N HCl followed by addition of a neutral protease (e.g. denazyme AP) to be produced by Aspergillus (e.g. Aspergillus oryzae), and then hydrolysis at 40 deg.C for 24hr followed by keeping the system at 90 deg.C for 30min to deactivate the protease. The resulting liquor is put to spray drying, thus obtaining the objective epsilon-polylysine with a polymerization degree of 2-19, little in harsh taste, and useful as an additive for foods and beverages.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing ε-polylysine having a degree of polymerization of 2 to 19.

0002

[Prior art] ε-polylysine with a degree of polymerization of 20 to 25 is
It is known to have antibacterial activity against various fungi such as gram-positive bacteria, gram-negative bacteria, fungi, lactic acid bacteria, and yeast, and antiphage activity against bacteriophages. It is proposed to be used as a food, etc. Further, as a result of research conducted by the present inventors, it was discovered that ε-polylysine having the above degree of polymerization has an effect of suppressing and reducing an increase in serum and liver cholesterol levels.

By the way, ε-polylysine having the above degree of polymerization has a persistent strong acrid taste (a strong unpleasant taste). Therefore, not only when directly ingested, but also when added in small amounts to foods and drinks, it has an acrid taste, making it difficult or unpleasant to ingest, and can also affect the texture and flavor of foods and drinks. I'm making it a thing. In particular, when adding ε-polylysine having the above degree of polymerization to foods and drinks as a cholesterol suppressant, it is desirable to add 1% or more from the viewpoint of the expression of effects, but adding 1% or more does not improve the taste of the food and drinks. They tend to make foods taste harsh and significantly reduce the original texture and flavor of foods and drinks.

0004

[Contents of the invention] The present inventors aimed to make the taste of ε-polylysine non-aggressive while maintaining the above-mentioned useful properties such as antibacterial action, antiphage action, and cholesterol suppressing action. I have continued my research. the result,
ε-polylysine with a degree of polymerization of 19 or less has a considerably less harsh taste, and has the same antibacterial, antiphage, and cholesterol-inhibiting effects as ε-polylysine with a degree of polymerization of 20 to 25; ε− below
It has been found that polylysine can be obtained by hydrolyzing the above-mentioned ε-polylysine having a degree of polymerization of 20 to 25 or a higher degree of polymerization using a neutral protease.

Therefore, the present invention is a method for producing ε-polylysine having a polymerization degree of 2 to 19 by hydrolyzing ε-polylysine having a polymerization degree of 20 or more with a neutral protease. ε-polylysine is represented by the following chemical formula 1.

[0006]

[Chemical formula 1]

In the present invention, any ε-polylysine having a degree of polymerization of 20 or more can be used as a raw material. In that case, if the ε-polylysine raw material is mostly composed of ε-polylysine with a degree of polymerization of 20 or more, it may be a single ε-polylysine with only one degree of polymerization, or it can be made of ε-polylysine with various degrees of polymerization. It may be a mixture of ε-polylysine, and may further contain a small amount of ε-polylysine having a degree of polymerization of 19 or less depending on the case. As ε-polylysine having a degree of polymerization of 20 or more, ε-polylysine with a degree of polymerization of 20 to 25, which is commercially available from Chisso Corporation, etc., can be used to efficiently convert ε-polylysine with a degree of polymerization of 2 to 19. can be manufactured,
Moreover, it is desirable because it is easy to obtain.

[0008] Hydrolysis treatment with neutral protease is
It is preferable to use the raw material ε-polylysine having a degree of polymerization of 20 or more in the form of an aqueous solution or an aqueous dispersion. and,
As a result of the research conducted by the present inventors, it has been found that, among many neutral proteases, neutral protease produced by Aspergillus bacteria has a target degree of polymerization of ε of 2 to 19. - advantageous for the production of polylysine, especially Aspergillus sojae
Fungus, Aspergillus oryzae
oryzae), and Aspergillus meleus (
It was found that a neutral protease produced by Aspergillus melleus) is suitable.

Therefore, in a desirable embodiment of the present invention, a neutral protease produced by a bacterium of the genus Aspergillus is used, and the degree of polymerization is 2.
It includes a method for producing ε-polylysine having a polymerization degree of 2 to 19 from ε-polylysine having a polymerization degree of 0 or more. Here, "neutral protease produced by Aspergillus genus" refers to Aspergillus (Aspergillus).
Protease is produced by bacteria of the genus Illus and exhibits reactivity in the neutral pH range of 6 to 8, that is, it refers to a protease that hydrolyzes peptide bonds.

[0010] The above-mentioned Aspergillus (Asp.
Among the neutral proteases produced by bacteria of the genus Aspergillus, Aspergillus sojae
The neutral protease produced by the bacterium
Enzyme [Seishin Pharmaceutical Co., Ltd.], PROTEASE7026 (
Aspergillus oryzae (As
Neutral protease produced by Pergillus oryzae) is Denazyme AP [Nagase Sangyo Co., Ltd.],
Protin FN [Daiwa Kasei Co., Ltd.], Protease A and Protease M [Amano Pharmaceutical Co., Ltd.], Sumiteam LP
[Shin Nippon Chemical Co., Ltd.], Orientase ON [Ueda Chemical Co., Ltd.], PROTEASE4032 (SIG
MA), etc., and Aspergillus meleus (As
pergillus melleus) is commercially available as Protease P [Amano Pharmaceutical Co., Ltd.] and is easily available. Among these proteases, Denazyme AP is particularly preferred since it has good decomposition efficiency.

[0011] The neutral protease may be used in a free state or immobilized on a carrier. Further, the hydrolysis treatment using a neutral protease may be carried out in a continuous method or in a batch method. Depending on whether the neutral protease is used in a free or immobilized state, whether the hydrolysis treatment is carried out in a continuous or batch process, etc.
Type of neutral protease, amount used, temperature during treatment,
The treatment may be carried out by appropriately selecting various conditions such as pressure, pH, treatment time, etc. Generally, the larger the amount of neutral protease used, the faster ε-polylysine with the desired degree of polymerization can be obtained. Aspergillus
s) When using a neutral protease produced by a bacterium belonging to the genus s), the pH of the solution is usually adjusted to about 6 to 8, and the hydrolysis is preferably carried out at a temperature of about 35 to 45°C.

[0012] ε-polylysine having a degree of polymerization of 20 or more is hydrolyzed by a neutral protease, and gradually becomes ε-polylysine with a low degree of polymerization of 19 or less. In that case, if the hydrolysis is excessive, it will eventually become lysine, which is an amino acid, and the desired degree of polymerization will decrease to 2.
~19 ε-polylysine is not obtained. Therefore, during hydrolysis treatment, the reaction solution is sampled over time during the hydrolysis process, and the proportion of ε-polylysine with a degree of polymerization of 2 to 19 in the product is determined by reverse phase chromatography or other appropriate methods. It is desirable to analyze to determine whether to continue or stop the hydrolysis reaction.

Usually, the raw material ε- with a degree of polymerization of 20 or more is used.
The hydrolysis reaction is preferably stopped when about 80% by weight or more, preferably about 90% by weight or more of the polylysine has been hydrolyzed. The hydrolysis reaction can be stopped by inactivating the neutral protease by heating or other suitable means. When neutral protease is inactivated by heating, it is generally recommended to heat to about 80 to 100°C.

For example, ε-polylysine with a degree of polymerization of 20 to 25 is prepared as an aqueous solution with a concentration of 50 mg/ml and a pH of about 6 to 8,
About 125 units/ml of Aspergillus (A
When a neutral protease produced by a bacterium of the genus Spergillus is added and hydrolyzed at a temperature of about 35 to 45°C, the protease is deactivated after about 24 to 48 hours, and the ε-polylysine used as a raw material is Approximately 95 to 9
9% by weight is hydrolyzed to yield a product containing about 94-98% by weight of ε-polylysine with a degree of polymerization of 2-19.

[0015] ε- with a degree of polymerization of 2 to 19 contained in the above liquid.
Components other than polylysine include a small amount of lysine and ε-polylysine having a degree of polymerization of 20 or more. The above liquid may be dried, for example, by spray drying, freeze drying, or other suitable drying means to obtain a powder containing a large amount of ε-polylysine having a desired degree of polymerization of 2 to 19. The ε-polylysine having a degree of polymerization of 2 to 19 obtained as described above may contain a small amount (usually about 10% or less) of ε-polylysine having a degree of polymerization of 20 or more so as not to cause an astringent taste.

If it is desired to obtain a product with higher purity, the liquid containing ε-polylysine with a degree of polymerization of 2 to 19 obtained above is subjected to one of the separation methods such as ion exchange, ultrafiltration, and gel filtration.
By treating one or more of them in combination, the desired ε-polylysine can be produced individually or with two or more degrees of polymerization.
It can be separated and recovered in the form of a mixture with any degree of polymerization between 19 and 19.

The above-obtained ε-polylysine with a degree of polymerization of 2 to 19 has the same effects as ε-polylysine with a degree of polymerization of 20 or higher, as well as an effect of suppressing and lowering the increase in cholesterol levels, an antibacterial effect, and an antiphage effect. There is. Moreover, the degree of polymerization is 20
Unlike the above-mentioned ε-polylysine, it has no harsh taste, so it is easy to ingest and can be effectively used as a cholesterol suppressant, antibacterial agent, antiphage agent, etc. by itself. When ε-polylysine with a degree of polymerization of 2 to 19 is administered as such, an aqueous solution; a solid preparation (tablet, pill, granule, capsule, etc.) supported on a carrier such as starch, talc, or calcium carbonate, etc. Any dosage form can be taken. In addition, it can be added to various foods and drinks (e.g. bread, cookies, biscuits, noodles, chewing gum, drinks, etc.), and in that case, it does not have an acrid taste, so it does not impair the texture or flavor of the food or drink. Never.

[0018]

[Example] <<Example 1>> After dissolving 200 g of ε-polylysine manufactured by Chisso Corporation (containing 50% by weight of ε-polylysine with a degree of polymerization of 20 to 25) in 2000 ml of water, 6
The pH of the aqueous solution was adjusted to 7.0 using N hydrochloric acid. A part of this ε-polylysine aqueous solution was collected and subjected to reverse phase chromatography [column used: Waters μ Bon
dapak C18: Made by Nippon Waters: Inner diameter 3.
9 mm, length 30 mm]. Next, using an aqueous solution containing 0.1% trifluoroacetic acid (liquid A) and an 80% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid (liquid B), in a mixed solution of liquids A and B. In,
Elution was performed at a flow rate of 1.0 ml/min with a concentration gradient in which the concentration of solution B linearly increased from 0% to 20% over 60 minutes. When this effluent was detected with UV light at a wavelength of 220 nm, the chromatogram shown in FIG. 1 was shown.

Denazyme AP [manufactured by Nagase Sangyo Co., Ltd.: Aspergillus oryzae (Aspe.
5 g of neutral protease produced by S. rgillusoryzae) was added and reacted at 40°C for 24 hours, at which point the mixture was kept at 90°C for 30 minutes to inactivate the protease. As a result, about 95% by weight of the raw material ε-polylysine having a degree of polymerization of 20 to 25 was hydrolyzed. The hydrolyzate thus obtained was spray-dried to produce a powdered product 190
I got g. This hydrolyzed solution was subjected to reversed phase chromatography and a chromatogram was taken in exactly the same manner as the ε-polylysine aqueous solution before hydrolysis treatment, and the results shown in FIG. 2 were obtained. The powdered product obtained in Example 1 contained about 47% by weight of ε-polylysine having a degree of polymerization of 2 to 19.

<<Example 2>> Hydrolysis treatment was carried out in the same manner as in Example 1 except that the hydrolysis treatment with Denazyme AP was carried out at 40°C for 48 hours, and the resulting hydrolyzed liquid was spray-dried. However, the powdered product 192
I got g. This hydrolyzed solution was subjected to reverse phase chromatography in the same manner as in Example 1 above, and a chromatogram was taken, and the results shown in FIG. 3 were obtained. The powdered product obtained in Example 2 contained about 49% by weight of ε-polylysine having a degree of polymerization of 2 to 19.

Sensory (taste) test The powdered products obtained in Examples 1 and 2, and the above-mentioned ε- manufactured by Chisso Corporation before being subjected to hydrolysis treatment.
Polylysine (50% ε-polylysine with a degree of polymerization of 20 to 25)
Grapefruit juices A to C having the formulations shown in Table 1 below were prepared using each of the following. Each grapefruit juice was consumed by 13 panelists, and the quality of the taste was tested according to the following evaluation criteria. The results are shown in Table 1.

[0022] Here, the taste score in Table 1 is 13.
This is the average score given by the named panelists. [Evaluation Criteria] 5 points: A very strong acrid taste and an extremely poor taste. 4 points: A strong acrid taste and a very poor taste. 3 points.
・2 points with a harsh taste and inferior taste ・1 point with a slightly astringent taste and a slightly inferior taste...
・0 points with a slight acrid taste and a slightly inferior taste...no acrid taste and a good taste

[0023]

[Table 1]

From the results in Table 1, it can be seen that the grapefruit juices A and B containing ε-polylysine with a degree of polymerization of 2 to 19 produced in Examples 1 and 2 of the present invention are different from the control grapefruit juices containing no ε-polylysine. It has a good taste with almost no harsh taste as juice, or has less harsh taste, but has a degree of polymerization of 20 or more.
- It can be seen that grapefruit juice C containing polylysine has a strong harsh taste and is inferior in taste.

[Reference Example] Cholesterol Suppressing Effect Test Four groups of 5-week-old Fisher female rats were prepared, each with 7 rats, and each group was preliminarily housed in separate wire mesh cages for 4 days.
For one day, each group was given free access to feed and water having the composition shown in Table 2. The breeding environment conditions were: humidity 60±10%;
The temperature was 23.0±1.0° C., and the lighting period was from 10:00 p.m. to 10:00 the next day every day. As is clear from Table 2, the first
The feed for the rats in the group (standard group) was a standard feed that did not contain cholesterol, lard, and sodium cholate, and the feed for the rats in the second group (control group), the third group, and the fourth group (all inventive groups) The feed for is a high cholesterol feed containing cholesterol, lard and sodium cholate. Among the high-cholesterol feeds of the second to fourth groups, the feeds of the third and fourth groups further contain ε-polylysine with a degree of polymerization of 2 to 19 obtained in Example 1 and Example 2. are doing.

[0026]

[Table 2]

[0027] The initial body weight at the start of rearing, final body weight at the end of rearing, weight gain, feed intake during the rearing period, and feed efficiency of the rats in Groups 1 to 4 above were investigated, and the results are shown in Table 3 below.
It was as shown in . The values in Table 3 are average values per rat, and the feed efficiency is expressed by the following formula. Feed efficiency = (weight gain/feed intake) x 100

[
0028

[Table 3]

From the results in Table 3 above, it can be seen that both the rats in the first group fed the standard diet and the rats in groups 2 to 4 fed the high-cholesterol diet had good growth, and that the rats in the first group fed the standard diet had good growth. Although there is a significant difference between rats in Group 1 and rats in Groups 2 to 4, there is no significant difference in weight gain between rats in Group 1 and rats in Groups 2 to 4. I understand.

During the above breeding period, blood was collected from the tail vein on the 4th and 7th day, and from the inferior aorta on the 11th day, and the total cholesterol in the blood was measured using Richmoncl W. As a result of quantitative determination using the method of et al., the results shown in Table 4 below were obtained.

[0031]

[Table 4]

Furthermore, after fasting for 14 hours from 19:00 on the end of the 11-day rearing period to 9:00 the next day, the liver was dissected under ether anesthesia, the liver was removed and its weight was measured, and the method of Folch et al. Lipids were extracted from the excised livers and analyzed for total cholesterol, free cholesterol, phospholipids, and neutral lipids. The results are shown in Table 5 below.

[0033]

[Table 5]

From the results in Tables 4 and 5 above, the rats of the third and fourth groups fed the high cholesterol feed containing ε-polylysine with a degree of polymerization of 2 to 19 produced by the method of the present invention, Although the amount of cholesterol in the blood and liver was higher compared to the rats of the first group fed the standard diet, the amount of cholesterol in the blood was higher than that of the rats of the second group fed the high cholesterol diet that did not contain ε-polylysine. The amount of cholesterol in the liver and liver was reduced, indicating that ε-polylysine with a degree of polymerization of 2 to 19 has a suppressive effect on cholesterol levels.

Example 3 Using various proteases shown in Table 6 below, ε-polylysine having a degree of polymerization of 20 to 25 was hydrolyzed in the same manner as in Example 1. At that time, the pH of the ε-polylysine-containing aqueous solution is determined from Table 6, which is said to be optimal for the activity of each protease.
The pH value shown in was adopted. The hydrolyzed solutions obtained in each case were subjected to reverse phase chromatography in the same manner as in Example 1, and chromatograms of the products contained therein were taken. As a result, the hydrolysis ability of each protease for medium degree of polymerization ε-polylysine with a degree of polymerization of 20 to 25 was as shown in Table 6.

[0036] In Table 6, the hydrolytic ability of protease was determined according to the following criteria. Judgment criteria for hydrolyzing ability of protease +...ε-polylysine was hydrolyzed. -...ε-polylysine was not hydrolyzed at all.

[0037]

[Table 6]

From the results in Table 6, Aspergillus (Asp.
Neutral protease produced by bacteria of the genus S. ergillus is capable of converting ε-polylysine with a polymerization degree of 2 to 19 from ε-polylysine with a polymerization degree of 20 or more.
It can be seen that it is particularly suitable as a protease for producing ε-polylysine.

[0039]

Effect of the invention: By the method of the present invention, the degree of polymerization is 2 to 19.
ε-polylysine can be smoothly produced. In particular, as a protease, Aspergillus
When a neutral protease produced by a bacterium belonging to the genus S. llus is used, the target ε-polylysine can be obtained in high yield. The ε-polylysine with a degree of polymerization of 2 to 19 produced by the method of the present invention has a considerably less harsh taste, and also has cholesterol level increase suppressing and lowering effects, antibacterial effects, and antiphage effects, and can be used as is or It can be effectively used by adding it to food and drinks.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a chromatogram obtained by subjecting a commercially available solution containing ε-polylysine with a degree of polymerization of 20 to 25 to reverse phase chromatography, and then detecting the effluent obtained by elution using UV light at a wavelength of 220 nm.

[Figure 2] The hydrolyzate obtained in Example 1 was subjected to reverse phase chromatography, and the effluent was then eluted with a wavelength of 2
It is a figure showing a chromatogram detected with 20 nm UV.

[Figure 3] The hydrolyzate obtained in Example 2 was subjected to reverse phase chromatography, and the effluent was then eluted with a wavelength of 2
It is a figure showing a chromatogram detected with 20 nm UV.

Claims (2)

[Claims] Claim 1: Hydrolyzing ε-polylysine with a degree of polymerization of 20 or more with a neutral protease, the degree of polymerization can be reduced to 2.
A method for producing ε-polylysine of ~190. 2. The production method according to claim 1, wherein the neutral protease is a neutral protease produced by a bacterium belonging to the genus Aspergillus.