JP2986764B2 - Method for preparing high-fischer-ratio peptide with low bitter amino acid composition close to human milk - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the preparation of a peptide having a high Fisher's ratio, in which a plurality of proteins are used as raw materials, and these raw materials are separately treated and then blended at a specific ratio. .

More specifically, the present invention uses casein and whey protein, which are milk proteins, as raw materials, and these raw materials are individually enzymatically hydrolyzed under specific conditions to specifically only aromatic amino acids. By selectively removing and then mixing both proteins thus obtained in a specific ratio, a peptide mixture having a low bitterness, an amino acid balance close to human milk, and a high Fisher ratio, and a method for producing the same are provided. Things. The present invention also relates to a composition containing the peptide mixture having a high Fischer ratio thus obtained, particularly a nutritional supplement composition for patients with liver disease.

[0003]

2. Description of the Related Art It has long been known that amino acids in the body show abnormalities during liver damage, especially during the decompensation of cirrhosis. I
ber et al. ( J. Clin . Lab . Med ., 50 : 4.
17-425, 1957) can be used to produce aromatic amino acids AAA (Tyr, Phe) and methionine (Met) during liver failure.
Etc. reported increasing. On the other hand, Zinman et al. ( Amer . J. Dig . Dis ., 14 : 118-1).
26, 1969) report a decrease in the branched chain amino acid BCAA (Val, Leu, Ile) during the decompensation of cirrhosis. In 1974, Fischer et al. (Amer. J.
Surg . , 127 : 40-47, 1974) is a BC
By positively linking the amino acid imbalance of reduced AA and increased AAA, ie, reduced BCAA / AAA molar ratio, to the development of hepatic encephalopathy and correcting the amino acid imbalance, ie, BCAA as a nutritional source
High content, low AAA and Met content, BCAA
It is reported that hepatic encephalopathy is improved by providing a nitrogen source having a high / AAA molar ratio (Fisher ratio).

[0004] For this reason, amino acid preparations in which purified amino acids mainly Val, Leu and Ile are mixed to prepare a high BCAA / AAA molar ratio are known. That is,
Originally, proteins are used after being decomposed into free amino acids by the action of proteases in vivo. Therefore, it is considered that free amino acids can be easily absorbed in vivo, and have a good nutritional effect especially in postoperative patients having poor digestive absorption ability. JP-A-64-83017 and JP-A-3-22366 disclose amino acid preparations for patients with liver failure.

[0005] However, such amino acid preparations have drawbacks such as being difficult to eat due to their strong chemical taste and being expensive, so that the price of the product itself is expensive. JP-A-56-68374 discloses an example in which a purified amino acid is added to fruit juice or agar to facilitate ingestion. Also, since the amino acid mixture is an electrolyte, the osmotic pressure increases,
Formulation at high concentrations is very difficult.

On the other hand, it is known that oligopeptides, which are hydrolysates of proteins, are taken into cells in the small intestinal tract, decomposed into free amino acids in the cells and absorbed. Then, it is possible to enzymatically decompose animal and plant proteins such as egg white and casein into a hydrolyzate having a high peptide content and use this as a nitrogen source.

However, in this case, the protein is decomposed with an enzyme to form a peptide or an oligopeptide. Therefore, the BCAA / AAA molar ratio of this peptide mixture depends on the molar ratio of the constituent amino acids of the protein itself. Become. In other words, it can be said that the nutritional value as a nitrogen source largely depends on the raw protein.

Furthermore, if a protein having a low AAA content, ie, a high BCAA / AAA molar ratio, is used, the protein source is suitable as a nitrogen source for patients with liver disease, and it can be decomposed with enzymes. In this case, the peptide is in the form of a peptide, so that the digestibility is further improved. Therefore, a peptide mixture obtained by enzymatically decomposing a protein having a high BCAA / AAA molar ratio has a high BCAA / AAA molar ratio and has good digestibility and absorbability, and thus is considered to be suitable as a nitrogen source for liver disease patients. However, in daily available foods or food ingredients, the B
The CAA / AAA molar ratio is not very high. JJP
As shown in EN 3 (2) 109-117 (1981), the BCAA / AAA molar ratio is approximately 0.8-3.0 for plant proteins, and 2.1-3.0 for animal proteins. It is about 4.

[0009]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the state of the art in order to efficiently create a peptide mixture having a low bitterness and a high Fisher ratio.

[0010]

SUMMARY OF THE INVENTION The present invention has been made to achieve the above object, and as described above,
At this time, there is no naturally derived food with a high Fisher ratio, and amino acid preparations are not only expensive, but they are not originally foods. It was made in view of the high natural product orientation.

In view of this situation, the present inventors decided to prepare a safe and low-cost peptide having a high Fischer ratio, which is derived from natural products or foods and has no bitterness. After some consideration, it did not succeed.

Therefore, the present inventors made a change in idea,
Instead of using a single raw material, we first focused on blending multiple things. Then, the present inventors studied the selection of the raw materials used, the processing method of each raw material, etc., confirmed the removal of allergens and bitterness, determined the optimal mixing ratio, integrated these research results, and finally The first successful creation of the desired high Fischer ratio peptide mixture has led to the present invention.

[0013] That is, the present invention focuses on milk proteins from among many natural raw materials, further selects casein and whey protein from among them, uses both of them as raw materials, and separately uses them. Enzymatic hydrolysis into
It decomposes proteins and loses antigenicity, then specifically adsorbs and removes hydrophobic components, especially aromatic amino acids, which inhibit the increase of Fisher ratio and also contributes to bitterness. The present invention relates to a method for preparing a peptide mixture having a low bitterness, an amino acid balance close to that of human milk, and a high Fisher ratio by mixing the same.

[0014]

DETAILED DESCRIPTION OF THE INVENTION In the present invention, a plurality of raw materials, namely, casein and whey protein, are used instead of a single raw material as in the prior art. Casein includes α, β,
In addition to various caseins such as κ, calcium caseinate, sodium caseinate, potassium caseinate, magnesium caseinate and the like are appropriately used. In addition, casein phosphopeptide (hereinafter referred to as C) obtained from trypsin digest of casein
PP may be used as a part or whole of casein, since it has a higher Fischer ratio of 10 or more than casein and has no bitterness.

As whey protein (sometimes called whey protein), acid whey and sweet whey, which are by-products when producing casein and cheese from milk, are used as raw materials, and all purified whey proteins are used as whey protein. Is done. For example, whey protein includes purified whey protein, fractionated whey protein, and processed products thereof (concentration,
Which have been subjected to processes such as dilution, pasting, and drying). The purified whey (whey) protein includes whey (whey) protein concentrate and whey (whey) protein isolate.

Whey protein concentrate (Whey Prot)
Ein Concentrate (hereinafter sometimes referred to as “WPC”) refers to a dried product obtained by concentrating whey produced as a by-product when producing cheese or casein and separating lactose that crystallizes. In this WPC, 35-80% of its content is protein, of which about 60% is β-lactoglobulin, about 20% is α-lactalbumin, and about 13% is immunoglobulin, Usually neutral.

Whey protein isolate (Whey Prot)
ein Isolate (hereinafter sometimes referred to as “WPI”) is distinguished from WPC, in which whey protein is adsorbed on an anion exchange column, eluted by pH adjustment, and then protein is passed through an ultrafiltration membrane. Is a native whey protein that has been dried after being concentrated, and has a much lower level of ash, fat and carbohydrates. In particular, the fat content is as low as 1% or less.

[0018] The whey (whey) protein fraction refers to all fractions obtained by de-lactating acid whey and sweet whey, and examples thereof include α-lactalbumin and β-lactoglobulin. .

The peptide mixture having a high molar ratio of BCAA / AAA of the present invention can be obtained by enzymatically decomposing the protein having a high molar ratio of BCAA / AAA to liberate and remove AAA and the peptide portion containing AAA. be able to.

In the present invention, these proteins are heated in an aqueous system, or acid or alkaline, in order to facilitate hydrolysis by an enzyme as described later and to facilitate release of AAA and a peptide portion containing AAA. It is preferable that the denatured product be denatured by being placed under conditions. This facilitates the action of the enzyme on the protein, and thus facilitates the release of AAA or the peptide portion containing AAA.

[0021] Endoproteases and exoproteases are used as enzymes for hydrolyzing proteins. It is difficult to release AAA or a peptide portion containing AAA using only the endoprotease, and it is not possible to reduce the molecular weight of the protein using only the exoprotease, and it is not possible to obtain a desired peptide mixture.

As shown in the present invention, an endoprotease and an exoprotease are allowed to act in combination,
Further, the peptide mixture of the present invention can be obtained by performing the adsorbent treatment described below. The peptide mixture thus obtained has little bitterness, astringency, or smell of amino acids, and is suitable as a nitrogen source for liver disease patients.

The endoprotease used in the present invention may be any enzyme derived from animals, plants, or microorganisms, as long as it acts on the C-terminal or N-terminal peptide bond of AAA or a peptide portion containing AAA. I do not care. For example, pepsin or chymotrypsin produced from pig or bovine offal is suitable for use in the present invention because it mainly cleaves the peptide bond at the C-terminal side of AAA in protein.

In the present invention, one or more endoproteases can be used in combination. Since only one type of endoprotease does not necessarily act on the C-terminal or N-terminal peptide bonds of all AAAs or peptide portions containing AAA contained in a protein, it is preferable to use a combination of different types of endoproteases. .

The exoprotease used in the present invention may be any enzyme derived from animals, plants, or microorganisms, as long as it acts sequentially on the C-terminal or N-terminal peptide bond of AAA or a peptide portion containing AAA. But it doesn't matter.

The degree of enzymatic degradation is adjusted by adjusting the working temperature, the working time, and the substrate / enzyme ratio so that the average molecular weight of the peptide mixture solution after the enzymatic decomposition is 5,000 or less, preferably 200 to 3,000.

After completion of the enzyme treatment, the enzyme is deactivated. Then, the adsorbent treatment may be performed immediately. However, if the unnecessary solid content is removed in advance by performing a filtration treatment before that, the treatment can be performed more efficiently, and an excellent peptide can be obtained. Various filtration treatments are appropriately performed as the filtration treatment. For example, the filtration treatment can be performed with a microfiltration membrane having a molecular weight cut-off of 0.05 to 1 μm, preferably 0.08 to 0.8 μm.

Here, according to the present invention, when casein or the like is hydrolyzed with an endoprotease, the resulting decomposed product has a molecular weight close to a normal distribution and a small average molecular weight, and does not show antigenicity. It has the drawback of strong bitterness.

Therefore, as a result of examination of the removal of the bitter taste, it was found that the peptide fraction having a strong bitter taste tends to contain a large amount of the peptide having a strong hydrophobicity,
It was found that yr and Phe tended to be large. Then, as a result of various studies on methods for specifically selecting and adsorbing and removing these hydrophobic components to remove bitterness, attention was paid to adsorbent treatment.

That is, in the present invention, the above-mentioned enzyme-treated product, particularly the permeate obtained by filtration if necessary, is treated with an adsorbent. As the adsorbent, any adsorbent capable of specifically adsorbing a hydrophobic component such as AAA such as Tyr and Phe can be used. Synthetic adsorbent Sepabead SP206 (manufactured by Mitsubishi Chemical), diamond Ion HP20 (manufactured by Mitsubishi Chemical Corporation) and the like are exemplified.

In the present invention, the casein-derived peptide and the whey protein-derived peptide thus obtained are blended. The mixing ratio of the two is 0.5 to 3 parts by weight, preferably 1 to 2 parts by weight, with respect to 1 part by weight of the former.

Each of the peptides derived from casein and whey protein produced by the above-mentioned steps can be subjected to various treatments such as concentration, sterilization or drying of the treatment solution in the final step, if necessary. The same applies to the peptide mixture obtained above.

Thus, the branched-chain amino acids (BCA
A peptide mixture having a molar ratio of A) / aromatic amino acid (AAA), that is, a Fisher ratio of 8 to 20, is obtained. Moreover, the high Fischer ratio peptide mixture according to the present invention has less bitterness, an amino acid balance similar to human milk, and is excellent.

The peptide mixture of the present invention can be used as an oral preparation for patients with liver disease or as a nitrogen source in therapeutic diets for diet.

The nutritional supplement composition for a liver disease patient of the present invention contains the above-mentioned peptide mixture, except that a known oral preparation for a liver disease patient or diet is used except for using a nitrogen source comprising the peptide mixture. As in the case of therapeutic foods, nitrogen compounds as various protein sources, carbohydrates, lipids, vitamins, minerals, other additives, etc. are added and mixed as necessary, and manufactured or used in combination with these. No problem.

As a protein source, milk whey proteins having a high amino acid score, animal proteins such as ovalbumin and egg white globulin, and vegetable proteins such as soy protein can be used.

As the carbohydrate, known monosaccharides, oligosaccharides, polysaccharides, dietary fiber and the like can be used. For example, glucose, maltose, sucrose, lactose, isomaltose, starch, dextrin and the like can be exemplified.

As the lipid, known coconut oil, corn oil, cottonseed oil, lard, palm oil, peanut oil, safflower oil, soybean oil, sunflower oil, coconut oil, MCT oil, fish oil, etc., or the fatty acid composition is adjusted. For this purpose, there may be mentioned, for example, an adjusted fat obtained by mixing various kinds of the above fat.

The composition according to the present invention can be used not only for patients with liver disease as described above, but also for the sick and babies with impaired liver function, It can be used for people with reduced liver function, and of course, it can be used by healthy people to prevent liver function deterioration. Therefore, the composition according to the present invention is used not only for patients with liver disease but also for those with illness, not only for use in the pharmaceutical type, but also for beverages, foods, nutritional foods, functional foods, foods for specified health uses, and food and drink types such as drinks. It can be widely used for healthy persons, persons after illness, and healthy persons.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples unless it departs from the gist.

[0041]

Example 1 Separated whey protein (WPI) as starting material
Was used to perform the enzymatic degradation treatment described below to prepare a high Fisher ratio peptide (HFP).

The WPI serving as a substrate is 3 to 10 (w / w)%
After dissolution, the pH was adjusted to 6.0 to 8.0 with a solution in which NaOH and KOH were mixed at a ratio of about 2: 1. This aims at considering the mineral balance in the finally obtained HFP and omitting the desalting treatment. Then, after denaturation by heat treatment at 90 ° C. for 15 minutes, the plate was sterilized at 120 ° C. for 15 minutes by a plate-type sterilizer.
Sterilized for 2 seconds.

After cooling to 50 ° C., Amano N (manufactured by Amano Pharmaceutical), a neutral endoprotease, was added to the substrate in 1 /
100 and kept for 5 hours. 90 ° C,
After inactivating the enzyme for 15 minutes, the mixture was cooled to 50 ° C. Next, Amano M (manufactured by Amano Pharmaceutical Co., Ltd.), which is an acidic exoprotease, was added so as to be 1/100 with respect to the substrate, and kept at 50 ° C. for 4 hours. The optimal pH of Amano M is 3.5 to 4.5, but the pH after Amano N decomposition
Even in the pH range of 5.0 to 7.0, the activity was sufficient, so the pH was not particularly adjusted here.

Next, after inactivating the enzyme at 90 ° C. for 15 minutes, the enzyme was cooled to 20 to 40 ° C., and the permeate was passed through a microfiltration membrane having a fraction particle size of 0.1 to 0.45 μm. Was collected.

The synthetic adsorbent Sepabeads SP206 (manufactured by Mitsubishi Chemical Corporation) was added in an amount 6 times or more the total solid content in the permeate, and the mixture was gently stirred for 30 minutes in a path (this operation was performed in a column. It was also possible.) Then, this was filtered, and the filtrate was concentrated to obtain a HFP concentrate.
This was further freeze-dried and spray-dried to obtain powdered HFP.

[0046]

Example 2 Casein (Na-Case) was used as a starting material.
Inate and Ca-Caseinate) were subjected to the following enzymatic degradation to prepare a high-Fisher-ratio peptide (HFP) having a low bitterness.

Na-Caseinate 3 serving as a substrate
5 parts by weight and 7 parts by weight of Ca-Caseinate
(W / w)%, and then the solution was mixed with NaOH and KOH at a ratio of about 1: 4 to obtain a pH of 6.0 to 6.0.
It was adjusted to 8.0. Then, 1 plate sterilizer
Sterilization treatment was performed at 20 ° C. for 15 seconds.

After cooling to 50 ° C., Amano N (manufactured by Amano Pharmaceutical), a neutral endoprotease, was added to the substrate in 1 /
250 and kept for 24 hours. 90
After inactivating the enzyme under the conditions of 15 ° C. and 15 minutes, 20 to 40
° C, and the fractionated particle size is 0.1-0.45μ
The permeated liquid was collected with a microfiltration membrane of m.

The synthetic adsorbent Sepabeads SP206 (manufactured by Mitsubishi Chemical Corporation) was added in an amount 6 times or more the total solid content in the permeate, and the mixture was gently stirred for 30 minutes in a path (this operation was performed in a column. It was also possible.) Then, this was filtered, and the filtrate was concentrated to obtain a HFP concentrate.
This was further freeze-dried and spray-dried to obtain powdered HFP.

Na-Ca treated according to the above embodiment
seinate / Ca-Caseinate and WPI
Table 1 below shows the amino acid composition and Fisher ratio of

[0051]

[Table 1]

[0052]

Embodiment 3 Lyophilized W treated according to the above embodiment
6 kg of PI and freeze-dried Na-Caseinate C
4 Kg of a-Caseinate was mixed to obtain a peptide mixture having a low bitter taste, an amino acid balance similar to human milk, and a Fisher ratio of 9.7.

[0053]

Example 4 Lyophilized Na-Caseina of Example 3
1kg of te ・ Ca-Caseinate 4kg
Was replaced with CPP (produced by the method of Japanese Patent Publication No. Hei 7-24597), and the others were the same as in Example 3 except that the amino acid balance was similar to human milk with less bitterness, and the Fisher ratio was 10.8. Was obtained. The amino acid composition and the Fisher ratio of the CPP used were as shown in Table 2 below.

[0054]

[Table 2]

[0055]

Industrial Applicability According to the present invention, a flavorful peptide mixture as a nitrogen source suitable for patients with liver disease can be produced. The peptide mixture can be widely used as a nitrogen source for various therapeutic preparations used for patients with liver disease, therapeutic diets for diet therapy, and the like. In addition, it can be widely used for people with reduced liver function, and of course, it can be widely used for health by healthy persons for the purpose of preventing liver function deterioration.

Since the composition for a patient with liver disease of the present invention mainly contains a peptide mixture as a nitrogen source, it has good absorbability from the gastrointestinal tract and has a lower osmotic pressure than the amino acid mixture. Therefore, diarrhea and the like can be avoided, and even when taken orally, the flavor is good and the burden on the patient is small. In addition, it can significantly improve the amino acid pattern and the BCAA / AAA molar ratio in the plasma of a liver disease patient.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Tomoko Ueda 1-21-3 Sakaecho, Higashimurayama-shi, Tokyo Meiji Dairy Products Co., Ltd. Nutrition Science Research Institute (56) References JP-A-2-300137 (JP, A) JP-A-3-272694 (JP, A) JP-A-5-276896 (JP, A) JP-A-7-264992 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) A23L 1 / 305 A23J 3/08-3/10 A23J 3/34

Claims (6)

(57) [Claims] 1. After hydrolyzing casein and whey protein individually to adsorb and remove hydrophobic components, the ratio of the former to the former is 0.5 to 3, preferably 1 to 2. A method of preparing a peptide mixture having a low bitterness, an amino acid balance similar to human milk, and a branched chain amino acid / aromatic amino acid molar ratio of 8 to 20, characterized by mixing. 2. Whey protein purified whey protein,
The method according to claim 1, wherein at least one selected from a whey protein fraction and the processed product is used. 3. The purified whey protein is whey protein concentrate (WPC) or whey protein isolate (WPI), and the whey protein fraction is β-lactoglobulin or α-lactalbumin. The method according to claim 1, wherein: 4. A peptide mixture prepared by the method according to claim 1. 5. A composition comprising the peptide mixture according to claim 4. 6. The composition according to claim 5, wherein the composition is a nutritional supplement composition for a patient with liver disease.