JP3202093B2 - Oligopeptide mixture and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a mixture of oligopeptides and a method for producing the same. Specifically, the oligopeptide mixture obtained in the present invention has excellent absorbability in the digestive tract,
It is extremely useful as a nutrient source at the time of invasion and intestinal dysfunction, and also has excellent heat stability, and is extremely useful as a tube and oral nutritional supplement, especially when the method of the present invention is used. Oligopeptide mixtures with properties can be produced very safely and economically in high yields.

[0002]

2. Description of the Related Art Animal protein, soybean, wheat, etc. contained in milk and eggs for use in nutritional components and functional foods.
Various attempts have been made to hydrolyze plant proteins and the like contained in corn and the like with a protease (such as a protease) into low-molecular-weight peptides. In particular,
Dipeptides and tripeptides in which two or three amino acids are bonded are attracting attention as materials for tube feeding, oral nutritional supplements, functional drinks, and the like because of their excellent absorbability in the digestive tract.

[0003] However, it has been difficult to efficiently produce an oligopeptide mixture having a high dipeptide or tripeptide content in view of the difficulty in selecting a protease and strict control of an enzyme reaction. In particular, since wheat protein is hardly soluble in water, it is difficult to be decomposed by proteases.Therefore, various methods for producing high molecular weight peptides by hydrolysis of wheat protein have been developed, but low protein containing a large amount of dipeptide or tripeptide has been developed. At present, a mixture of oligopeptides of a molecular weight has not yet been obtained.

In recent years, with the advancement of clinical nutrition, specific amino acids such as branched-chain amino acids and glutamine are extremely effective against abnormal metabolism of proteins and amino acids at the time of stress load such as invasion of living organisms, and good medical care is provided. Has been shown to be effective [Okada; “Chemistry and Biology”, 27,
585 (1989)]. The branched-chain amino acids have already been used as nutritional supplements for liver failure and the like, and applications in peptide form have been attempted. Glutamine is also important as an energy source for the intestinal tract.It improves nitrogen balance during invasion and stress, improves small intestinal function such as preventing gastrointestinal mucosal atrophy during central parenteral nutrition, gastric ulcer and duodenum. Its efficacy as a drug for ulcers has been confirmed [Yoshimura et al., “Ayumi of Medicine”, 149, 385 (1989),
Suzue; "Clinical Nutrition", 80,289 (1992), Okuma; "Infusion and Nutrition", 13, 1 (1989)].

[0005] However, although glutamine is useful as a material for infusions, tube feeding and oral nutrition, it is unstable to heat in the form of amino acids and cannot exert its function sufficiently. , Acetylation, etc., and conversion to dipeptides or tripeptides by chemical synthesis for use [WWSouba; J. Parent. Ent. Nutr., 9, 608 (1985); -51399, JP-A-62-151156, etc.]. However, these conventional methods have not been put to practical use yet because they are chemical methods and have problems in terms of safety , and the use of expensive amino acids as raw materials increases costs.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a safe and inexpensive oligopeptide mixture having a high content of dipeptides and tripeptides excellent in digestive absorption in the gastrointestinal tract and having a high glutamine content. That we can provide.

[0007]

Means for Solving the Problems As a result of continuous research conducted by the present inventors to solve the above-mentioned problems, the present inventors have identified wheat protein under specific pH and temperature conditions using alkaline protease produced by Bacillus bacteria. It has been found that upon hydrolysis over time, a beneficial oligopeptide mixture with a high content of dipeptides and tripeptides and a high content of glutamine can be efficiently obtained. In addition, the present inventors have found that when the alkaline protease treatment described above is further subjected to a hydrolysis treatment with a neutral protease produced by a microorganism or a plant under specific conditions, the above-described beneficial oligopeptide mixture can be obtained at a higher yield. And obtained the present invention based on those findings.

That is, the present invention provides: (1) pH 9.0 to 10.0 and a temperature of 50 to 60 using an alkaline protease (hereinafter sometimes simply referred to as “alkaline protease”) produced by a bacterium belonging to the genus Bacillus. Hydrolyzed at 5 ° C. for 5 to 30 hours.
Having a weight average molecular weight of 200 to 1000; and a total content of dipeptide and tripeptide of 50
A method for producing an oligopeptide mixture having a free amino acid content of 10% by weight or less; and a glutamine content of 30% by weight or more; and (2) Bacteria belonging to the genus Bacillus. PH 9.0 to 10.0 using alkaline protease produced by
After hydrolyzing at 0 to 60 ° C. for 2 to 10 hours, the pH of the mixture is further increased by using a neutral protease derived from microorganisms or plants (hereinafter sometimes simply referred to as “neutral protease”).
It is hydrolyzed at 0 to 8.0 at a temperature of 40 to 60 ° C. for 5 to 20 hours, and has the following characteristics;
A total of dipeptide and tripeptide is 50% by weight or more; a free amino acid content is 10% by weight or less; and a glutamine content is 30% by weight or more. How to do it.

Further, the present invention provides the following characteristic obtained by the method (1) or (2); the weight average molecular weight is 200 to 1000; and the total content of dipeptide and tripeptide is 50% by weight. % Or more;
Has a free amino acid content of 10% by weight or less; and
A mixture of oligopeptides derived from wheat protein having a glutamine content of 30% by weight or more; The present invention also includes a tube and oral nutritional supplement containing an oligopeptide mixture having the above characteristics.

The wheat protein used in the production method of the present invention is mainly composed of gluten, and is known to contain proteins such as albumin and globulin.

In the present invention, when the method (1) is carried out, the wheat protein is hydrolyzed with an alkaline protease at pH 9.0 to 10.0 at a temperature of 5 to 10.
The reaction is carried out at 0 to 60 ° C. for 5 to 30 hours, preferably 10 to 20 hours, and the resulting oligopeptide mixture is recovered to obtain the target oligopeptide mixture.

[0012] When the method is carried out by the method (2), the wheat protein is subjected to pH adjustment using an alkaline protease.
After hydrolysis at 9.0 to 10.0 at a temperature of 50 to 60 ° C. for 2 to 10 hours, preferably 5 to 10 hours, the pH is further increased to 6.0 to 8.0, preferably 6.0 using a neutral protease. 5 to 7.5, temperature 40 to 60 ° C, preferably 50 to
The target oligopeptide mixture can be obtained by performing the hydrolysis treatment at 60 ° C. for 5 to 20 hours, preferably 10 to 15 hours.

When the method (2) is adopted, the pH of the solution containing the product obtained by hydrolysis with the alkaline protease is adjusted to the above-mentioned range where the neutral protease acts, and the neutral protease is used as it is. Even if the hydrolysis is continued, or the product obtained by hydrolysis with an alkaline protease is once recovered from the liquid, and the product is dispersed or dissolved again in an aqueous medium adjusted to pH 6.0 to 8.0. It may be hydrolyzed by a neutral protease.

According to the method (1) of the present invention,
An oligopeptide mixture having the following characteristics can be obtained in a high yield of 50% or more. However, when the method (2) is adopted, the oligopeptide mixture having the characteristics of Peptide mixture can be obtained,
Moreover, this oligopeptide mixture has a higher dipeptide and tripeptide content.

In each of the above methods (1) and (2), the hydrolysis treatment with an alkaline protease produced by a bacterium belonging to the genus Bacillus disperses or dissolves wheat protein in an aqueous medium such as water. It is better to do it in a state,
Although it is necessary to appropriately adjust the concentration depending on the type of protease to be used and the like, it is usually preferable to perform the hydrolysis treatment with the concentration of wheat protein in the aqueous medium being about 5 to 15% by weight.

As the alkaline protease, any alkaline protease produced by a bacterium belonging to the genus Bacillus can be used, and examples thereof include subtilisin and the like. These alkaline proteases are "Alcalase" manufactured by Novo Industries Japan, Ueda Chemical Co., Ltd. It can be obtained as "Orientalase 22" manufactured by Kogyo Co., Ltd., "Protin A" manufactured by Daiwa Kasei Co., Ltd., or the like. Only one alkaline protease may be used, or a plurality of alkaline proteases may be used in combination as long as the alkaline proteases do not adversely affect each other. When a plurality of alkaline proteases are used, the hydrolysis may be performed in the presence of a plurality of alkaline proteases at the same time, or the hydrolysis may be performed sequentially by using one kind at a time.

The alkaline protease may be used in a free state or in an immobilized state. In each case, the alkaline protease is used in an amount of 0.05 to 2 per 100 g of dried wheat protein.
g (about 10,000 to 400,000 units), preferably 0.1 to 1 g
(Approximately 20,000 to 200,000 units).

When the oligopeptide is produced by the method (1), the hydrolysis treatment with an alkaline protease is usually carried out at a hydrolysis degree of about 70 to 95%.
After performing the above conditions until the state described above, it is preferable to inactivate the alkaline protease by any method such as heat treatment or lowering the pH, and to recover the generated oligopeptide in the form of a mixture.

In the case of the method (2), the hydrolysis treatment with an alkaline protease is usually carried out under the above-mentioned conditions until the degree of hydrolysis is about 60 to 80%, and Adjust the pH to the neutral range of 6.0-8.0, or once the product is recovered,
Dispersion or dissolution in an aqueous medium of 8.0 and further hydrolysis using neutral protease under the conditions described above.

As the neutral protease used in the method (2), any neutral protease derived from microorganisms or plants can be used, and examples thereof include metalloproteases and papain. Proteases are obtained as "Protease Amano N" manufactured by Amano Pharmaceutical Co., "Aloase AP-10" manufactured by Yakult, "Orientase 90N" manufactured by Ueda Chemical Industry Co., Ltd., "Protin PC10F" manufactured by Daiwa Kasei Co., Ltd. be able to. Even if only one kind of neutral protease is used,
Alternatively, as long as neutral proteases do not adversely affect each other, a plurality of types may be used in combination. When a plurality of neutral proteases are used, the hydrolysis may be performed in the presence of a plurality of neutral proteases at the same time, or the hydrolysis may be performed sequentially by using one kind at a time.

As in the case of the alkaline protease, the neutral protease may be used in a free state or may be used after being immobilized. In each case, 0% is used per 100 g of dried wheat protein used first. 0.05 to 2 g
(About 20,000 to 800,000 units), preferably 0.1 to 1 g
(Approximately 40,000 to 400,000 units).

The hydrolysis treatment with a neutral protease is usually carried out at a pH of 6.0 to 8.0 and a temperature of 40 to 60 ° C. until the degree of hydrolysis is about 70 to 95%.
After conducting the reaction for 5 to 20 hours under the conditions described above, the neutral protease is inactivated by any method such as heating or lowering or raising the pH, and the produced wheat protein is recovered from the liquor. The resulting oligopeptide mixture can be obtained.

Here, the degree of hydrolysis of wheat protein in this specification is 0.75M trichloroacetic acid (T
CA), and is represented by the following equation 1.

[0024]

## EQU1 ## Degree of hydrolysis (%) = {(0.75 M TCA dissolved protein) / (total protein)} × 100

Further, the protease activity (uni
t) are all measured by the following method. As a substrate for measuring protease activity, Hammerstein casein 1 manufactured by Merck, USA
% Solution, using the modified Anson-Hagiwara method [edited by Shiro Akahori “Enzyme Research Method”, Vol. 2, p. 237 (Jan. 10, 1961)
Sun, published by Asakura Shoten)]. The reaction was carried out at 30 ° C. for 30 minutes, and the amount of enzyme required to release 1 μg of tyrosine equivalent amount per minute was defined as 1 unit.

The method for recovering the oligopeptide mixture obtained by the above-mentioned method (1) or (2) is not particularly limited, and may be any method. For example, since the oligopeptide mixture obtained by the method (1) or (2) is soluble in the aqueous medium used for the hydrolysis treatment, the insoluble matter is separated by centrifugation after the hydrolysis treatment,
It is possible to employ a method of obtaining a target oligopeptide mixture by collecting and removing the liquid component by separating it by a usual solid-liquid separation method such as filtration and decantation and drying it.

When it is desired to obtain a higher-purity oligopeptide mixture, the liquid after removing insolubles is treated with an adsorbent such as activated carbon or diatomaceous earth to remove impurities contained in the liquid by adsorption. Then, the remaining liquid is further concentrated as necessary, and then dried by any method such as spray drying, freeze drying, and heat drying to collect the oligopeptide mixture as a solid. When heating and drying at that time,
Temperature at which the product does not heat denature (usually about 70-80 ° C or less)
And the drying treatment is preferably performed until the water content in the obtained solid content becomes about 10% by weight or less.

By performing the method (1) or the method (2) of the present invention,
Characteristic, that is, the weight average molecular weight is 200 to 100
0; the total content of dipeptide and tripeptide is 50% by weight or more based on the weight of the oligopeptide mixture;
Oligopeptide mixtures according to the invention having a free amino acid content of not more than 10% by weight based on the weight of the oligopeptide mixture; and a glutamine content of not less than 30% by weight based on the weight of the oligopeptide mixture can be obtained.

Here, the weight average molecular weight, dipeptide and tripeptide contents, glutamine content and free amino acid content of the oligopeptide mixture of the present invention were measured as follows.

Weight average molecular weight : PolyHYDROX from PolyLC
High-performance liquid chromatography using a YETHYL Aspartamide column, eluent 50 mM formic acid, flow rate 0.5 ml
/ Min, detection was measured under conditions of ultraviolet absorption at 220 nm.
At that time, data analysis was performed on oligopeptide 6 of known molecular weight.
Kinds (Bradykinin = 1060, Gly-Gly-Tyr-Arg AcOH H ₂ O =
518, Ile-Ala-Pro = 299, Leu-Gly-Gly = 246, Gly-Gly =
132, Gly = 75) to prepare a calibration curve, which was carried out by a GPC analysis system manufactured by System Instruments.

Dipeptide and tripeptide contents : The total amount of peptides having a weight average molecular weight of 125 to 397 was measured by the same method as the above-mentioned method for measuring the weight average molecular weight, and the total amount of dipeptide and tripeptide was measured. The ratio (% by weight) to the weight of the oligopeptide mixture obtained in each example was determined.

Glutamine content : The oligopeptide mixture was hydrolyzed with hydrochloric acid according to a conventional method, and amino acid analysis was performed. The amount of free glutamine was B, and the amount was determined by AB.

Free amino acid content : After removing protein from the sample, it was converted to Phenyl isothiocyanate, and
The amino acid composition was analyzed using an ICO-TAG system, and the content was determined as the content of free amino acid (% by weight) relative to the weight of the oligopeptide mixture.

The oligopeptides of the present invention having the above-mentioned characteristics are well soluble in water, do not form precipitates, have little unpleasant odor peculiar to free amino acids, and have other properties commonly used as foods and pharmaceuticals. It has good compatibility with substances, has a high dipeptide and tripeptide content, and has a large amount of glutamine which is extremely useful as a material for infusions, tube feeding and oral nutrition. Therefore, it can be effectively used as a tube or oral nutritional supplement, or as a functional ingredient, in foods and drinks such as various drinks and soups and feeds.

[0035]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.

Example 1 Powdered wheat protein (Bunge Bio
products Pty. Ltd. “Special Fine Dry Gluten”) 13
3.5 kg was dissolved in 870 liters of 0.05 N ammonia water with stirring (pH 10.0).
500g of 2BF (1000units / g of wheat protein)
Was added and reacted at 55 ° C. for 5 hours. Next, the pH of the solution was adjusted to 7.0 with hydrochloric acid, and then orientase 9 was added.
500 g (4000 units / 1 g of wheat protein) of 0N was added and reacted at 60 ° C. for 15 hours. 50% at that time
About 15 liters of L-lactic acid was added to adjust the pH of the solution to 4.5, and then heated at 90 ° C. for 20 minutes to inactivate the protease. After cooling, 10 kg of activated carbon ("Takecol 50W" manufactured by Takeda Pharmaceutical Co., Ltd.) was added, and the mixture was stirred for 1 hour, and then subjected to a filter press treatment using diatomaceous earth as a filter aid to obtain a transparent filtrate. The filtrate was concentrated under reduced pressure, sterilized with a plate heater, and then spray-dried to obtain 82 kg of a powdery oligopeptide mixture (61.4% yield of oligopeptide mixture relative to wheat protein).

When the properties of the oligopeptide mixture obtained above were measured by the above method, the weight average molecular weight of the oligopeptide mixture was 420, and the total content of dipeptide and tripeptide relative to the weight of the oligopeptide mixture was 59.8% by weight. , Glutamine content was 33.9% by weight and free amino acid content was 7.2% by weight. In addition,
For reference, Table 1 shows the amino acid composition of the oligopeptide mixture obtained in Example 1.

Example 2 133.5 kg of the same powdered wheat protein used in Example 1 was dissolved in 870 liters of 0.02 N aqueous ammonia with stirring, and the pH was adjusted to 9.8 with sodium hydroxide. Adjust and orientase 22
1 kg of BF (2000 units / g of wheat protein) was added and reacted at 60 ° C. for 0.5 hour. After readjusting the pH to 9.5 with sodium hydroxide, the reaction was further performed at 60 ° C. for 10 hours. At that time, about 15 liters of 50% L-lactic acid was added to adjust the pH of the solution to 4.5, and heated at 90 ° C. for 20 minutes to inactivate the protease. Example 1 after cooling
After adding 10 kg of the same activated carbon as used in Step 1 and stirring for 1 hour, a filter press treatment was performed using diatomaceous earth as a filter aid to obtain a transparent filtrate. After concentrating the filtrate under reduced pressure,
The mixture was sterilized with a plate heater and then spray-dried to obtain 80 kg of a powdery oligopeptide mixture (59.9% yield of oligopeptide mixture relative to wheat protein).

When the characteristics of the oligopeptide mixture obtained above were measured by the above-mentioned method, the weight average molecular weight of the oligopeptide mixture was 540, and the total content of dipeptide and tripeptide relative to the weight of the oligopeptide mixture was 50.4% by weight. , Glutamine content was 34.3% by weight and free amino acid content was 5.8% by weight. In addition,
For reference, Table 1 shows the amino acid composition of the oligopeptide mixture obtained in Example 2.

[0040]

[Table 1]

From the results shown in Table 1, the above (2) of the present invention was obtained.
In both cases of Example 1 corresponding to the method of Example 1 and Example 2 corresponding to the method of the above (1), the total content of dipeptide and tripeptide and glutamine content are all high, and the free amino acid content is extremely low. Example 1 in which a useful oligopeptide mixture can be obtained in high yield from wheat protein, especially after hydrolysis with an alkaline protease followed by hydrolysis with a neutral protease
According to the method described above, it can be seen that an oligopeptide mixture having a higher total content of dipeptide and tripeptide can be obtained.

[0042]

Since the oligopeptide mixture of the present invention contains a large amount of dipeptides and tripeptides, it has excellent digestive and absorptive properties in the gastrointestinal tract, and is suitable for the elderly, infants, sick, etc., having a reduced digestive and absorptive capacity. Very useful as a material. In addition, the oligopeptide mixture of the present invention contains a large amount of glutamine, so that it is useful as an energy source during invasion or intestinal dysfunction.In addition, since glutamine exists not in a free form but in a peptide form, it can be heated. It is stable to processing and can be used for a wide range of applications. Since the oligopeptide mixture of the present invention has a low free amino acid content, it does not have the unpleasant taste peculiar to free amino acids, has excellent oral administration receptivity, and has a low osmotic pressure, so that it does not easily cause symptoms such as diarrhea. Furthermore, according to the method of the present invention, a highly safe oligopeptide mixture can be economically produced from wheat protein, which is itself a useful food material, in extremely high yield with good productivity.

Claims (4)

(57) [Claims] The present invention relates to a method for producing wheat protein using an alkaline protease produced by a bacterium belonging to the genus Bacillus at pH 9.0 to 10.0;
Is hydrolyzed at a temperature of 50 to 60 ° C. for 5 to 30 hours to have the following characteristics: a weight average molecular weight of 200 to 1000; a total content of dipeptides and tripeptides of 50% by weight or more; A method for producing an oligopeptide mixture having a glutamine content of 10% by weight or less; and a glutamine content of 30% by weight or more. 2. A method for producing wheat protein using an alkaline protease produced by a bacterium belonging to the genus Bacillus at pH 9.0 to 10.0;
After hydrolyzing at a temperature of 50 to 60 ° C. for 2 to 10 hours, it is further hydrolyzed with a neutral protease derived from a microorganism or a plant at a pH of 6.0 to 8.0 and a temperature of 40 to 60 ° C. for 5 to 20 hours. A weight average molecular weight of 200 to 1000; a total content of dipeptides and tripeptides of 50% by weight or more; a free amino acid content of 10% by weight or less; and a glutamine content of 30% by weight. % Or more; a method for producing an oligopeptide mixture comprising: 3. The following characteristic obtained by the method according to claim 1 or 2; the weight average molecular weight is 200 to 1000; the total content of dipeptides and tripeptides is 50% by weight or more; 10% by weight or less; and a glutamine content of 30% by weight or more; 4. A tube or oral nutritional supplement containing the oligopeptide mixture according to claim 3.