JPS6258714B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing an oligopeptide mixture with excellent nutritional value from soybean whey in high yield. More specifically, the present invention provides an oligopeptide mixture that has an excellent nutritional improvement effect on malnourished people and patients undergoing medical treatment. (Prior Art) A method for producing soybean protein from defatted soybeans from which oil is extracted is well known. In this manufacturing process, okara (mainly composed of polysaccharides) and soybean whey (mainly composed of small sugars) are produced as by-products, and the effective use of these is a major industrial challenge. Among them, there are many studies on soybean whey, one of which is research on soybean whey protein contained in soybean whey. As shown in Table 1 below, although this soybean whey protein is plant-based, it has superior utilization efficiency compared to other plant proteins, and its nutritional improvement effects are comparable to those of animal-based lactalbumin. It has been found that something is.

[Table] PER is expressed as the ratio of body weight gain to ingested protein within a certain rearing period, and is an index of the utilization efficiency of ingested protein. On the other hand, many studies have been conducted on the nutritional value of proteins, and many new findings regarding the digestion and absorption of proteins have been obtained. For example, low molecular weight peptides such as di- or tripeptides rather than amino acids are rapidly and efficiently absorbed in the intestinal tract. A method for producing peptides containing this dei or tripeptide as a main component is disclosed, for example, in Japanese Patent Publication No. 57-45560. A method for obtaining a low molecular weight peptide having an average molecular weight of 700 or less as a main component with a yield of 80% or more is known. However, there is no known method for obtaining an oligopeptide mixture with a uniform molecular weight of 500 to 1500 Daltons from soybean whey protein in high yield, as in the present invention. At the same time, it is not known that such oligopeptide mixtures have excellent nutritional improvement effects. (Purpose) The present inventors aimed to produce from soybean whey in high yield an oligopeptide mixture that has an excellent nutritional improvement effect on malnourished people and patients receiving medical treatment. (Progress) While aiming to further enhance the nutritional improvement effect of soybean whey protein obtained from soybean whey, the present inventors discovered that soybean whey protein, which is a high molecular compound, requires many digestive enzymes in the digestive system. It is a heavy burden for malnourished people and patients undergoing medical treatment, and conversely, amino acids and di- or tripeptides require almost no digestive enzymes and are somewhat unnatural as a diet.
Furthermore, the increase in osmotic pressure overpromotes the secretion of digestive juices, causing abdominal bloating and diarrhea, and the taste of these proteins, amino acids, and low We conducted research on oligopeptides, which are different from molecular peptides and require digestive enzymes, but have very little burden and can be used as natural foods. As a result, the molecular weight obtained from soy whey protein
We have found that an oligopeptide mixture of 500 to 1500 Daltons has a superior nutritional improvement effect on malnourished animals compared to a protein or amino acid mixture (all three have the same amino acid composition). Therefore, the present inventor conducted research with the aim of producing an oligopeptide mixture with a molecular weight of 500 to 1,500 Daltons (preferably 700 to 1,000 Daltons) with a high yield and excellent nutritional improvement effects. However, the
As disclosed in 57-45560, oligopeptides with an average molecular weight of 820 or 950 can only be obtained with a low yield of 16.5% or 20.8% by simply hydrolyzing raw protein with trypsin or alkaline protease. . Therefore, as a result of further intensive research on various methods, the present inventors first obtained soybean whey protein from soybean whey in a high yield, and then, as a second step, soybean whey protein was treated with thiol protease or carboxyl protease. From soybean whey, a polypeptide mixture is obtained by limited hydrolysis with protease under certain special conditions, and then, as a third step, the resulting polypeptide mixture is specifically hydrolyzed with serine protease or proline-specific peptidase. By combining the process of obtaining whey protein with the two different enzymatic reactions of limited hydrolysis and specific hydrolysis, the present invention was completed based on the knowledge that the desired oligopeptide mixture can be obtained in high yield. . (Structure) The present invention comprises (1) (a) a step of obtaining soybean whey protein from soybean whey; (b) limited hydrolysis of soybean whey protein in an aqueous system using thiol protease or carboxyl protease at pH 7 to 11 to produce a polypeptide mixture; (c) specifically hydrolyzing the obtained polypeptide mixture with a serine protease or a proline-specific peptidase.
1000 Daltons) The soybean whey used in the present invention is obtained by extracting the defatted soybean with an aqueous solvent, removing okara from the defatted soybean by a known method to obtain soymilk, and then using a method such as acid precipitation. It can be obtained by separating soybean protein, or by directly extracting defatted soybeans with an acidic aqueous solution (PH is around the isoelectric point of soybean protein). Generally, soybean whey obtained in the soybean protein production process can be used as is. In step (a) of the present invention, soybean whey protein can be obtained by heating soybean whey to thermally denature and insolubilize soybean whey protein, and then separating it using means such as centrifugation. It can also be obtained by separating soybean whey protein from soybean whey using a membrane separation means such as an ultrafiltration membrane. It can also be obtained by precipitating and separating soybean whey with a polar solvent such as alcohol. Soybean whey protein prepared by these methods has an advantage over isolated soybean protein in that it is rich in sulfur-containing amino acids such as cysteine and methionine, which are limiting amino acids. The enzyme used in step (b) of the present invention can be an enzyme that can undergo limited hydrolysis in the presence of water, such as thiol proteases such as papain, bromelain, and huicin, and carboxyl proteases such as pepsin. be able to. The action conditions of the enzyme used in step (b) of the present invention are different from those in which free amino acids and low-molecular-weight peptides are easily produced, such as in ultimate hydrolysis.
It is important to use conditions that facilitate the production of polypeptides, in other words, conditions that allow for limited hydrolysis. For this purpose, the substrate concentration is suitably relatively high compared to the ultimate hydrolysis conditions, usually 5 to 40% w/w. As the substrate concentration increases, stable limited hydrolysis can be performed in the PH range of the present invention. The optimum pH for hydrolysis of the enzyme used in step (b) of the present invention is 5, but the pH for limited hydrolysis in the present invention is higher than this, 7 to 11, preferably 8.
A value between 10 and 10 is appropriate. When the pH is less than 7 or more than 11, the desired limited hydrolysis is difficult to occur. By the step (b) of the present invention, a polypeptide mixture mainly having a molecular weight of 1,500 to 9,000 daltons can be obtained with a high yield of 85% or more. The generation of free amino acids can be almost completely eliminated. The amino acid composition of the resulting polypeptide mixture is almost the same as that of soybean whey protein. This is compared to the fact that a low molecular weight peptide with an average molecular weight of 420 can be obtained with a yield of only 21.6% by hydrolysis using papain (a type of thiol protease) as disclosed in the aforementioned Japanese Patent Publication No. 57-45560. This shows that step (b) is a different method from ordinary hydrolysis in terms of molecular weight and yield. The polypeptide mixture obtained in step (b) may be subjected to membrane separation (for example, using a membrane with a molecular size of about 1000) to remove low-molecular substances, if desired. RO (Reverse
Osmosis) and UF (Urtra Filter) can be used. Furthermore, separation means using acids, polar organic solvents, etc. can also be used. The polypeptide mixture thus obtained in step (b) can be diluted, concentrated, or dried as it is. In step (c) of the present invention, the previously obtained polypeptide mixture can be specifically hydrolyzed using a serine protease or a proline-specific peptidase. The conditions for specific hydrolysis are usually an enzyme/substrate ratio of 0.01 to 5, a protein (substrate) concentration of 1 to 10%, and a pH of 1 to 10%.
is the optimum to effective pH range for hydrolysis, temperature is 10 to 60
℃, the time can be as short as 1 to 300 minutes (preferably 5 to 240 minutes) depending on the reaction rate of specific hydrolysis. Specific means that a certain site is specifically hydrolyzed, rather than being hydrolyzed randomly. In the present invention, step (a), step (b) and step (c)
By combining the steps, you can achieve a high yield (44% or more for serine protease/soybean whey protein), and if desired, by repeating the fractionation-specific hydrolysis described below, you can achieve an even higher yield and increase the molecular weight.
Oligopeptide mixtures of 500 to 1500 daltons can be obtained. This is because the hydrolysis using trypsin (a type of serine protease) disclosed in the aforementioned Japanese Patent Publication No. 57-45560 produces an oligopeptide with an average molecular weight of 820.
Compared to the fact that the yield was only 16.5%, it can be seen that this method is superior in terms of yield.
Furthermore, the short action time of the enzyme is also a major industrial advantage. In step (c) of the present invention, if desired, the target oligopeptide mixture and other non-oligopeptide mixtures can be separated. As a method for fractionation, known methods such as membrane separation and solvent precipitation can be used, but from an industrial standpoint, fractionation using an organic solvent is preferred. In this case, after adding an organic solvent (for example, a polar organic solvent such as ethanol) to a concentration of 50 to 80 w/w% in the entire system, the precipitate and supernatant are separated using a separation method such as centrifugation. After separation, the supernatant can be subjected to solvent removal (for example, evaporation), and optionally concentrated or dried to obtain an oligopeptide mixture. Furthermore, the precipitate is reduced by repeating the specific hydrolysis-fractionation operation in which the precipitate is subjected to specific hydrolysis using the enzyme in step (c) in the same manner as in the case of the polypeptide mixture. The yield of supernatant or oligopeptide mixture can be increased. That is, in step (c), the polypeptide mixture obtained in step (b) is specifically hydrolyzed with serine protease or proline-specific peptidase,
By repeating the specific hydrolysis and fractionation operation of separating the oligopeptide mixture into an oligopeptide mixture and a non-oligopeptide mixture, and then specifically hydrolyzing the non-oligopeptide mixture again with serine protease or proline-specific peptidase, one or more times, A high yield of oligopeptide mixtures with uniform molecular weights (e.g. 61% for serine protease) with low free amino acid production and almost no polypeptides.
or more/soybean whey protein). The oligopeptide mixture obtained in this way has almost the same level of amino acid composition as soybean whey protein, and is nutritionally deficient compared to high-molecular soybean whey protein and low-molecular amino acid mixture (with the same amino acid composition). The nutritional improvement effect for animals is extremely good. (Example) Embodiments of the present invention will be described below with reference to Examples. Example 1

【table】

[Table] ｜
Soy whey ultrafiltration concentrated protein
5.83g (5% moisture, 75% protein = dry matter)
(Protein recovery rate 67.3%)

[Table] Soy whey alcohol precipitated protein
480g (moisture 3.2%, protein 83.2% dry matter)
(Protein recovery rate 62.5%)
The amino acid composition of soybean whey protein obtained by each of the above methods is shown in Table 2 below. However, soybean whey protein by heating, soybean whey protein by ultrafiltration membrane, and soybean whey protein by alcohol precipitation.

【table】

【table】

【table】

【table】

[Table] When the oligopeptide mixture thus obtained was subjected to Biogel P-2 chromatography to determine its molecular weight distribution, it was found to be an oligopeptide mixture with a peak around 900 daltons, with only 4% free amino acids. I found out that there isn't. The constituent amino acid composition of this oligopeptide mixture is shown in Table 3 for comparison with that of the raw material whey protein. They have almost the same amino acid composition. However, OPM is an oligopeptide mixture obtained from soy whey alcohol precipitated protein.

【table】

[Table] (Rat breeding test) OPM, soybean whey protein, and amino acid mixture (amino acid composition is the same as soybean whey protein) shown in Table 3 above were used as the N source, and N containing other lipids, carbohydrates, vitamins, and minerals was used. HAPER at level 1.5%
A type feed was prepared. The test rats were normal rats at the maximum growth stage (5 weeks of age) and protein-deficient rats (both
Wistar (male) was used. In the latter case, a protein-free diet was administered to 6-week-old rats for 4 weeks, and the rats were completely anemic. These rats were kept at a temperature of 22 ± 1°C and a humidity of 50–55°C.
%, artificial lighting with a 12 hour light/dark cycle (7am
The animals were given each feed ad libitum for 3 weeks under conditions (lights on at ~7pm), and weight gain, N intake, and excretion were measured.
PER (Protein Efficiency Ratio) was calculated. As a result, the oligopeptide mixture produced from soybean whey showed excellent nutritional improvement effects even in malnourished rats.

[Table] (Effects) As detailed above, the present invention has made it possible to produce an oligopeptide mixture with excellent nutritional value from soybean whey. In other words, oligopeptide mixtures and diets using the same have an excellent nutritional improvement effect even for malnourished people and people undergoing medical treatment, such as those who have undergone gastrointestinal resection and those with digestive secretion deficiency. It has become easier to provide food, and it not only contributes to improving people's nutrition, but also greatly contributes to the development of industry.

Claims (1)

[Claims] 1 (a) Step of obtaining soybean whey protein from soybean whey, (b) Process of obtaining soybean whey protein from soybean whey using thiol protease or carboxyl protease in an aqueous system.
(c) specific hydrolysis of the obtained polypeptide mixture with serine protease or proline-specific peptidase. Preferably 700 to 1000
Dalton) method for producing oligopeptide mixture.