JP5695826B2 - Enzyme processing method for horse or sheep myocardium, food material, and health food for immunostimulation, tonicity, and metabolism promotion - Google Patents

Description

The present invention relates to a technique for producing a low molecular weight peptide by hydrolyzing horse or sheep myocardium and providing it as a functional health / food material.

  Animal extracts and dry powder with built-in animals are used for nutritional and health foods. Although the effects of ingesting these are due to the usefulness of the proteins contained in them, the resulting protein has a large molecular weight, and even if it is ingested by humans, it is absorbed and functionally qualitatively and quantitatively. There was a problem. Therefore, the present inventors studied the tonic action and immunity improvement effect of animal myocardium. As a result, it was clarified that immunity activation and tonic improvement were promoted by continuously ingesting a myocardial peptide mixture obtained from animal myocardium.

  In addition, patent documents 1-6 are mention | raise | lifted as a prior art document relevant to this invention. Patent Document 1 discloses “a method for producing a substance having an antilipidemic effect” in which a mammal's heart or the like is polished and self-digested before being processed. Patent Document 2 discloses that a chicken extract is obtained by heating chicken fry with atmospheric pressure heating water to obtain a chicken extract, adding water to the extraction residue and heating with pressurized hot water to obtain a chicken extract, and then mixing both extracts. Is disclosed. Patent Document 3 discloses a “concentrated extract production method” in which an extract extracted with hot water to an extract solids concentration of about 0.5 to 10% is concentrated under conditions of a superheating temperature of 90 ° C. or less and an evaporation temperature of 60 ° C. or less. It is disclosed. Patent Literature 4 includes (i) a bird gull or a step of hot water extraction of a bird gull and its meat, (ii) a step of removing oil from the extract, and (iii) an extract from which oil has been removed. There is disclosed a “bird extract manufacturing method” comprising a step of mincing a bird gall, (iv) a step of enzymatically decomposing a composition comprising an oil-removed extract and a bird gull mince. Patent Document 5 discloses a “liver lipid metabolism improving agent” comprising a heart peptide mixture obtained by enzymatic treatment of animal heart. Patent Document 6 discloses “avian and livestock fermented processed foods” obtained by the action of koji fungus in the presence of saccharides on wild and livestock meat.

Japanese Patent Publication No. 48-6863 JP-A-2-42955 JP-A-2-276552 JP-A-6-165654 JP 2000-256200 A JP 2001-136933 A

  By the way, as described above, protein has a large molecular weight, and even if it is ingested by humans, there is a problem in terms of absorption and function in the body. That is, there is a solution in which a protein containing an animal myocardium elastic protein connectin, myoglobin, cytochrome C, etc. is peptideized and taken as food or health food. However, it is clear that degrading proteins into peptides to improve immunostimulation, tonicity, and metabolism-promoting effects should be improved when controlling animal myocardial hydrolysis. For this reason, there was a problem in terms of uniformity, flavor, absorbability, and functionality of the product (peptide-containing product).

Accordingly, the present invention provides a horse improved reaction control, or, it is an object to provide a like enzyme treatment method sheep myocardium.

  In order to obtain a peptide mixture by hydrolyzing a protein containing connectin, myoglobin, cytochrome C and the like of animal myocardium, the present inventors have obtained an alcalase, a cysteine protease, a neutralase, a serine protease, a peptidase, a pancreatin, a protease, and a ribase. Regarding the above, studies were made on the addition of one or two of these enzymes or the simultaneous addition of these enzymes.

  Therefore, as a result of various studies, the present inventors adjusted two kinds of degrading enzymes according to the characteristics of the enzymes, the first stage, the second stage and the individual degrading enzymes individually, the optimum decomposition temperature, the optimum pH value. It was found that stable decomposition can be achieved by setting (hydrogen ion concentration value) and causing it to act at an optimum relative activity. Furthermore, by improving the functionality of the product, the peptide bond involving the carboxyl group of tyrosine, phenylalanine, and tryptophan is degraded by chymotrypsin degrading enzyme, thereby increasing the functionality of the peptide and adding functionality to various foods. I found that I can do it.

The present inventors, based on the above findings, horses, or sheep myocardium to improve the reaction efficiency when hydrolysis, equine, or, for the sheep heart muscle, by performing the step hydrolysis process Focusing on the improvement in reaction controllability, the present invention has been completed. That is, the present invention retains the properties of the myocardium and the properties of the enzyme (eg, ease of reaction control, continuity, product homogenization conditions) in hydrolyzing the myocardium of horses or sheep. However, it is characterized in that the myocardium of the horse or sheep is efficiently hydrolyzed.

Moreover, immunostimulatory those containing peptide mixture derived from the generated myocardium those mainly, tonic, and completed the present invention by paying attention to possible to manufacture a functional food as metabolism improving accelerator .

ADVANTAGE OF THE INVENTION According to this invention, the enzyme processing method etc. of the horse or sheep myocardium which improved reaction control can be provided.

It is a figure which shows the swimming duration of horse myocardium powder. It is a figure which shows NK activity of horse myocardium powder. Graph at the time of one week administration It is a figure which shows NK activity of horse myocardium powder. Graph after 2 weeks of administration It is a figure which shows NK activity of horse myocardium powder. Graph after 4 weeks of administration

Hereinafter, modes (embodiments) for carrying out the present invention will be described.
In practicing the present invention, in the first stage reaction, the degradation reaction conditions were set for the properties of the serine protease enzyme, the enzyme degradation was performed, and the enzyme was inactivated after the completion of the enzyme degradation. Furthermore, in the second stage reaction, the degradation reaction conditions were set in the characteristics of the cysteine protease enzyme and the degradation was completed. Then, the enzyme was deactivated in the same manner as in the first stage.

Furthermore, after the second stage, in the third stage reaction, peptide bonds involving tyrosine, phenylalanine and tryptophan carboxyl groups are degraded by chymotrypsin enzyme to improve the function of myocardial protein as a substrate or add new functionality. It was.
The peptide bond is a bond formed by dehydration condensation of amino acids among amide bonds (dehydrogen bond between amine and organic acid). A macromolecular substance in which a large number of amino acids are condensed is a protein.

The stepwise combination of degrading enzymes may be performed by serine protease and cysteine protease and chymotrypsin or serine protease and chymotrypsin, or a combination of cysteine protease and chymotrypsin. The food which is the object of the present invention can be blended with the powder alone or appropriately as long as the composition is not impaired. EXAMPLES Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
Incidentally, in Examples 1 and 2 described later, enzymes shown in Table 1 below were selectively used. In Example 2, the third stage reaction = the second stage reaction.
Supplementally, a serine protease can be suitably used in the first stage reaction. In the second stage reaction, cysteine protease can be preferably used. In the third stage reaction, chymotrypsin can be preferably used.

  In addition, the NK cell activity in Example 5 described later (“NK activity (%)” in FIG. 2) indicates the activity of natural killer cells (NK cells). Here, NK cells are a kind of cytotoxic lymphocytes that act as a major factor of innate immunity, and are particularly important for rejection of tumor cells and virus-infected cells. NK cells, unlike T cells, do not need to be sensitized beforehand in order to kill these malignant cells. That is, they kill tumor cells and virus-infected cells without antigen sensitization (injury). Therefore, it is named NK (natural killer cell).

(About enzymes)
The “serine protease” used in the first stage reaction is a protease (proteolytic enzyme) having a serine residue that performs a nucleophilic attack as a catalytic residue. The “cysteine protease” used in the second stage reaction is a proteolytic enzyme that uses the thiol group of cysteine as a nucleophilic group at the catalytic site. In addition, "chymotrypsin" used in the third stage reaction is a kind of endopeptidase, a serine protease, and a digestive enzyme contained in pancreatic juice, and is a peptide bond on the carboxyl group side of an aromatic amino acid. Is hydrolyzed.

(About amino acids)
"Tyrosine" is a kind of amino acid and has a phenol moiety in the side chain. Also called 2-amino-3-hydroxyphenylpropionic acid or p-hydroxyphenylalanine. The molecular formula is “C ₉ H ₁₁ NO ₃ ”. “Phenylalanine” is also a kind of amino acid and has a benzyl group in the side chain. The molecular formula is “C ₉ H ₁₁ NO ₂ ”. “Tryptophan” is also a kind of amino acid, and is 2-amino-3- (indolyl) propionic acid. The molecular formula is “C ₁₁ H ₁₂ N ₂ O ₂ ”. All of these have a carboxyl group (COOH).

(About protein)
“Connectin”, also called titin, is an elastic protein that connects the Z-line and M-line of the striated muscle with a single molecule, and has a very large molecular weight. In addition, “myoglobin” is a heme protein having a molecular weight of about 17500, which is mainly present in the heart muscle and skeletal muscle, and stores oxygen molecules until they are necessary for metabolism in the muscle. Further, “cytochrome C” is a molecule having a molecular weight of about 12400 present in muscle and is a kind of heme protein containing heme iron having a redox function.

Embodiment 1
In the first embodiment, as will be described later, the first-stage reaction, the subsequent second-stage reaction, and the subsequent third-stage reaction were performed using a container with a temperature control function for horse myocardium. .
In the first stage reaction, serine protease was used as the enzyme. Serine protease has the characteristics that the optimum pH-activity value in relative activity is pH 7.0, and the temperature-activity optimum is 50-55 ° C., so it can be suitably used for the first stage reaction.
In the second stage reaction, cysteine protease was used as the enzyme. Cysteine protease can be suitably used for the second stage reaction since it has the characteristics that the optimum pH-activity value in relative activity is pH 6.5 and the temperature-activity optimum is 80 ° C.
In the third stage reaction, chymotrypsin was used as an enzyme. Chymotrypsin has the characteristics that the optimum pH-activity value in relative activity is pH 7.5, and the temperature-activity optimum is 50 ° C., so it can be suitably used for the third stage reaction.
The use of 4 times the amount of water is due to consideration of protein diffusivity, dispersibility, and added enzyme reactivity. Productivity decreases when there is much water. On the other hand, when there is little water, the reactivity of an enzyme will fall.

<< Embodiment 2 >>
In the second embodiment, as described later, for the sheep myocardium, the first-stage reaction and the subsequent second-stage (= third-stage) reaction were performed using a container with a temperature control function.
In the first stage reaction, serine protease was used as the enzyme. Serine protease has the characteristics that the optimum pH-activity value in relative activity is pH 7.0, and the temperature-activity optimum is 50-55 ° C., so it can be suitably used for the first stage reaction.
In the second stage (third stage) reaction, chymotrypsin was used as an enzyme. Chymotrypsin has the characteristics that the optimum pH-activity in relative activity is pH 7.5 and the temperature-activity optimum is 50 ° C., so it can be suitably used for the second stage reaction.
The use of three times the amount of water is the same as described above.

  Hereinafter, examples embodying the embodiment will be described. Needless to say, the present invention is not limited to this embodiment.

Example 1 corresponding to Embodiment 1 will be described. In Example 1, 50 kg of equine myocardium was homogenized and placed in an appropriate amount of a stainless steel container (reaction tank) with a temperature control function. Is set to 50-55 ° C. and is adjusted to pH 7.0 after temperature stabilization. 3 to 5 hours later, the pH is confirmed and adjusted again, 1 kg of serine protease is added, and enzymatic degradation is performed for 10 hours. After the decomposition, the enzyme is inactivated by maintaining at 80 ° C or higher for 40 minutes (second stage), then cooled and set to 80 ° C, adjusted to pH 6.5 after temperature stabilization, and the pH is confirmed again after 2 to 5 hours Prepare, add 1 kg of cysteine protease, enzymatically degrade for 10 hours, and hold at 80 ° C. or higher for 40 minutes to inactivate the enzyme. (Third stage) Next, cool, set to 50 ° C., adjust to pH 7.5 after temperature stabilization, add 400 g of chymotrypsin, perform enzymatic degradation for 3-5 hours, then hold at 80 ° C. or higher for 40 minutes to lose the enzyme. After removing the insoluble matter, the solution was freeze-dried to obtain about 43 kg of powder.
The reaction from the first stage to the third stage was performed in the same reaction tank.

Example 2 corresponding to Embodiment 2 will be described. In Example 2, 2 kg of sheep myocardium was homogenized and placed in a glass 5 liter container (reaction tank) with a temperature control function, to which 3 times the amount of water was added, and (first stage) the temperature was 50- Set to 55 ° C, adjust to pH 7.0 after temperature stabilization, adjust pH again after 2-4 hours, add serine protease 40g and perform enzymatic degradation for 6-8 hours, then hold at 80 ° C or higher for 40 minutes Inactivate the enzyme. (Third stage (= second stage)) Next, the temperature was lowered to 50 ° C., adjusted to pH 7.5 after temperature stabilization, 10 g of chymotrypsin was added, and enzymatic degradation was performed for 3 to 6 hours. The enzyme was inactivated by holding for 30 minutes to remove insoluble matters, and then freeze-dried to obtain about 1.6 kg of powder.
In Example 2, the reaction at each stage was performed in the same reaction tank.
Incidentally, in Example 2, the second step by cysteine protease does not exist, but this is due to the difference in the molecular weight of the total protein including connectin due to the difference in the target animals (horse and sheep).

freeze drying;
250 g of Paindex (registered trademark) # 100 manufactured by Matsutani Chemical Industry Co., Ltd. was added to the liquid before freeze-drying obtained in Example 2 and freeze-dried to obtain about 1.8 kg. The paindex is a high-purity starch decomposition product obtained by hydrolyzing and purifying starch. By adding a starch degradation product, this becomes an excipient, which improves the preservability, solubility and flavor of the raw material product, and can be made into a highly functional food by blending with other raw materials.

Anti-fatigue test;
Experiments with mice were conducted to evaluate the physical anti-fatigue action of horse myocardium powder. 30 mice (ddy, male, 6 weeks old) were divided into 5 groups: control group (Cont), ginseng extract powder (commercially available) group (100 mg), equine myocardium powder 50, 100, 200 mg / kg group. After breeding, each powder was dispersed in water and orally administered to mice at each dose once a day for 2 weeks. In the second week, a weight of 10% of the mouse body weight was loaded and a forced swimming experiment (the time until the head was completely submerged in water for 5 seconds was measured) was performed. Results At the measurement of the second week after administration, the horse myocardial powder administration group showed an increase in swimming duration in a concentration-dependent manner as compared to the control group (FIG. 1). In addition, as a nourishing tonic material, as compared with ginseng extract powder, horse myocardial powder was found to have an extended duration, suggesting an effect on physical fatigue.
All groups are fed the same diet. Moreover, mg / kg is the dose converted into the body weight of a mouse | mouth.

Immunity enhancing action;
Twenty-four mice (male and female, 4 weeks old) were divided into 4 groups of control group, 50 mg / kg group, 100 mg / kg group and 200 mg / kg group, and NK of spleen of mice after 1 week, 2 weeks and 4 weeks Cell activity was measured. NK cells tended to increase as the dose of equine myocardial powder increased from 1 week after administration, and showed a significantly higher value in the 200 mg / kg group administered for 2 weeks and 4 weeks than the control group (Fig. 2 to 4). From the measurement results, it was shown that NK activity was increased by administering 200 mg / kg per mouse for 2 weeks or more. In addition, it was suggested that continuous ingestion of equine myocardium powder may improve immunity.

In the present invention, this composition of peptided horse or sheep myocardium has high dispersibility and is used as a material for functional, health foods, regardless of form, such as solids, tablets, beverages, powders, granules, etc. In addition, it can be widely used as a highly functional food by blending with other foods.

Claims (4)

A method for enzymatic treatment of horse or sheep myocardium, in which an enzyme is processed stepwise on the basis of the properties of the enzyme in the same reaction tank for enzymatic degradation of the horse or sheep myocardium ,
Before SL enzymes are serine proteases, cysteine proteases, or a horse, characterized in that either serine proteases and cysteine proteases, or enzymatic treatment method for sheep myocardium. The amino acid sequence of polypeptides derived from the horse or sheep myocardium according to claim 1 or the sheep myocardium by the enzyme treatment method, wherein tymotrypsin enzyme is used to produce tyrosine, phenylalanine, and tryptophan. A method for enzymatic treatment of horse or sheep myocardium , comprising enzymatically decomposing a peptide bond involving a carboxyl group. According to claim 1, wherein the horse, or a horse obtained by treatment with the enzyme treatment method sheep myocardium, or food material, which comprises a peptide mixture derived from sheep myocardium.   A health food for promoting immunostimulation, tonicity and metabolism, comprising the food material according to claim 3.