KR100566938B1 - Process for preparing crude peptide from amyda japonica - Google Patents

Abstract

 The present invention relates to a method for producing Zarazopeptide, which comprises the step of enzymatically digesting the slaughtered plant, inoculating and inoculating the lactic acid bacteria. The method for producing Zara zopeptide of the present invention comprises the steps of: (i) adding a protease to the slaughtered Zara; (Ii) heating the reactants to inactivate the protease, then inoculating and incubating the lactic acid bacteria; (Iii) filtrating or centrifuging the electric culture to obtain a culture, followed by mixing with crystalline cellulose and filtering to obtain crystalline cellulose to which the peptide is adsorbed; And (iii) drying the obtained crystalline cellulose. When the production method of the present invention is used, functional peptides can be produced in high yields from growth, and thus will be widely contributed to economic production of functional peptides.

Grow up, peptide, protease, lactic acid bacteria

Description

Process for Preparing Crude Peptide from Amyda japonica}

The present invention relates to a method for producing zopeptide. More specifically, the present invention relates to a method for preparing Zara zopeptide, which comprises the step of enzymatically digesting the slaughtered plant, inoculating and inoculating the lactic acid bacteria.

In addition to nutritional functions as a nitrogen source, peptides have physiological activities such as taste, antioxidant and antimicrobial effects, and have bioregulatory functions such as immunomodulatory function, hormone and neurotransmitter and antihypertensive effect. And attention in the food industry. Functional peptide materials can be divided into peptides prepared by hydrolysis of synthetic products and natural food materials by the polymerization of amino acids, and exhibit various physiological activities depending on the peptide preparation conditions. On the other hand, the production of functional peptides can be classified into enzymatic hydrolysis using proteolytic enzyme, synthetic method using reverse reaction of enzymatic hydrolysis on organic solvent, method using recombinant strain, chemical synthesis method, and the like. Economical and mass-producible enzyme hydrolysis is the most widely used.

On the other hand, Zara ( Amyda japonica ) is a reptile belonging to the turtle tree Jaraaceae , and has been used as a tonic and aphrodisiac since ancient times, and in particular, oriental medicine has used tortoiseshell, the spine of Zara, as a valuable herbal medicine. For example, agree that "the tortoises control organ abnormalities, protect the body and blood, and reinforce physiological functions and strength." It is described. However, tortoiseshell contains abundant protein and fat and is easily deteriorated at room temperature. However, in one case, the tortoiseshell is treated with steam, and then dried in the shade to be preserved. In order to improve the preservation by processing a short shell for a short time. For example, Korean Patent Registration No. 302054 discloses a processing method of a tortoiseshell, comprising the steps of hot water treatment and drying of the tortoiseshell, followed by baking at a high temperature in a kiln, and powdering.

In order to utilize the above-described functionalities of Zara, efforts have been made to prepare peptides derived from Zara by enzymatic hydrolysis using proteolytic enzymes. However, the yield is low due to a yield of about 50%. Because of the enormous cost and time required, there is no economic feasibility and no peptide is produced from growing. If a method for producing a peptide at higher yield is developed, it is expected to generate enormous added value, but there is no achievement.

Therefore, there is a constant need to develop a method for producing peptides in high yield using jara.

Thus, the present inventors have made diligent research to develop a method for producing peptides with high yield using Zara. As a result, the slaughter of Zara was hydrolyzed using proteolytic enzymes, and then used as a medium to produce lactic acid bacteria. When inoculated and cultured to obtain a peptide, which was isolated by adsorption on crystalline cellulose, it was confirmed that the peptide can be produced in a higher yield, the present invention was completed.

After all, the main object of the present invention is to provide a method for producing a peptide in high yield from growing.

In the method for producing Zara zopeptide of the present invention, protease N or protease P is added to the slaughter of Zara, and under conditions of 40 to 60 ° C. and pH 7 to 8 Reacting for 90 minutes; (Ii) heating the reactants to inactivate proteolytic enzymes, and then inoculating and incubating L. acidophilus or L. salivarius; (Iii) Filtration or centrifugation of the electric culture to obtain a culture medium, and then mixed with the electric culture medium and crystalline cellulose in a ratio of 3: 7 to 5: 5 (v / v) and filtered, crystal cellulose adsorbed peptide To obtain; And (iii) drying the obtained crystalline cellulose. At this time, the culture conditions of Lactobacillus ashdophyllus (L. acidophilus) or Lactobacillus salivarius (L. salivarius) is not particularly limited, a temperature of 30 to 40 ℃ and pH 4.0 to 6.0 which are commonly used culture conditions It is preferable to use a method of culturing for 12 to 36 hours under the condition of, and may further include a step of grinding the dried crystalline cellulose to a size of 40 to 80 mesh in order to improve the ease of production.

The present inventors found that the peptide production yield was very low as a result of producing a peptide from the slaughter product using a conventional enzymatic hydrolysis method to prepare a functional peptide from Zara. In other words, enzyme hydrolysis is typically performed by adding an alkaline solution to the slaughter to degrease, hydrolyzing using conventional proteolytic enzymes, filtering using activated clay and diatomaceous earth, and concentrating and freeze-drying the filtrate. Including the process, when the method was grown and applied to the slaughter, it was confirmed that the yield of the peptide produced is very low.

Thus, the present inventors came to pay attention to the method using microbial fermentation while studying various methods in order to improve the production yield of zopeptide. In particular, when inoculated and cultured with lactic acid bacteria such as L. acidophilus, L. salivarius, Lactobacillus L. salivarius, the reaction product produced by enzymatic digestion of the slaughter By various enzymes, macromolecule proteins that were not degraded by proteolytic enzyme treatment before lactic acid inoculation were easily decomposed into peptides of smaller size, and as a result, the yield of peptide production was increased.

In addition, conventionally, the hydrolyzed reactant was filtered through activated clay and diatomaceous earth to remove impurities, and the filtrate was concentrated and dried. However, when using such a conventional method, an extremely low yield from the hydrolyzed reactant was used. As the peptides were obtained, the researchers searched for a variety of methods to overcome them. Attention was directed to obtaining a peptide using a substance capable of specifically adsorbing the peptide. As a result of searching for a substance, it was confirmed that when crystalline cellulose was used, the peptide was most effectively obtained. That is, when the hydrolyzed reactant is mixed with the crystalline cellulose, it was confirmed that the peptide contained in the hydrolyzed reactant is selectively adsorbed to the crystalline cellulose, thereby simplifying the preparation process of the peptide.

"Zara zopeptide" in the present invention means that the peptide prepared from the slaughter of Zara by the above-described manufacturing method is adsorbed to the crystalline cellulose, the electro-zapeptide shows the effect of improving blood sugar level and hangover .

When the production method of the present invention is used, functional peptides can be produced in high yields from growth, and thus will be widely contributed to economic production of functional peptides.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Establishment of Peptide Preparation Conditions Using Slaughterhouses

The purpose of this study was to determine the hydrolysis conditions (reaction temperature, reaction pH and reaction time), selection of cultured microorganisms, culture conditions of cultured microorganisms and mixing ratio of culture and crystalline cellulose using protein enzymes, which can improve the yield of peptides. .

Example 1-1 : Establishment of Protease Reaction Conditions

First, washed Zara (1 kg) was slaughtered, chopped, and then put into a mixer with 3 L of Tris buffer (pH 8.0) and ground for 2 hours to grow to obtain a pulverized product.

Electrolysate was added to protease P (Sigma Chem. Co., USA) to 0.5% (w / v) to the ground, and the temperature range of 10 to 80 ℃, pH range of pH 4 to 10 and After hydrolysis in the time range of 10 to 120 minutes, and then heated at 121 ℃ for 15 minutes to inactivate the protease. Subsequently, a cut off of 10,000 filtration membranes was passed to obtain a peptide fraction, and then its concentration was measured, and the growth rate of the electrophoresis was compared with the protein concentration of the pulverized product to determine the yield of the peptide (see Table 1a). , Table 1b and Table 1c).

Comparison of Production Yields of Peptides According to Reaction Temperature when Proteolytically Hydrolyzed for pH 8.0 and 60 min Reaction Time Reaction temperature (℃) Peptide yield (%) 10 20 30 40 50 60 70 80 8 10 13 23 28 35 12 5

As shown in Table 1a, the production yield of the peptide was increased when it is 40 ℃ or more, it can be seen that the production yield of the peptide rather decreased when the reaction at 70 ℃ or more.

Comparison of production yields of peptides according to reaction pH conditions when protein hydrolysis is performed at a reaction temperature of 50 ° C. and a reaction time of 60 minutes Reaction pH Peptide yield (%) 4 5 6 7 8 9 10 -5 11 22 28 10-

As shown in Table 1b, the production yield of the peptide was increased when the pH is 7 or more, and the yield of the peptide was lowered when the reaction was carried out at pH 9 or more.

Comparison of production yields of peptides according to reaction time when protein hydrolysis at pH 8.0 and reaction temperature of 50 ℃ Response time (minutes) Peptide yield (%) 10 20 30 40 50 60 70 80 90 10 110 120 5 8 11 22 25 28 31 34 36 36 36 36

As shown in Table 1c, the production yield of the peptide was increased when the reaction was performed for 30 minutes or more, and the production yield of the peptide was no longer increased when the reaction was conducted for 90 minutes or more.

In summary, when the hydrolysis was carried out for 30 to 90 minutes at a temperature of 40 to 60 ℃ and pH 7 to 8, it was found that the yield of the peptide is excellent, but the best yield was only 36% In addition, it was found that additional decomposition process is required.

Example 1-2 : Selection of cultured microorganisms and establishment of culture conditions

Washed Zara (1 kg) was slaughtered, chopped, and then placed in a mixer with 3 L of Tris buffer (pH 8.0) and ground for 2 hours to give a grind. Subsequently, proteolytic enzyme P was added to 100 ml of the milled product so as to be 0.5% (w / v), and hydrolyzed for 90 minutes under the conditions of 50 ° C. and pH 8 to obtain a primary degradation product. E. coli, Saccharomyces cerevisiae, Shijosaccharomyces cerevisiae, Monascus phyllosus, Aspergillus duckweed, Lactobacillus ashdophyllus and Lactobacillus salivarius 1 x 10 ⁵ cells were inoculated each, incubated at 37 ° C. for 24 hours, and then centrifuged to obtain a supernatant. The cut off 10,000 was then passed through a filtration membrane to obtain a peptide fraction, the concentration of which was then measured, and the yield of peptide was measured by comparison with the protein concentration of the pulverized product. At this time, as a control, a primary degradation product in which no strain was cultured was used (see Table 2).

Comparison of Production Yields of Peptides According to Culture Strains Culture strain Peptide yield (%) Control Escherichia coli Saccharomyces cerevisiae Shijo Saccharomyces cerevisiae Monascus Philosos Aspergillus Ducks Lactobacillus Ashdophyllus Lactobacillus Salivarius 36 40 52 48 53 55 72 76

As shown in Table 2a, when most of the strains were cultured, the production yield of the peptide was improved compared to the control, except for Lactobacillus ashidophyllus and Lactobacillus salivarius, the production yield of the peptide was not significantly increased. It was found that.

Example 1-3 Selection of Peptide Adsorbents

Washed Zara (1 kg) was slaughtered, chopped, and then placed in a mixer with 3 L of Tris buffer (pH 8.0) and ground for 2 hours to give a grind. Subsequently, proteolytic enzyme P was added to 100 ml of the milled product so as to be 0.5% (w / v), and hydrolyzed for 90 minutes under the conditions of 50 ° C. and pH 8 to obtain a primary degradation product. Lactobacillus salivarius was inoculated into 100 ml of the first degradation product obtained above, incubated for 24 hours at 37 ° C. and pH 5.0, and centrifuged to obtain a supernatant. Subsequently, a cutoff of 10,000 filtration membranes was passed to obtain a peptide fraction, and then its concentration was measured. Subsequently, the peptide fraction was mixed with adsorbents such as corn starch, potato starch, sweet potato starch, crystalline cellulose, xanthan gum, gellan gum or carrageenan gum at 1: 1 (v / v), filtered and filtered. The crystalline cellulose fraction to which the peptide of the peptide fraction was adsorbed was obtained, respectively. Subsequently, the peptide concentration of the peptide fraction and the peptide concentration of the electrofiltered liquid component were compared, and the content of peptide adsorbed to each adsorbent was compared (see Table 3a).

Comparison of Peptide Adsorption Rate by Adsorbents Adsorption material Peptide Adsorption Rate (%) Corn Starch Potato Starch Sweet Potato Starch Crystalline Cellulose Xanthan Gum Gelan Gum Carrageenan Gum 68 72 69 88 54 56 58

As shown in Table 3a, the peptide showed a higher adsorption rate on starch (corn starch, potato starch, sweet potato starch, crystalline cellulose) than gums (xanthan gum, gellan gum, carrageenan gum), and crystal cellulose among starches. The highest adsorption rate was obtained when mixed with.

Example 1-4 : Determination of the Mixing Ratio of the Culture and Crystalline Cellulose

Washed Zara (1 kg) was slaughtered, chopped, and then placed in a mixer with 3 L of Tris buffer (pH 8.0) and ground for 2 hours to give a grind. Subsequently, proteolytic enzyme P was added to 100 ml of the milled product so as to be 0.5% (w / v), and hydrolyzed for 90 minutes under the conditions of 50 ° C. and pH 8 to obtain a primary degradation product. Lactobacillus salivarius was inoculated into 100 ml of the first degradation product obtained above, incubated for 24 hours at 37 ° C. and pH 5.0, and centrifuged to obtain a supernatant. Subsequently, a cutoff of 10,000 filtration membranes was passed to obtain a peptide fraction, and then its concentration was measured. The peptide fraction and crystalline cellulose were then separated into 9: 1, 8: 2, 7: 3, 6: 4, 5: 5, 4: 6, 3: 7, 2: 8 or 1: 9 (v / v). The mixture was filtered and filtered to obtain a crystalline cellulose fraction in which the filtered liquid component and the peptide fraction of the peptide were adsorbed, respectively. Subsequently, the peptide concentration of the peptide fraction was compared with the peptide concentration of the electrofiltered liquid component, and the content of peptide adsorbed to the crystalline cellulose was compared (see Table 3b).

Comparison of Peptide Adsorption Rate on Crystalline Cellulose by Mixing Ratio of Peptide Fraction and Crystalline Cellulose Mixing ratio of peptide fraction and crystalline cellulose (v / v) Peptide adsorption rate to crystalline cellulose (%) 9: 1 8: 2 7: 3 6: 4 5: 5 4: 6 3: 7 2: 8 1: 9 18 30 41 56 88 92 96 96 96

As shown in Table 3b, when the peptide fraction and the crystalline cellulose were mixed at 5: 5 (v / v) or more, the peptide adsorption rate to the crystalline cellulose was significantly increased, and the peptide fraction and the crystalline cellulose were 3: 7 (v). / v), it was found that the peptide adsorption rate to the crystalline cellulose does not increase any more.

Example 2 Preparation of Zopeptide Using Grow Slaughterhouse

Under the conditions determined in Examples 1-1 to 1-3 above, crude peptides were prepared from the slaughter and the production yield thereof was measured.

Example 2-1 Preparation of Peptides Using Protease P

Washed Zara (1 kg) was slaughtered, chopped, and then placed in a mixer with 3 L of Tris buffer (pH 8.0) and ground for 2 hours to give a grind. Subsequently, proteolytic enzyme P was added to 100 ml of the pulverized product so as to be 0.5% (w / v), and hydrolyzed for 50 minutes under the conditions of 50 ° C. and pH 8 to obtain a primary degradation product. Lactobacillus salivarius was inoculated into 100 ml of the first degradation product obtained above, incubated for 24 hours under conditions of 30 ° C. and pH 5.0, and the mixture was mixed with 4: 6 (v / v) of the cultivated cell culture and filtered. Thus, the crystalline cellulose fraction to which the peptide of the culture was adsorbed was obtained. Subsequently, the cellulose cellulose fraction obtained by adsorbing the peptide was hot-air-dried, powdered to 80 mesh, and then the yield of the peptide was measured. The yield was 91.2% (w / w) relative to the ground product. It was confirmed that the peptide can be prepared.

Example 2-2 Preparation of Peptides Using Protease N

Washed Zara (1 kg) was slaughtered, chopped, and then placed in a mixer with 3 L of Tris buffer (pH 8.0) and ground for 2 hours to give a grind. Subsequently, protease N (Sigma Chem. Co., USA) was added to 100 ml of the ground product to make 0.5% (w / v), and hydrolyzed for 70 minutes under the conditions of 40 ° C and pH 7. To obtain a primary decomposition product. Lactobacillus salivarius was inoculated into 100 ml of the obtained first degradation product, incubated for 30 hours under conditions of 40 ° C. and pH 6.0, and the electric culture and crystalline cellulose were mixed at 3: 7 (v / v) and filtered. Thus, the crystalline cellulose fraction to which the peptide of the culture was adsorbed was obtained. Subsequently, the cellulose cellulose fraction obtained by adsorbing the peptide was hot-air-dried, powdered to 60 mesh, and then the yield of the peptide was measured. The yield was 93.4% (w / w) relative to the ground product. It was confirmed that the peptide can be prepared.

Comparative Example 1 Preparation of Peptides Using Conventional Enzymatic Hydrolysis

Peptides were prepared from slaughtered plants using the method disclosed in Korean Patent No. 333837, which discloses a method for preparing a functional peptide using conventional fish meat, and the yield thereof was measured.

That is, slaughtered washed jars are obtained, 5 kg of samples excluding tofu and intestines are obtained, washed with water, and then 20 L of 0.1% (w / v) NaHCO ₃ solution containing 0.05% (w / v) NaCl is added thereto. Triturated with colloid mill and centrifuged at 5,000 xg for 20 minutes to obtain a precipitate. Subsequently, the precipitate was mixed with twice the volume of water, and the proteinase papain was added to 0.5% (w / v) and hydrolyzed at 70 ° C. and pH 6.0 for 4 hours. Promod 192p enzyme was added to 0.5% (w / v), and then hydrolyzed for 2 hours at 50 ° C. and pH 4.5 for 2 hours, and then ripened for 2 minutes. Subsequently, the reaction product was sequentially applied to activated clay and diatomaceous earth, filtered under reduced pressure, concentrated under vacuum and lyophilized to obtain a protein hydrolyzate, which was dissolved in water to 10% (w / v), Peptides were finally prepared by passing through a cutoff of 10,000 filtration membranes, and their yield was about 56%.

As described above, in the case of using the conventional manufacturing method, the yield of the peptide was only 56%. However, as shown in Examples 2-1 and 2-2, when the manufacturing method of the present invention was used, the peptide was 90% or more. As the production yield was shown, it was found that the production method of the present invention was much superior to the conventional method in terms of production yield.

Example 3 Effect of Zopeptides Prepared from Zara

Already, the present inventors have conducted various studies regarding the functionality of Zarabala, and as a result, it was found that a composition containing Zarabala and two leaf lobes could improve blood sugar levels. The patent application was filed in Korean Patent Application No. 2005-23236 dated, and the research result was obtained that the composition containing Zara's Tortoiseshell and Vitamin C can have hangover relief effect. No. 2005-23293 filed a patent. The present inventors confirmed whether or not the peptides prepared in the present invention can improve blood glucose levels and exhibit hangover relief.

Example 3-1 : Improvement of Blood Glucose Level of Zopeptide

First, STZ (streptozotocin, A0130, Sigma) is dissolved in a buffer solution (0.1M citrate buffer, pH 4.0) at a concentration of 250mg / ㎖, and 0.2ml / kg intraperitoneal injection in 300-350g rats to induce diabetes I was. After 7 days, mice with a blood glucose level of 300 mg / dl or more were selected using a blood glucose meter (Medisense Precision Q.I.D., USA). The electropeptide diabetic rats were fed the peptides prepared in Example 2-1 for 300 weeks / kg for 4 weeks, and the blood glucose level was measured at 0, 1, 2, 3 and 4 weeks. After the measurement, the blood glucose level was measured by dividing the blood glucose level at the time point 0 weeks passed, and compared with the change in blood glucose level over time. At this time, the diabetic rats that were not administered anything were used as the positive control group, and the diabetic rats which did not cause diabetes were used as the negative control group. The blood glucose level was positive control group: 415.5mg / dl, negative control group: 110.8 mg / dl and experimental group: 412.8 mg / dl (see Table 4a).

Changes in Blood Glucose Levels According to Duration of Administration of Zopeptides Prepared from Jara (Unit:%) Elapsed time (weeks) 0 One 2 3 4 Positive control 100 103.4 108.2 114.2 121.6 Negative control 100 101.3 103.5 101.7 98.6 Experimental group 100 99.5 91.4 77.4 60.0

As shown in Table 4a, as compared with the positive control group, the experimental group was found that the blood glucose level is significantly reduced, it was confirmed that the peptide produced in the present invention can improve the blood glucose level.

Example 3-2 Hangover Relief Effect of Zopeptide

After 5 minutes / kg oral administration of an aqueous 38% (v / v) ethanol solution in a rat body of 300 to 350 g, 60 minutes elapsed, the oreptides prepared in Example 2-2 were orally administered in an amount of 10 mg / kg. After 0, 1, 2, 3, 6 and 8 hours had elapsed, 0.2 ml of blood was collected. The collected blood was reacted with trichloroacetic acid for 5 minutes, and then centrifuged at 2,000 rpm for 5 minutes to obtain plasma. Subsequently, 0.1 mL of the obtained plasma was mixed with 2.9 mL of the NAD-ADH solution included in the alcohol concentration measurement kit (# 332, Sigma Chem. Co., USA), reacted at 37 ° C for 10 minutes, and the absorbance was measured at 340 nm. Blood alcohol concentration in blood was calculated by substituting the measured values into the standard curve. At this time, the control group was used rats that did not receive the peptide (see Table 4b).

Changes in Alcohol Concentration in Blood According to the Duration of Zopeptide Administration (Unit: mg / dl) Elapsed time (hours) Blood alcohol level in the control group Blood Alcohol Concentration in Experimental Group 0 84.2 ± 7.0 84.2 ± 7.1 One 77.3 ± 9.5 8.3 ± 6.4 2 61.8 ± 5.8 5.6 ± 4.5 3 45.0 ± 3.8 2.4 ± 2.1 6 18.0 ± 2.1 0 8 0 0

As shown in Table 4b, it was confirmed that the experimental group exhibited an excellent blood alcohol removal effect as compared to the control group, the peptides prepared in the present invention showed a hangover relief effect.

 As described and demonstrated in detail above, the present invention provides a method for preparing Zara zopeptide, which comprises the step of enzymatically digesting Zara extract, inoculating and inoculating the lactic acid bacterium. When the production method of the present invention is used, functional peptides can be produced in high yields from growth, and thus will be widely contributed to economic production of functional peptides.

Claims (2)

(Iii) adding protease N or protease P to the slaughtered shoots and reacting for 30 to 90 minutes under conditions of 40 to 60 ° C and pH 7 to 8; (Ii) heating the reactants to inactivate proteolytic enzymes, and then inoculating and culturing L. acidophilus or L. salivarius; (Iii) Filtration or centrifugation of the electric culture to obtain a culture medium, and then mixed with the electric culture medium and crystalline cellulose in a ratio of 3: 7 to 5: 5 (v / v) and filtered, crystal cellulose adsorbed peptide To obtain; And, (Iii) A method for producing zopeptide, comprising the step of drying the obtained crystalline cellulose. The method of claim 1, Further comprising the step of grinding the dried crystalline cellulose to the size of 40 to 80mesh Zappa peptide production method.