TWI662961B - USE AND BUFFERED COMBINAITION OF A MULTI-pH-VALUE BUFFER FORMULATION AND PROTEIN HYDROLYSIS EFFICIENCY ENHANCER FOR BOTH PEPSIN AND TRYPSIN ENHANCER - Google Patents

Abstract

The present invention is a buffer composition for promoting the proteolytic efficiency of pepsin and trypsin, comprising at least one acid component, wherein the at least one acid component is an organic acid; at least one salt component, wherein the at least one salt component is An organic salt, the at least one acid component and the at least one salt component have a conjugate relationship, and the at least one acid component and the at least one salt component form a buffering formula; and a proteolytic efficiency gaining agent, wherein the protein hydrolysis efficiency The gain agent is one of ascorbic acid, a salt thereof, and a combination thereof.

Description

Buffer composition of multiple pH buffering formula and pepsin and trypsin co-proteolytic efficiency gaining agent and use thereof

The present invention relates to a buffer composition which contributes to enhancing the proteolytic efficiency and absorption efficiency of human pepsin and trypsin. More specifically, the present invention relates to a buffer composition for increasing the proteolytic efficiency of pepsin and trypsin in the range of acid-base (pH 5 - 7.5) and its use.

After the protein enters the digestive tract of the mammal, it is hydrolyzed by pepsin in the gastric juice, trypsin in the pancreatic juice, chymotrypsin, carboxypeptidase, aminopeptidase in the intestinal juice, and dipeptidase. Finally, it becomes an amino acid, which is then absorbed by intestinal epithelial cells.

The gastric juice contains pepsinogen and gastric acid, and pepsinogen is activated in the stomach through gastric acid to become pepsin. After the food passes through the stomach, the pancreas secretes pancreatic juice and is transported through the pancreatic duct to the duodenum upstream of the small intestine. The trypsinogen in the pancreatic juice is secreted into the duodenum to promote intestinal activation. The trypsinogen is activated by trypsin (actokinase) to trypsin, which can decompose the protein into oligopeptides. Trypsin also activates other proproteinases and trypsinogens that are transformed into the gut.

Since the 1950s, animal husbandry has widely added antibiotic growth promoters to animal feed. However, with the growing concern about animal health and drug residues, the EU has banned the use of antibiotics as feed additives since 2006. The management of drug-containing feed additives has become increasingly strict. In order to replace antibiotics, non-pharmaceutical additives such as acidulants have been rapidly developed in recent years. The acidifying agent can lower the pH of the feed in the gastrointestinal tract and increase the activity of the pepsin, thereby making the digestion of the protein more efficient. However, since the acidifying agent rapidly lowers the pH of the feed and stomach contents, it inhibits the normal secretion of gastric acid, and the long-term use may cause the animal to have a digestive ability to decrease and a slower growth effect.

In order to help digestion and absorption of the digestive tract, the applicant developed a non-pharmaceutical additive that can enhance the proteolytic efficiency and absorption efficiency of pepsin and trypsin in the human gastrointestinal tract, which can make up for the shortcomings of the current products. A brief description.

The main object of the present invention is to enhance the proteolytic efficiency and absorption efficiency of human pepsin and trypsin. In order not to affect the normal physiological function of the gastrointestinal tract, the present invention provides a multi-pH buffering formula combination which can be optimally operated in different pH environments of the gastrointestinal tract, thereby enhancing the overall hydrolysis efficiency and absorption efficiency of the protein in the gastrointestinal tract. .

The multiple pH buffering formula combination of the invention cooperates with different pH environments of the gastrointestinal tract, and can significantly increase the proteolytic efficiency of the main protease of the gastrointestinal tract in an acid-base environment (pH 5~7.5). In order to achieve the above effects, the multiple pH buffering formula combination of the present invention comprises at least one acid component, at least one salt component, and a proteolytic efficiency gaining agent, wherein the at least one acid component is an organic acid, and the at least one salt component is an organic salt. The proteolytic efficiency enhancing agent is ascorbic acid, a salt thereof or a combination thereof. The acid component forms a buffering formula with the salt component to make the protein The hydrolysis efficiency gain agent exhibits a stable high activity in the buffer formulation.

The present invention provides a buffer composition which can comprehensively enhance the proteolytic efficiency of pepsin and trypsin in the gastrointestinal tract, comprising: at least one acid component, at least one salt component, and a proteolytic efficiency gain agent, wherein the at least one acid component is An organic acid, the at least one salt component is an organic salt, the at least one acid component has a conjugate relationship with the at least one salt component, and the at least one acid component and the at least one salt component form a buffering formula, and the protein is hydrolyzed. The efficiency gain agent is one of ascorbic acid, a salt thereof, and a combination thereof.

The above objects and advantages of the present invention will become more apparent to those skilled in the <RTIgt;

The first panel shows the effect of pH on the activity of three digestive enzymes in the gastrointestinal tract.

Figure 2A is a graph showing the proteolytic efficiency measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 2.0 for the pepsin (control group) and the experimental group containing different concentrations of ascorbic acid.

The second B panel is a graph showing the proteolytic efficiency measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 5.0 for the pepsin (control group) and the experimental group containing different concentrations of ascorbic acid.

The second C-graph is a graph showing the proteolytic efficiency measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 6.0 for the pepsin (control group) and the experimental group containing different concentrations of ascorbic acid.

Figure 3A is a graph showing that trypsin (control group) and an experimental group containing different concentrations of ascorbic acid were measured at pH 5.0 at 10 minutes, 1 hour, 2 hours, and 3 hours. Proteolytic efficiency.

The third panel B is a graph showing the proteolytic efficiency measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 6.0 of the trypsin (control group) and the experimental group containing different concentrations of ascorbic acid.

The third C-graph is a graph showing the proteolytic efficiency measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 7.5 in trypsin (control group) and the experimental group containing different concentrations of ascorbic acid.

In designing the multiple pH buffering formulation combinations of the present invention, the pH environment of the gastrointestinal tract must be considered. Please refer to the first figure, which is documented in Essentials of Human Physiology (2017). As shown in the figure, the three main digestive enzymes in the gastrointestinal tract have different pH optimum environments. For example, pepsin has the best activity in pH 1.5~2 environment, while trypsin has the best pH 7.5~8 environment. active. In fact, during the digestion of the gastrointestinal tract, the pH does not remain constant, but varies between different pH values. When the food enters the upper part of the stomach, the pH value is about 4.0~6.5. When entering the lower part of the stomach, the pH value is about 1.5~4.0; similarly, the pH of each part of the small intestine is also different in the duodenum. The pH value is about 6.0 to 7.5, and the pH in other segments of the small intestine is about 5.0 to 7.5. Although the protein has better hydrolysis efficiency under the optimum pH environment of pepsin and trypsin, it can not reach the optimum pH environment, or in individuals with insufficient gastric acid secretion (such as newborn babies or the elderly, even the first weaning) Piglets), if it can improve the proteolytic efficiency of pepsin and trypsin in the gastrointestinal tract in an environment of pH 5~7.5, especially pH 5~6, can improve the overall hydrolysis of protein in the gastrointestinal tract. Efficiency and absorption efficiency.

The buffer formulation combination provided in the present invention comprises at least one acid component, at least one salt Ingredients and a protein hydrolysis efficiency gain agent. Since the buffer formulation combination of the present invention can be added to a human protein nutritional supplement, the components used must be non-toxic to the organism, the acid component is preferably an organic acid, the salt component is preferably an organic salt, and the protein hydrolysis efficiency gain agent is ascorbic acid. , its salts or a combination thereof. Further, phosphoric acid and phosphate are widely used food additives, and therefore are also covered by the acid component and the salt component of the present invention.

The acid component has a conjugation relationship with the salt component, such as an organic acid and its conjugate salt, or an organic salt and its conjugate acid, which are formulated into a buffer formulation. The pH of the buffer formulation is determined by the dissociation constant (pKa) of the acid component and the ratio of the acid component to the salt component. Generally, the pH is in the range of about ±1 pKa. The pKa values of commonly used organic acids, phosphoric acid and ascorbic acid are shown in Table 1 (see Lab Manual for Zumdahl/Zumdahl's Chemistry, 6th Edition). Among these acid components, citric acid has three different pKa values and is an organic acid, and thus is an optimum choice for the acid component in the buffer formulation of the present invention.

The buffer formulation formed by selecting the appropriate acid component and the salt component can stabilize the pH change of the gastrointestinal environment in a certain pH range, and ensure that the protein hydrolysis efficiency gain agent can exert its proper function in the gastrointestinal tract.

The acid component used in the present invention includes formic acid, acetic acid, propionic acid, butyric acid, malic acid, fumaric acid, lactic acid, citric acid, and phosphoric acid. Preferably, the acid component is an organic acid. In a preferred embodiment, the organic acid is citric acid. The salt component used in the present invention is an organic salt or a phosphate. Preferably, the organic salt or phosphate refers to an alkali metal salt or an alkaline earth metal salt. In a preferred embodiment, the organic salt is one of sodium citrate and potassium citrate.

The buffer formulation of the present invention may also comprise a plurality of organic acids and their conjugate salts, or a plurality of organic salts with their conjugate acids. The organic acid may also be combined with phosphoric acid as the acid component in the buffer formulation of the present invention, and the organic salt may also be combined with phosphate as the salt component in the buffer formulation of the present invention. Buffer formulations suitable for the gastrointestinal pH environment are all within the scope of the present invention.

The proteolytic efficiency-enhancing agent used in the present invention is ascorbic acid, a salt thereof or a combination thereof. Preferably, the salt of ascorbic acid is one of a sodium salt, a potassium salt, a calcium salt, a magnesium salt and a combination thereof.

Ascorbic acid (L-ascorbate), also known as vitamin C, is a compound that is soluble in water and readily absorbed. Since ascorbic acid is easily present in an aqueous solution when it is present in an aqueous solution alone, the buffer formulation of the present invention can maintain the activity of ascorbic acid, a salt thereof or a combination thereof in an environment having a pH of more than 4, in order to achieve transacid Alkaline environment enhances pepsin and trypsin The purpose of the enzyme's proteolytic efficiency.

In another aspect, the buffer composition of the present invention can be used as a buffering composition for helping to comprehensively enhance the efficiency of proteolytic hydrolysis of pepsin and trypsin in the gastrointestinal tract, for example, an additive added to a protein nutritional supplement such as a formula, at pH. The proteolytic activity of pepsin and trypsin is enhanced in a 5~7.5 environment, and the proteolytic efficiency of pepsin and trypsin is significantly improved in a pH of 5-6 environment.

According to the concept of the present invention, at least one acid component, at least one salt component and a proteolytic efficiency gain agent are included in the composition, wherein at least one acid component forms a buffering formula with at least one salt component, and the buffering formula enables protein hydrolysis efficiency. The gain agent maintains high activity in an acid-base environment. In one embodiment, the proteolytic efficiency gain agent is distributed in the buffer formulation. In another embodiment, the buffer formulation is uniformly mixed with the proteolytic efficiency gain agent.

In the present invention, the proteolytic efficiency-enhancing agent is ascorbic acid, a sodium salt thereof, a potassium salt, a calcium salt, a magnesium salt or a combination thereof. In a preferred embodiment, the proteolytic efficiency enhancing agent is ascorbic acid, especially L-ascorbic acid, having a concentration between 60 ppm and 1000 ppm. Preferably, the concentration of ascorbic acid is between 60 ppm and 240 ppm, more preferably 240 ppm. Of course, the ascorbic acid of the present invention may have a higher concentration as long as it conforms to the daily intake of humans (up to about 2 g per day).

In accordance with the teachings of the present invention, the dosage forms of the above buffer compositions include powders, granules, lozenges, microparticles, liquids, capsules or sustained release dosage forms.

The following is an embodiment of the present invention

(1) Experimental methods

1. Casein pepsin enzymatic hydrolysis experiment (pH=2.0)

1.1 Control group: Four test tubes were taken, and four control solutions containing casein (Casein, 3.85 mg/mL) and pepsin (Pepsin, 385 ppm) were prepared in a citric acid buffer solution having a pH of 2.0. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity was expressed as a Tyrosine calibrated line previously established at the same pH value, and expressed as a total amino acid equivalent (Total Amino Acid Equivalent (TAAE) μg/mL).

1.2 Experimental group (60ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 60 ppm ascorbic acid, pepsin (385 ppm) with a pH 2.0 citric acid buffer solution. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

1.3 Experimental group (120ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 120 ppm ascorbic acid, pepsin (385 ppm) with a pH 2.0 citric acid buffer solution. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% was added. The chloroacetic acid (TCA) solution was mixed well, allowed to stand at room temperature for 10 minutes, and centrifuged at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

1.4 Experimental group (240ppm ascorbic acid): Four tubes were prepared and four samples containing casein (Casein, 3.85 mg/mL), 240 ppm ascorbic acid, pepsin (385 ppm) were prepared with a pH 2.0 citric acid buffer solution. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

2. Casein pepsin enzymatic hydrolysis experiment (pH=5.0)

2.1 Control group: Four test tubes were taken, and four control solutions containing casein (Casein, 3.85 mg/mL) and pepsin (Pepsin, 385 ppm) were prepared in a citric acid buffer solution having a pH of 5.0. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. Down test Determine the intensity of the fluorescence. The obtained fluorescence intensity was expressed as a Tyrosine calibrated line previously established at the same pH value, and expressed as a total amino acid equivalent (Total Amino Acid Equivalent (TAAE) μg/mL).

2.2 Experimental group (60ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 60 ppm ascorbic acid, pepsin (385 ppm) with a pH 5.0 citric acid buffer solution. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

2.3 Experimental group (120ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 120 ppm ascorbic acid, pepsin (385 ppm) with a pH 5.0 citric acid buffer solution. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). . 2.4 Prepare a new test tube, and take 20 μL of the supernatant from the tube after centrifugation and add 2.4 mL of phthalate. Formaldehyde (OPA) reagent, after standing for 2 minutes, the fluorescence intensity was measured by a fluorescence spectrometer at EX. 340 nm and EM.455 nm.

2.4 Experimental group (240ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 240 ppm ascorbic acid, pepsin (385 ppm) with a pH 5.0 citric acid buffer solution. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

3. Casein pepsin enzymatic hydrolysis experiment (pH=6.0)

3.1 Control group: Four test tubes were taken, and four control solutions containing casein (Casein, 3.85 mg/mL) and pepsin (Pepsin, 385 ppm) were prepared in a citric acid buffer solution having a pH of 6.0. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity was expressed as a Tyrosine calibrated line previously established at the same pH value, and expressed as a total amino acid equivalent (Total Amino Acid Equivalent (TAAE) μg/mL).

3.2 Experimental group (60ppm ascorbic acid): Take four tubes and use a lemon with a pH of 6.0. Acid buffer solution Four experimental solutions containing casein (3.81 mg/mL), 60 ppm ascorbic acid, pepsin (Pepsin, 385 ppm) were prepared. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

3.3 Experimental group (120ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 120 ppm ascorbic acid, pepsin (385 ppm) with a pH 6.0 citric acid buffer solution. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

3.4 Experimental group (240ppm ascorbic acid): Four tubes were prepared, and four samples containing casein (Casein, 3.85 mg/mL), 240 ppm ascorbic acid, pepsin (385 ppm) were prepared in a citric acid buffer solution with a pH of 6.0. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. Minutes, then at 3000 rpm Centrifuge for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

4. Casein enzymatic hydrolysis test of casein (pH=5.0)

4.1 Control group: Four test tubes were taken, and four control solutions containing casein (Casein, 3.85 mg/mL) and trypsin (Trypsin, 385 ppm) were prepared in a citric acid buffer solution having a pH of 5.0. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity was expressed as a Tyrosine calibrated line previously established at the same pH value, and expressed as a total amino acid equivalent (Total Amino Acid Equivalent (TAAE) μg/mL).

4.2 Experimental group (60ppm ascorbic acid): Four tubes were prepared and four samples containing casein (Casein, 3.85 mg/mL), 60 ppm ascorbic acid, trypsin (Trypsin, 385 ppm) were prepared with a pH 5.0 citric acid buffer solution. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The resulting fluorescence intensity is previously in the same pH The value is calculated as the tyrosine (Tyrosine) calibration line established under the same ascorbic acid concentration and expressed as the relative equivalent of total amino acid (Total Amino Acid Equivalent (TAAE) μg/mL).

4.3 Experimental group (120ppm ascorbic acid): Four tubes were prepared, and four samples containing casein (Casein, 3.85 mg/mL), 120 ppm ascorbic acid, trypsin (Trypsin, 385 ppm) were prepared with a pH 5.0 citric acid buffer solution. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

4.4 Experimental group (240ppm ascorbic acid): Four tubes were prepared, and four samples containing casein (Casein, 3.85 mg/mL), 240 ppm ascorbic acid, trypsin (Trypsin, 385 ppm) were prepared with a pH 5.0 citric acid buffer solution. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

5. Casein enzymatic hydrolysis test of casein (pH=6.0)

5.1 Control group: Take four tubes and prepare four portions with citric acid buffer solution with pH 6.0. A control solution containing casein (3.85 mg/mL) and trypsin (Trypsin, 385 ppm) was included. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity was expressed as a Tyrosine calibrated line previously established at the same pH value, and expressed as a total amino acid equivalent (Total Amino Acid Equivalent (TAAE) μg/mL).

5.2 Experimental group (60ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 60 ppm ascorbic acid, trypsin (Trypsin, 385 ppm) in a citric acid buffer solution with a pH of 6.0. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity was expressed in terms of a Tyrosine calibrated line previously established at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent (TAAE) μg/mL.

5.3 Experimental group (120ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 120 ppm ascorbic acid, trypsin (Trypsin, 385 ppm) in a citric acid buffer solution with a pH of 6.0. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. Minutes, then at 3000 rpm Centrifuge for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

5.4 Experimental group (240ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 240 ppm ascorbic acid, trypsin (Trypsin, 385 ppm) in a citric acid buffer solution with a pH of 6.0. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

6. Casein enzymatic hydrolysis of casein (pH=7.5)

6.1 Control group: Four test tubes were taken, and four control solutions containing casein (Casein, 3.85 mg/mL) and trypsin (Trypsin, 385 ppm) were prepared in a citric acid buffer solution having a pH of 7.5. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a Tyrosine calibration line established in advance at the same pH value. The content is expressed in terms of total amino acid relative equivalents (Total Amino Acid Equivalent (TAAE) μg/mL).

6.2 Experimental group (60ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 60 ppm ascorbic acid, trypsin (Trypsin, 385 ppm) in a citric acid buffer solution with a pH of 7.5. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

6.3 Experimental group (120ppm ascorbic acid): Four tubes were used to prepare four samples containing casein (Casein, 3.85 mg/mL), 120 ppm ascorbic acid, trypsin (Trypsin, 385 ppm) in a citric acid buffer solution with a pH of 7.5. Solution. The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

6.4 Experimental group (240ppm ascorbic acid): Take four tubes and prepare four parts containing casein (Casein, 3.85mg/mL) and 240ppm anti-bad with citrate buffer solution with pH 7.5. Experimental solution of blood acid, trypsin (Trypsin, 385 ppm). The test tube was reacted at a constant temperature of 37 ° C, and four tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of 10% trichloroacetic acid (TCA) solution was added thereto, uniformly mixed, and allowed to stand at room temperature for 10 hours. In minutes, centrifuge again at 3000 rpm for 10 minutes. Four new tubes were taken, and 20 μL of the supernatant at the above four time points were added, respectively, 2.4 mL of o-phthalaldehyde (OPA) reagent was added, and after standing for 2 minutes, the fluorescence spectrometer was used at EX.340 nm and EM.455 nm. The fluorescence intensity was measured. The obtained fluorescence intensity is expressed as a tyrosine (Tyrosine) calibration line established in advance at the same pH value and the same ascorbic acid concentration, and expressed as a relative amino acid equivalent amount (Total Amino Acid Equivalent (TAAE) μg/mL). .

Experimental result

Please refer to Table 2 and Figure 2A, which show the proteolytic efficiency of pepsin (control) and the experimental group containing different concentrations of ascorbic acid at pH 2.0. Since pH 2.0 is the optimum environment for pepsin, the hydrolysis efficiency between groups is not much different, but it can be seen that the group containing ascorbic acid has better hydrolysis efficiency than the control group.

a. The numerical unit shown in the above table is Total Amino Acid Equivalent (TAAE) μg/mL.

Please refer to Tables 3 and B, which shows the proteolytic efficiency of pepsin (control) and the experimental group containing different concentrations of ascorbic acid at pH 5.0. Not a stomach egg at pH 5.0 The optimal environment for the white enzyme, so pepsin is less efficient in this environment, but it can be observed that 240 ppm of ascorbic acid significantly increases the hydrolysis efficiency of pepsin.

a. The unit of value shown in the above table is TAAE μg/mL

Please refer to Table 4 and Figure 2 C, which shows the proteolytic efficiency of pepsin (control group) and the experimental group containing different concentrations of ascorbic acid at pH 6.0. At pH 6.0, it is also not the optimum environment for pepsin, but it can be seen that the group containing ascorbic acid has better hydrolysis efficiency than the control group.

a. The unit of value shown in the above table is TAAE μg/mL

Please refer to Tables 5 and 3, which shows the proteolytic efficiency of trypsin (control group) and the experimental group containing different concentrations of ascorbic acid at pH 5.0. Not a pancreatic egg at pH 5.0 The optimum environment of the white enzyme, therefore, the hydrolysis efficiency of trypsin in this environment is poor, but it can be observed that 240 ppm of ascorbic acid significantly enhances the hydrolysis efficiency of trypsin.

a. The unit of value shown in the above table is TAAE μg/mL

Please refer to Tables 6 and 3B, which shows the proteolytic efficiency of trypsin (control group) and the experimental group containing different concentrations of ascorbic acid at pH 6.0. At pH 6.0, the optimum environment for non-trypsin was also observed, but it can be seen that the group containing ascorbic acid has better hydrolysis efficiency than the control group.

a. The unit of value shown in the above table is TAAE μg/mL

Please refer to Tables 7 and C, which show the proteolytic efficiency of trypsin (control) and the experimental group containing different concentrations of ascorbic acid at pH 7.5. Because the pH is 7.5 for the egg The optimum environment of white enzymes, so the hydrolysis efficiency between the groups is not much different, but it can be seen that the group containing ascorbic acid has better hydrolysis efficiency than the control group.

a. The unit of value shown in the above table is TAAE μg/mL

Comparing the relative concentrations of the various pH environments, the concentration of the amino acid measured in the optimum environment is the highest, and the concentration is relatively decreasing as the environmental conditions become worse. Both pepsin and trypsin have the same tendency. In the environment of pH 2~6, the optimized (pH 2) control group was used as a comparison index, and it was observed that ascorbic acid had a significant gain effect on pepsin proteolytic efficiency in pH 5, pH 6 and pH 2. Under the environment of pH 5~7.5, the optimized (pH 7.5) control group was used as a comparison index, and it was observed that ascorbic acid had a significant gain effect on trypsin proteolytic efficiency at pH 5, pH 6 and pH 7.5. The buffer combination of this case was observed from pH2 (polar-high hydrogen ion concentration) to pH5 (weak-high hydrogen ion concentration) and pH6 (weakly neutral-high hydrogen ion concentration) to pH 7.5 (low hydrogen ion concentration). Both showed significant gains in proteolytic efficiency, which were significantly pH-independent.

Since the buffer composition of the present invention does not contain a pharmaceutical ingredient, it is less susceptible to drug resistance and food safety.

The invention is a difficult and innovative invention, and has profound industrial application value, and is submitted in accordance with the law. Moreover, the invention can be modified by those of ordinary skill in the art, However, it does not depart from the scope of the invention as claimed.

Claims (11)

A use of a buffer composition for preparing a gaining agent for enhancing a protein hydrolysis efficiency of pepsin and trypsin in a human body, the buffer composition comprising: an acid component, wherein the acid component is a citric acid; a salt component, wherein the salt component is a citrate; and a common proteolytic efficiency gaining agent of pepsin and trypsin, wherein the common proteolytic efficiency gaining agent of the pepsin and trypsin is ascorbic acid, a salt thereof And one of the combination thereof, wherein the acid component and the salt component have a conjugate relationship, and the acid component and the salt component form a buffering formula, and wherein the buffering formula stabilizes the pepsin in an environment of pH 5 to 7.5 The co-proteolytic efficiency of the trypsin enhances the activity of the agent to enhance the proteolytic efficiency of the pepsin and the trypsin in the human body. The use of the buffer composition is added to a protein nutritional supplement, which is a formula or a high protein nutritional supplement, as claimed in claim 1. The use of claim 1, wherein the buffer formulation is uniformly mixed with the co-proteolytic efficiency gain agent of the pepsin and trypsin. The use according to claim 1, wherein the concentration of the pepsin and trypsin co-proteolytic efficiency-enhancing agent is between 60 ppm and 1000 ppm. A buffer composition for improving a protein hydrolysis efficiency of a pepsin and a trypsin in a human body, comprising: an acid component, wherein the acid component is a citric acid; a salt component, wherein the salt component is a citrate; And a common proteolytic efficiency gaining agent for pepsin and trypsin, wherein the stomach The co-proteolytic efficiency-enhancing agent of protease and trypsin is one of ascorbic acid, a salt thereof and a combination thereof, wherein the acid component and the salt component have a conjugate relationship, and the acid component and the salt component form a buffer formula And wherein the buffering formula stabilizes the activity of the pepsin and trypsin co-proteolytic efficiency-enhancing agent in an environment of pH 5 to 7.5, thereby improving the proteolytic efficiency of the pepsin and the trypsin in the human body. The buffer composition of claim 5, wherein the citrate is one of an alkali metal salt and an alkaline earth metal salt. The buffer composition of claim 5, wherein the citrate is one of sodium citrate and potassium citrate. The buffer composition of claim 5, wherein the concentration of the ascorbic acid is between 60 ppm and 1000 ppm. The buffer composition of claim 5, wherein the salt of ascorbic acid is one of a monosodium salt, a monobasic salt, a monocalcium salt, a magnesium salt, and a combination thereof. The composition of claim 5, wherein the buffer composition is selected from the group consisting of powders, granules, troches, microparticles, liquids, capsules, and sustained release dosage forms. The buffer composition of claim 5, wherein the ascorbic acid is L-ascorbic acid.