TW201938150A - Use and composition of buffer formulation having multiple pH values and protein digestion enhancer - Google Patents

Abstract

The present invention provides a composition for helping protein digestion, including at least one acid component, at least one base component and a protein digestion enhancer, wherein the at least one acid component is one of an organic acid, a phosphoric acid and a combination thereof, the at least one base component is one of an organic base, a phosphate and a combination thereof, the at least one acid component and the at least one base component conjugate with each other to form a buffer formulation, and the protein digestion enhancer is one of an ascorbic acid, its salt and a combination thereof.

Description

Composition of multiple pH buffering formula and protein digestion aid and use thereof

The present invention relates to a composition for increasing digestion and absorption in humans or animals. In more detail, the present invention relates to a composition for improving the proteolytic efficiency of pepsin and trypsin in the range of acid and base and its use.

After entering the mammalian digestive tract, proteins are hydrolyzed by pepsin in gastric juice, trypsin in pancreatic juice, chymotrypsin, carboxypeptidase, aminopeptidase, and dipeptidase in intestinal juice. Finally, it becomes amino acid, which is then absorbed by the intestinal epithelial cells.

The gastric juice contains pepsinogen and pepsin. Pepsinogen is activated in the stomach through pepsin and becomes pepsin. After food passes through the stomach, the pancreas secretes pancreatic juice, which is transported through the pancreatic ducts to the duodenum upstream of the small intestine. Trypsinogen in the pancreatic juice is secreted into the duodenum and promotes intestinal activation. Among them, trypsinogen is activated into trypsin by enterokinase, which can break down proteins into oligopeptides. Trypsin can also activate other proteases and trypsinogens in the intestine.

In order to help the digestion and absorption of the digestive tract and achieve growth-promoting effects, the animal husbandry industry has added antibiotic-type growth promoters to animal feeds since the 1950s. However, with the emergence of animal health concerns such as drug resistance and drug residues, the EU has been comprehensive since 2006. The use of antibiotics as feed additives is banned, and countries have tightened their management of medicated feed additives. In order to replace antibiotics, non-drug additives such as acidifiers have developed rapidly in recent years. Acidifiers can reduce the pH of the feed in the gastrointestinal tract, increase the activity of pepsin, and thus make protein digestion more efficient. However, because acidifiers rapidly reduce the pH of feed and stomach contents, they instead inhibit the normal secretion of gastric acid. Long-term use will cause the adverse effects of reduced digestion and slower growth of animals.

In view of the above shortcomings of the existing antibiotic-type growth promoters and acidifiers, the applicant of this case has developed a non-drug additive that increases digestion and absorption efficiency in humans or animals, which can make up for the shortcomings of current products. The following is a brief description of this case.

The main purpose of the present invention is to increase the digestive and absorption efficiency of humans or animals. In order not to affect the normal physiological function of the gastrointestinal tract, the present invention provides a multiple pH buffering formula combination that can operate optimally under different pH environments of the digestive tract, thereby improving the overall digestive and absorption functions of the protein.

Different from the existing feed additives, the multiple pH buffering formula combination of the present invention can significantly improve the proteolytic efficiency of the main protease in the digestive tract under the condition of trans-acid and alkaline environment (pH 2 ~ 7.5). . In order to achieve the above effect, the multiple pH buffering formula combination of the present invention includes at least one acid component, at least one alkaline component, and a protein digestion aid, wherein the at least one acid component is an organic acid, phosphoric acid, or a combination thereof, and the at least one alkaline component is An organic base, a phosphate base, or a combination thereof, and the protein digestion aid is ascorbic acid, a salt thereof, or a combination thereof. The acid component and the alkali component form a buffering formula, so that the protein digestion assistant exhibits stable and high activity in the buffering formula.

The invention provides a composition for assisting digestion of a protein, comprising: at least one acid component, at least one alkaline component, and a protein digestion aid, wherein the at least one acid component is one of an organic acid, a phosphoric acid, and a combination thereof, the At least one alkali component is one of an organic base, a phosphate base, and a combination thereof, the at least one acid component and the at least one alkali component have a conjugate relationship, and the at least one acid component and the at least one alkali component form a buffer Formula, the protein digestion aid is one of ascorbic acid, its salts and combinations thereof.

The above objects and advantages of the present invention will become more immediately apparent to those having ordinary knowledge in the technical field after referring to the following detailed description and accompanying drawings.

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

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

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

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

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

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

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

When designing the multiple pH buffering formula combination of the present invention, the pH environment of the gastrointestinal tract must be considered. Please refer to the first picture, which is recorded in Essentials of Human Physiology (2017). As shown in the figure, each of the three main digestive enzymes in the gastrointestinal tract has its own optimal pH environment. For example, pepsin has the best activity at pH 2 ~ 6, while trypsin has the best activity at pH 5 ~ 7.5. active. In fact, during the digestion of food in the gastrointestinal tract, the pH value does not remain constant, but changes between different pH values. When food enters the upper half of the stomach, the pH value is about 4.0 to 6.5, and when it enters the lower half of the stomach, the pH value is about 1.5 to 4.0. Similarly, the pH value of each section of the small intestine is also different, in the duodenum The pH value is about 7.0 ~ 8.5, and the pH value in other sections of the small intestine is about 4.0 ~ 7.0. Although the protein has better hydrolysis efficiency under the optimal pH environment of pepsin and trypsin, when the optimal pH environment has not been reached, or in individuals with insufficient gastric acid secretion (such as newborn infants), In the environment (pH 2 ~ 7.5), especially in the pH 5 ~ 6 environment, improving the proteolytic efficiency of pepsin and trypsin in the digestive tract can improve the overall digestive and absorption functions of the protein.

The buffering formula combination provided in the present invention includes at least one acid component, at least one alkali component and a protein digestion aid. Since the buffer formulation combination of the present invention can be added to humans or animals Therefore, the ingredients used must be non-toxic to the organism. The acid component is preferably an organic acid, the alkali component is preferably an organic base, and the protein digestion aid is ascorbic acid, a salt thereof, or a combination thereof. In addition, phosphoric acid and phosphate are widely used food additives, and therefore are also covered by the acid and alkali components of the present invention.

The acid component and the base component have a conjugate relationship, for example, an organic acid and a conjugate base thereof, or an organic base and a conjugate acid thereof, and the two are formulated into a buffering formula. 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 alkali component. Generally, the pH value is in the range of ± 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 3 different pKa values and is an organic acid, so it is the best choice for the acid component in the buffer formulation of the present invention.

The buffer formula formed by selecting the appropriate acid and alkali components can have multiple pH values, so it can adapt to changes in the gastrointestinal environment and ensure that the protein digestion aids can play a role 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 alkali component used in the present invention is an organic base or a phosphate base. Preferably, the organic base or the phosphate base refers to an alkali metal salt or an alkaline earth metal salt. In a preferred embodiment, the organic base is one of sodium citrate and potassium citrate.

The buffer formulation of the present invention may also include multiple organic acids and their conjugate bases, or multiple organic bases and their conjugate acids. Organic acids can also be combined with phosphoric acid as the acid component in the buffer formulation of the present invention, and organic bases can also be combined with phosphate base as the alkali component in the buffer formulation of the present invention. As long as a buffering formula suitable for the pH environment of the gastrointestinal tract can be formulated, it is within the scope of the present invention.

The protein digestion aid 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-ascorbic acid), also known as Vitamin C, is a compound that is soluble in water and easily absorbed. Since ascorbic acid is alone in an aqueous solution, it is easily degraded in an environment with a pH greater than 4. The buffer formulation of the present invention can maintain ascorbic acid, its salts, or a combination thereof in an environment with a pH greater than 4, so as to achieve transacidity. The purpose of alkaline environment is to improve the proteolytic efficiency of pepsin and trypsin.

On the other hand, the composition of the present invention can be used as a composition to help digest protein, for example, it can be added to protein nutritional supplements such as formula milk powder or animal feed, and can increase pepsin and trypsin under the environment of pH 2 ~ 7.5 It is better to improve the digestive efficiency of pepsin and trypsin under the environment of pH 5 ~ 6.

According to the concept of the present invention, the above composition includes at least one acid component, at least one alkali component, and a protein digestion aid, wherein the at least one acid component and the at least one alkali component form a buffering formula, and the buffering formula enables the protein digestion aid Maintains high activity in a trans-acid-base environment. In one embodiment, the protein digestion aid is distributed in the buffering formula. In another embodiment, the buffering formula is uniformly mixed with the protein digestion aid.

In the present invention, the protein digestion aid is ascorbic acid, a sodium salt, a potassium salt, a calcium salt, a magnesium salt or a combination thereof. In a preferred embodiment, the protein digestion aid is ascorbic acid, especially L-ascorbic acid, which has 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, as long as the daily intake of the human body is met (up to about 2 g per day), the ascorbic acid of the present invention can have a higher concentration.

According to the concept of the present invention, the dosage form of the above composition includes powder, granule, lozenge, micron granule, liquid, capsule or sustained release dosage form.

The following are examples of the present invention

(I) Experimental method

1. Casein pepsin digestion experiment (pH = 2.0)

1.1 Control group: Take four test tubes and prepare four controls containing casein (Casein, 3.85mg / mL) and pepsin (385ppm) with a citric acid buffer solution with a pH value of 2.0. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity was converted into a content based on a tyrosine calibration curve previously established at the same pH value, and expressed as a relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL).

1.2 Experimental group (60 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 60 ppm ascorbic acid, and pepsin (385 ppm) in a citric acid buffer solution with a pH value of 2.0. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

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

1.4 Experimental group (240 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 240 ppm ascorbic acid, and pepsin (385 ppm) in a citric acid buffer solution with a pH value of 2.0. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

2. Casein pepsin digestion experiment (pH = 5.0)

2.1 Control group: Take four test tubes and prepare four control solutions containing casein (Casein, 3.85 mg / mL) and pepsin (Pepsin, 385 ppm) with a citric acid buffer solution with a pH value of 5.0. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a content based on a tyrosine calibration curve established at the same pH value, and expressed as a relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL).

2.2 Experimental group (60 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 60 ppm ascorbic acid, and pepsin (385 ppm) in a citric acid buffer solution with a pH value of 5.0. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

2.3 Experimental group (120 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 120 ppm ascorbic acid, and pepsin (385 ppm) in a citric acid buffer solution with a pH value of 5.0. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) . 2.4 Prepare a new test tube and pipette 20 μL of the supernatant from the centrifuged tube and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer to measure the fluorescence at EX.340nm and EM.455nm. strength.

2.4 Experimental group (240 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 240 ppm ascorbic acid, and pepsin (385 ppm) in a citric acid buffer solution with a pH value of 5.0. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

3. Casein pepsin digestion experiment (pH = 6.0)

3.1 Control group: Take four test tubes and prepare four control solutions containing casein (Casein, 3.85 mg / mL) and pepsin (Pepsin, 385 ppm) with a citric acid buffer solution with a pH value of 6.0. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455mn. The fluorescence intensity was measured. The obtained fluorescence intensity was converted into a content based on a tyrosine calibration curve previously established at the same pH value, and expressed as a relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL).

3.2 Experimental group (60ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85mg / mL), 60ppm ascorbic acid, and pepsin (385ppm) in a citric acid buffer solution with a pH value of 6.0. Solution. The test tube was set at 37 ° C for reaction. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and the same amount of 10% trichloroacetic acid (TCA) solution was added, mixed evenly, left at room temperature for 10 minutes, and centrifuged at 3000 rpm for 10 minutes. . Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

3.3 Experimental group (120ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85mg / mL), 120ppm ascorbic acid, and pepsin (385ppm) in a citric acid buffer solution with a pH value of 6.0. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

3.4 Experimental group (240 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 240 ppm ascorbic acid, and pepsin (385 ppm) in a citric acid buffer solution with a pH value of 6.0. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of o-phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer on the The fluorescence intensity was measured at EX.340nm and EM.455nm. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

4. Casein trypsin digestion experiment (pH = 5.0)

4.1 Control group: Take four test tubes and prepare four control solutions containing casein (Casein, 3.85 mg / mL) and trypsin (Trypsin, 385 ppm) with a citric acid buffer solution with a pH value of 5.0. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity was converted into a content based on a tyrosine calibration curve previously established at the same pH value, and expressed as a relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL).

4.2 Experimental group (60ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85mg / mL), 60ppm ascorbic acid, and trypsin (Trypsin, 385ppm) in a citric acid buffer solution with a pH value of 5.0. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

4.3 Experimental group (120 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 120 ppm ascorbic acid, and trypsin (Trypsin, 385 ppm) in a citric acid buffer solution with a pH value of 5.0 Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

4.4 Experimental group (240 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 240 ppm ascorbic acid, and trypsin (385 ppm) in a citric acid buffer solution with a pH value of 5.0 Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

5. Trypsin hydrolysis test of casein (pH = 6.0)

5.1 Control group: Take four test tubes and prepare four control solutions containing casein (Casein, 3.85 mg / mL) and trypsin (Trypsin, 385 ppm) with a citric acid buffer solution with a pH value of 6.0. The test tubes were reacted at a constant temperature of 37 ° C. The four test tubes were reacted for 10 minutes, 1 hour, 2 hours, After 3 hours, an equal amount of a 10% trichloroacetic acid (TCA) solution was added to each of them, and they were mixed uniformly, and allowed to stand at room temperature for 10 minutes, and then centrifuged at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity was converted into a content based on a tyrosine calibration curve previously established at the same pH value, and expressed as a relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL).

5.2 Experimental group (60 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 60 ppm ascorbic acid, and trypsin (385 ppm) in a citric acid buffer solution with a pH value of 6.0. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

5.3 Experimental group (120 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 120 ppm ascorbic acid, and trypsin (385 ppm) in a citric acid buffer solution with a pH value of 6.0 Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of o-phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer on the The fluorescence intensity was measured at EX.340nm and EM.455nm. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

5.4 Experimental group (240ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85mg / mL), 240ppm ascorbic acid, and trypsin (385ppm) in a citric acid buffer solution with a pH value of 6.0 Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

6. Casein trypsin digestion experiment (pH = 7.5)

6.1 Control group: Take four test tubes and prepare four control solutions containing casein (Casein, 3.85 mg / mL) and trypsin (Trypsin, 385 ppm) with a citric acid buffer solution with a pH value of 7.5. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity was converted into a content based on a tyrosine calibration curve previously established at the same pH value, and expressed as a relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL).

6.2 Experimental group (60ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85mg / mL), 60ppm ascorbic acid and trypsin (Trypsin, 385ppm) in a citric acid buffer solution with a pH of 7.5. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

6.3 Experimental group (120 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 120 ppm ascorbic acid, and trypsin (385 ppm) in a citric acid buffer solution with a pH of 7.5. Solution. The test tubes were reacted at a constant temperature of 37 ° C. Four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, and an equal amount of a 10% trichloroacetic acid (TCA) solution was added, mixed well, and left at room temperature for 10 hours. Minutes and centrifuge at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

6.4 Experimental group (240 ppm ascorbic acid): Take four test tubes and prepare four experiments with casein (Casein, 3.85 mg / mL), 240 ppm ascorbic acid, and trypsin (Trypsin, 385 ppm) in a citric acid buffer solution with a pH of 7.5. Solution. The test tubes were reacted at a constant temperature of 37 ° C. After four test tubes were reacted for 10 minutes, 1 hour, 2 hours, and 3 hours, respectively, an equal amount of 10% was added. Chloroacetic acid (TCA) solution, mixed well, left at room temperature for 10 minutes, and centrifuged at 3000 rpm for 10 minutes. Take four new test tubes, add 20 μL of the supernatant at the four time points, and add 2.4 mL of phthalaldehyde (OPA) reagent. After standing for 2 minutes, use a fluorescence spectrometer at EX.340nm and EM.455nm. The fluorescence intensity was measured. The obtained fluorescence intensity is converted into a tyrosine calibration curve established in advance at the same pH value and the same ascorbic acid concentration, and expressed as the relative equivalent of total amino acids (Total Amino Acid Equivalent (TAAE) μg / mL) .

Experimental results

Please refer to Tables 2 and 2A, which show the proteolytic efficiency of pepsin (control group) and the experimental group containing different concentrations of ascorbic acid at pH 2.0. Because pH 2.0 is the most suitable environment for pepsin, there is not much difference in hydrolysis efficiency between the groups, but it can still be seen that the group containing ascorbic acid has better hydrolysis efficiency than the control group.

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

Please refer to Tables 3 and 2B, which show the proteolytic efficiency of pepsin (control group) and the experimental group containing different concentrations of ascorbic acid at pH 5.0. At pH 5.0, pepsin is not the most suitable environment, so pepsin has poor hydrolysis efficiency under this environment, but 240 ppm ascorbic acid can be observed to significantly increase the hydrolysis efficiency of pepsin.

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

Please refer to Table 4 and the second graph C, which shows the proteolytic efficiency of pepsin (control group) and the experimental group containing different concentrations of ascorbic acid at pH 6.0. pH 6.0 is not the optimal environment for pepsin, 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 TAAE μg / mL

Please refer to Table 5 and the third graph A, which shows the proteolytic efficiency of trypsin (control group) and the experimental group containing different concentrations of ascorbic acid at pH 5.0. At pH 5.0, trypsin is not the most suitable environment, so trypsin has poor hydrolysis efficiency under this environment, but 240ppm ascorbic acid can be observed to significantly improve trypsin hydrolysis efficiency.

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

Please refer to Tables 6 and 3B, which show 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, it is not the optimal environment for trypsin, 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 TAAE μg / mL

Please refer to Tables 7 and 3C, which show the proteolytic efficiency of trypsin (control group) and the experimental group containing different concentrations of ascorbic acid at pH 7.5. Because pH 7.5 is the most suitable environment for trypsin, there is not much difference in hydrolysis efficiency between the groups, 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 TAAE μg / mL

Comparing the relative concentration of each pH environment, the amino acid concentration measured in accordance with the optimized environment is the highest, and the concentration gradually decreases with the deterioration of environmental conditions. Both pepsin and trypsin have the same trend. Under the pH 2 ~ 6 environment, using the optimized (pH 2) control group as a comparison index, it was observed that ascorbic acid has a significant gain effect on the pepsin proteolysis efficiency under pH 5, pH 6 and pH 2 environments. Under the pH 5 ~ 7.5 environment, using the optimized (pH 7.5) control group as a comparison index, it was observed that ascorbic acid has a significant gain effect on trypsin proteolysis efficiency at pH 5, pH 6 and pH 7.5.

Since the composition of the present invention contains no medicinal ingredients, there is less concern about drug resistance and food safety.

The invention is an incompetent and innovative invention with deep industrial application value. In addition, the present invention can be modified in any way by those with ordinary knowledge in the technical field, without departing from the scope to be protected by the scope of the appended patents.

Claims (15)

Use of a composition for increasing digestion and absorption in humans or animals. The composition includes at least one acid component, wherein the at least one acid component is one of an organic acid, a phosphoric acid, and a combination thereof. ; At least one alkali component, wherein the at least one alkali component is one of an organic base, a monophosphate base, and a combination thereof; the at least one acid component and the at least one alkali component have a conjugate relationship; and the at least one acid component and The at least one alkali component forms a buffering formula; and a protein digestion aid, wherein the protein digestion aid is one of ascorbic acid, a salt thereof, and a combination thereof. The use as described in item 1 of the patent application range, wherein the buffer formulation has multiple pH values. The use as described in item 1 of the patent application range, wherein the buffering formula is used to improve the stability of the ascorbic acid in a pH greater than 4 environment. A composition for assisting digestion of a protein, comprising: at least one acid component, wherein the at least one acid component is one of an organic acid, a phosphoric acid, and a combination thereof; at least one alkaline component, wherein the at least one alkaline component is an organic component One of a base, a monophosphate base, and a combination thereof, the at least one acid component and the at least one base component have a conjugate relationship, and the at least one acid component and the at least one base component form a buffering formula; and a protein digestion aid Agent, wherein the protein digestion aid is one of ascorbic acid, a salt thereof, and a combination thereof. The composition according to item 4 of the scope of patent application, which is added to a protein nutritional supplement, and the protein nutritional supplement is a formula milk powder or an animal feed. The composition according to item 4 of the scope of patent application, wherein the organic acid is selected from the group consisting of formic acid, ethyl Acid, propionic acid, butyric acid, malic acid, fumaric acid, lactic acid, citric acid and phosphoric acid, and the organic base and the base of the phosphoric acid are one of an alkali metal salt and an alkaline earth metal salt One. The composition according to item 6 of the scope of the patent application, wherein the organic acid is citric acid, and the organic base is one of sodium citrate and potassium citrate. The composition according to item 4 of the scope of patent application, wherein the buffering formula is uniformly mixed with the protein digestion aid. The composition according to item 4 of the patent application range, wherein the buffering formula has multiple pH values. The composition according to item 4 of the scope of patent application, wherein the concentration of the ascorbic acid is between 60 ppm and 1000 ppm. The composition according to item 4 of the scope of the patent application, wherein the ascorbic acid salt is one of a sodium salt, a potassium salt, a calcium salt, a magnesium salt and a combination thereof. The composition according to item 4 of the scope of patent application, wherein the composition is used to improve the digestive function of pepsin and trypsin under the environment of pH 2 ~ 7.5. The composition according to item 12 of the scope of patent application, wherein the composition is used to improve the digestive function of pepsin and trypsin under the environment of pH 5-6. The composition according to item 4 of the scope of patent application, wherein the dosage form of the composition is one selected from the group consisting of powder, granules, lozenges, micro granules, liquids, capsules, and sustained release dosage forms. The composition according to item 4 of the application, wherein the ascorbic acid is L-ascorbic acid.