US20210219569A1 - Use and composition of buffer formulation with multiple ph values and protein digestion enhancer - Google Patents

Use and composition of buffer formulation with multiple ph values and protein digestion enhancer Download PDF

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US20210219569A1
US20210219569A1 US16/306,754 US201816306754A US2021219569A1 US 20210219569 A1 US20210219569 A1 US 20210219569A1 US 201816306754 A US201816306754 A US 201816306754A US 2021219569 A1 US2021219569 A1 US 2021219569A1
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acid
composition
ascorbic acid
ppm
salt
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Ta-Lu Shen
Fu-An Chen
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Eco-Geo Bio-Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A23K10/26—Animal feeding-stuffs from material of animal origin from waste material, e.g. feathers, bones or skin
    • A23K10/28—Animal feeding-stuffs from material of animal origin from waste material, e.g. feathers, bones or skin from waste dairy products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1322—Inorganic compounds; Minerals, including organic salts thereof, oligo-elements; Amino-acids, peptides, protein-hydrolysates or derivatives; Nucleic acids or derivatives; Yeast extract or autolysate; Vitamins; Antibiotics; Bacteriocins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/325—Working-up of proteins for foodstuffs by hydrolysis using chemical agents of casein
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/174—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/03—Organic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/03—Organic compounds
    • A23L29/035—Organic compounds containing oxygen as heteroatom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins

Definitions

  • the present invention relates to a composition for increasing digestion and/or absorption efficacies in a human being or an animal, specifically to a use and a composition for enhancing the rate of proteolysis of pepsin and trypsin in a broad pH range across acid and base.
  • proteins enter the digestive tract of a mammal they are hydrolyzed to polypeptides and amino acids by pepsin of the stomach and trypsin, chymotrypsin, carboxypeptidase in the intestinal fluid, and then, the polypeptides and amino acids are absorbed by the intestinal epithelium.
  • Gastric juice contains gastric acid and pepsinogen secreted by the stomach wall is activated into pepsin by gastric acid.
  • the pancreas secretes pancreatic juice through the pancreatic duct to the duodenum, upstream of the small intestine.
  • Trypsinogen of the pancreatic juice is secreted into the duodenum, wherein the trypsinogen is activated into trypsin by enterokinase for hydrolysing proteins to oligopeptides. Trypsin also directly or indirectly activates zymogens including trypsinogen and other inactive digestion enzymes into active enzymes in the intestine.
  • antibiotic growth promoters have been widely used in animal feeds in the animal husbandry industry since the 1950 s .
  • animal health concerns such as drug resistance and drug residue
  • the European Union has completely banned the use of antibiotics as a feed additive since 2006, and the management of drug-containing feed additives is also becoming increasingly stringent in all countries.
  • non-drug additives such as acidulants have been well developed in order to replace antibiotics. Acidulants can only decrease the pH of the feed in the gastrointestinal tract in order to extra activate pepsinogen into pepsin in the beginning stage of gastric juice secretion as the secretion has not yet reached the full operation, thereby the digestion of proteins can become more efficient.
  • the acidulant does rapidly reduce the pH of the feed and also the pH of stomach contents, on the other hand, it gradually retards normal secretion of the gastric acid, and is going to have a negative effect of a declination in digestion capability and a growth slowdown of the animals during a long-term use.
  • the Applicant of the present application has developed a non-drug additive for increasing digestion and/or absorption efficacies in humans or animals, and the non-drug additive compensates for the defects of the existing products.
  • the summary of the present invention is described below.
  • the main object of the present invention is to increase digestion and/or absorption efficacies in humans or animals.
  • a multiple pHs buffer formulation composition that operates optimally under different pH conditions of the digestive tract is provided in the present invention, and thereby the overall protein digestion and absorption is naturally enhanced.
  • the multiple pHs buffer formulation composition of the present invention can adapt to various pH environments in the gastrointestinal tract, and significantly improve the proteolysis efficiency of the major proteases in the digestive tract under an environment across acid and base (pH 2-7.5).
  • the composition of multiple pHs buffer formulation of the present invention includes at least one acid component, at least one base component and a protein digestion enhancer, wherein the at least one acid component is one selected from a group consisting of an organic acid, a phosphoric acid and a combination thereof, the at least one base component is one selected from a group consisting of an organic base, a phosphate and a combination thereof, and the protein digestion enhancer is one selected from a group consisting of an ascorbic acid, a salt of the ascorbic acid and a combination thereof.
  • the acid component and the base component form a buffer formulation so as to steadily sustain the activity of the protein digestion enhancer in the buffer formulation
  • the present invention provides a composition for enhancing protein digestion, which includes at least one acid component, at least one base component and a protein digestion enhancer, wherein the at least one acid component is one selected from a group consisting of an organic acid, a phosphoric acid and a combination thereof, the at least one base component is one selected from a group consisting 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 selected from a group consisting of an ascorbic acid, a salt of the ascorbic acid and a combination thereof.
  • FIG. 1 shows the effect of pH on the activities of three digestive enzymes in the gastrointestinal tract.
  • FIG. 2A is a curve diagram showing the proteolytic efficiencies of pepsin (control group) and experimental groups containing different concentrations of ascorbic acid measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 2.0.
  • FIG. 2B is a curve diagram showing the proteolytic efficiencies of pepsin (control group) and experimental groups containing different concentrations of ascorbic acid measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 5.0.
  • FIG. 2C is a curve diagram showing the proteolytic efficiencies of pepsin (control group) and experimental groups containing different concentrations of ascorbic acid measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 6.0.
  • FIG. 3A is a curve diagram showing the proteolytic efficiencies of trypsin (control group) and experimental groups containing different concentrations of ascorbic acid measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 5.0.
  • FIG. 3B is a curve diagram showing the proteolytic efficiencies of trypsin (control group) and experimental groups containing different concentrations of ascorbic acid measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 6.0.
  • FIG. 3C is a curve diagram showing the proteolytic efficiencies of trypsin (control group) and experimental groups containing different concentrations of ascorbic acid measured at 10 minutes, 1 hour, 2 hours, and 3 hours at pH 7.5.
  • each of the three main digestive enzymes in the gastrointestinal tract has its own optimal pH environment.
  • pepsin has the optimal activity at pH around 2
  • trypsin has the optimal activity at pH around 8.
  • the pH of the food varies rather than being constant.
  • the pH of the various parts of the small intestine are also different.
  • the pH is about 7.0 to 8.5 in the duodenum, and the pH is about 4.0 to 7.0 in the other sections of the small intestine.
  • the overall digestion and absorption function of the protein can be enhanced if the proteolytic efficiency of pepsin and trypsin in the digestive tract can be improved across acid and base (pH 2 ⁇ 7.5), especially at the pH 5 ⁇ 6 when the optimal pH environment has not yet been achieved or in individuals who have insufficient secretion of gastric acid (such as newborn babies, the elderly and weaned piglets).
  • the buffer formulation composition provided in the present invention includes at least one acid component, at least one base component, and a protein digestion enhancer. Because the buffer formulation composition of the present invention can be applied to protein nutritional supplements for humans or animals, the components used in the buffer formulation must be non-toxic to the organism. Therefore, the acid component is preferably an organic acid, the base component is preferably an organic base, and the protein digestion enhancer is ascorbic acid, the salts of the ascorbic acid, or combinations thereof. In addition, as phosphoric acid and phosphates are widely used as food additives, therefore, they are also encompassed by the scope of the acid component and base component of the present invention.
  • the acid component has a conjugate relationship with the base component to form a buffer formulation, e.g., the buffer formulation may be formulated in an organic acid and its conjugate base, or an organic base and its conjugate acid.
  • the pH value of the buffer formulation is determined by the dissociation constant (pKa) of the acid component and the ratio of the acid component to the base component. In general, the pH value is in the range of ⁇ 1 pKa.
  • the pKa values of common 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 therefore it is the best choice for the acid component in the buffer formulation of the present invention.
  • the buffer formulation formed by the appropriate acid component and base component can have multiple pHs, so it can adapt to the changes in the gastrointestinal environment to have the protein digestion enhancer better perform 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.
  • the acid component is an organic acid.
  • the organic acid is the citric acid.
  • the base component used in the present invention is an organic base or phosphate, preferably, the organic base or the phosphate refers to an alkali metal salt or an alkaline earth metal salt.
  • the organic base is sodium citrate or potassium citrate.
  • the buffer formulation of the present invention may also include a plurality of organic acids and their conjugated bases, or a plurality of organic bases and their conjugated acids.
  • the organic acid may also be combined with the phosphoric acid as the acid component in the buffer formulation of the present invention, and the organic base may also be combined with the phosphate as the base component in the buffer formulation of the present invention.
  • Any buffer formulation that can be formulated as being suitable for gastrointestinal pH conditions is within the scope of the present invention.
  • the protein digestion enhancer used in the present invention is an ascorbic acid, a salt of the ascorbic acid or a combination thereof.
  • the salt of the ascorbic acid is one of a sodium salt, a potassium salt, a calcium salt, a magnesium salt or a combination thereof.
  • the ascorbic acid also known as vitamin C, is a water-soluble and easily absorbed compound.
  • the ascorbic acid is easily degraded in an environment where the pH is greater than 4.
  • the buffer formulation of the present invention can steadily maintain the activity of ascorbic acid, the salts of the ascorbic acid, or a combination thereof in the environment at pH greater than 4, so as to achieve the goal of enhancing the proteolytic efficiencies of pepsin and trypsin in the environment across acid and base.
  • the composition of the present invention can be used as a composition for enhancing protein digestion, e.g., an additive of the protein nutritional supplement such as a formulated milk powder or an animal feed, which enhances the proteolytic efficiencies of pepsin and trypsin in the environment with a pH of 2-7.5, and preferably, significantly enhances the proteolytic efficiencies of pepsin and trypsin in the environment at pH of 5-6.
  • an additive of the protein nutritional supplement such as a formulated milk powder or an animal feed
  • a pH of 2-7.5 e.g., a formulated milk powder or an animal feed
  • the above-mentioned composition includes at least one acid component, at least one base component and a protein digestion enhancer, wherein the at least one acid component and the at least one base component form a buffer formulation, and the buffer formulation enables the protein digestion enhancer to maintain high activity in an environment across acid and base.
  • the protein digestion enhancer is distributed in the buffer formulation.
  • the buffer formulation is evenly mixed with the protein digestion enhancer.
  • the protein digestion enhancer is ascorbic acid, its sodium salt, potassium salt, calcium salt, magnesium salt or a combination thereof.
  • the protein digestion enhancer is ascorbic acid, especially L-ascorbic acid, which has a concentration ranges from 60 ppm to 1000 ppm.
  • the concentration of the ascorbic acid ranges from 60 ppm to 240 ppm, and more preferably 240 ppm.
  • the concentration of the ascorbic acid of the present invention may be higher as long as it meets the recommended daily intake for the human body (up to about 2 g per day).
  • the dosage form of the above-mentioned composition includes powder, particle, tablet, micron-particle, liquid or capsule.
  • the composition is manufactured as a delayed or extended-release formula.
  • Control groups Prepare four control solutions containing casein (3.85 mg/mL) and pepsin (385 ppm) in pH 2.0 citric acid buffer solution and put them into four test tubes.
  • the four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% trichloroacetic acid (TCA) solution was added into each test tube and mixed with the control solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • TCA trichloroacetic acid
  • o-phthalaldehyde (OPA) reagent was added into each new test tube and held for 2 minutes, and the fluorescence intensity was measured at EX. 340 nm and EM. 455 nm by a fluorescence spectrometer. The obtained fluorescence intensity was calculated as the amount of tyrosine according to a tyrosine calibration line established in advance at the same pH value, and represented as total amino acid equivalent (TAAE, ⁇ g/mL).
  • Experimental groups 120 ppm ascorbic acid: Prepare four experimental solutions containing casein (3.85 mg/mL), 120 ppm ascorbic acid and pepsin (385 ppm) in pH 2.0 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the experimental solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Control groups Prepare four control solutions containing casein (3.85 mg/mL) and pepsin (385 ppm) in pH 5.0 citric acid buffer solution and put them into four test tubes.
  • the four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the control solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • mice 60 ppm ascorbic acid: Prepare four experimental solutions containing casein (3.85 mg/mL), 60 ppm ascorbic acid and pepsin (385 ppm) in pH 5.0 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the experimental solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Experimental groups 120 ppm ascorbic acid: Prepare four experimental solutions containing casein (3.85 mg/mL), 120 ppm ascorbic acid and pepsin (385 ppm) in pH 5.0 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the experimental solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supematants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Experimental groups (240 ppm ascorbic acid): Prepare four experimental solutions containing casein (3.85 mg/mL), 240 ppm ascorbic acid and pepsin (385 ppm) in pH 5.0 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the experimental solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Control groups Prepare four control solutions containing casein (3.85 mg/mL) and pepsin (385 ppm) in pH 6.0 citric acid buffer solution and put them into four test tubes.
  • the four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the control solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Experimental groups (240 ppm ascorbic acid): Prepare four experimental solutions containing casein (3.85 mg/mL), 240 ppm ascorbic acid and pepsin (385 ppm) in pH 6.0 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the experimental solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Control groups Prepare four control solutions containing casein (3.85 mg/mL) and trypsin (385 ppm) in pH 5.0 citric acid buffer solution and put them into four test tubes.
  • the four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the control solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Experimental groups 120 ppm ascorbic acid: Prepare four experimental solutions containing casein (3.85 mg/mL), 120 ppm ascorbic acid and trypsin (385 ppm) in pH 5.0 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the experimental solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Experimental groups (240 ppm ascorbic acid): Prepare four experimental solutions containing casein (3.85 mg/mL), 240 ppm ascorbic acid and trypsin (385 ppm) in pH 5.0 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the experimental solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Control groups Prepare four control solutions containing casein (3.85 mg/mL) and trypsin (385 ppm) in pH 6.0 citric acid buffer solution and put them into four test tubes.
  • the four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the control solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • mice 60 ppm ascorbic acid: Prepare four control solutions containing casein (3.85 mg/mL), 60 ppm ascorbic acid and trypsin (385 ppm) in pH 6.0 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the control solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supematants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Experimental groups 120 ppm ascorbic acid: Prepare four experimental solutions containing casein (3.85 mg/mL), 120 ppm ascorbic acid and trypsin (385 ppm) in pH 6.0 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the experimental solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Experimental groups (240 ppm ascorbic acid): Prepare four experimental solutions containing casein (3.85 mg/mL), 240 ppm ascorbic acid and trypsin (385 ppm) in pH 6.0 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the experimental solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Control groups Prepare four control solutions containing casein (3.85 mg/mL) and trypsin (385 ppm) in pH 7.5 citric acid buffer solution and put them into four test tubes.
  • the four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the control solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • mice 60 ppm ascorbic acid: Prepare four control solutions containing casein (3.85 mg/mL), 60 ppm ascorbic acid and trypsin (385 ppm) in pH 7.5 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the control solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • Experimental groups 120 ppm ascorbic acid: Prepare four experimental solutions containing casein (3.85 mg/mL), 120 ppm ascorbic acid and trypsin (385 ppm) in pH 7.5 citric acid buffer solution and put them into four test tubes. The four test tubes were respectively incubated at 37° C. for 10 minutes, 1 hour, 2 hours and 3 hours, an equal amount of 10% TCA solution was added into each test tube and mixed with the experimental solution uniformly, and the test tubes were held at room temperature for 10 minutes and then centrifuged at 3000 rpm for 10 minutes. 20 ⁇ L of the supernatants taken from the above-mentioned test tubes at four time points were put into four new test tubes.
  • FIG. 2A show the proteolytic efficiencies of pepsin (control group) and experimental groups containing different concentrations of ascorbic acid at pH 2.0. Because the optimal environment for the pepsin is at pH 2.0, there is no significant difference in the hydrolysis efficiencies among every group, but it can still be seen that the groups containing ascorbic acid have better hydrolysis efficiencies than the control group.
  • FIG. 2B show the proteolytic efficiencies of pepsin (control group) and experimental groups containing different concentrations of ascorbic acid at pH 5.0. Because the pepsin is not in the optimal environment as at pH 5.0, the proteolytic efficiency of the pepsin is lower under this environment, but it can be seen that 240 ppm of the ascorbic acid improves the proteolytic efficiency of the pepsin significantly.
  • FIG. 2C show the proteolytic efficiencies of pepsin (control group) and experimental groups containing different concentrations of ascorbic acid at pH 6.0.
  • the pepsin is also not in the optimal environment as at pH 6.0, but it can be seen that the experimental groups containing the ascorbic acid have better proteolytic efficiencies than the control group.
  • FIG. 3A show the proteolytic efficiencies of trypsin (control group) and experimental groups containing different concentrations of ascorbic acid at pH5.0. Because the trypsin is not in the optimal environment as at pH5.0, the proteolytic efficiency of the trypsin is lower under this environment, but it can be seen that 240 ppm of the ascorbic acid improves the proteolytic efficiency of the trypsin significantly.
  • FIG. 3B show the proteolytic efficiencies of trypsin (control group) and experimental groups containing different concentrations of ascorbic acid at pH6.0.
  • the trypsin is also not in the optimal environment as at pH6.0, but it can be seen that the experimental groups containing the ascorbic acid have better proteolytic efficiencies than the control group.
  • composition of the present invention does not contain any drug component, it can be used without the doubt in drug resistance, drug-residue and the concern for food safety.

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